biologia (pakistan) issn0006-3096 biological society ...m.anwar malik department of zoology, gc...
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BIOLOGIA (PAKISTAN) ISSN 0006-3096
BIOLOGICAL SOCIETY OF PAKISTANBiological Laboratories, GC University, Lahore, Pakistan
PRESIDENTM. Anwar Malik
Department of Zoology,GC University, Lahore, Pakistan
VICE PRESIDENTS
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BIOLOGIA(PAKISTAN)
Zaheer-ud-din KhanDep311ment of Botany,
GC University, Lahore, Pakistan
Farrukh HussainDep311ment of Botany.
Peshawar University, Peshawar, Pakistan
Vol. 54, No.1, 2008
BUSINESS MANAGERM. Sharif Mughal
Department of Zoology.GC University, Lahore, Pakistan
Farida Begum Yasmin NawazDepartment of Zoology, Department of Zoology,
University of Karachi, Karachi, Pakistan University of Balochistan, Quetta, PakistanShafiq-ur-RehmanDepartment of Botany,
University of AJK, Muzaffarabad, AJKGENERAL SECRETARY JOINT SECRETARY
Ikram-ul-I-iaq, SI Javed IqbalInstitute of Industrial Biotechnology, Govemment College, Multan,GC University, Lahore, Pakistan Pakistan
EDITORS
Editor-in"Chiej
AZIZULLAH
,
'Of
Nicola Lacerata (Italy)[email protected]
Arshad Ali (U.S.A)[email protected].
C.J. Secombes(u.K)[email protected]
B.Faye (France)[email protected]
Salih Dogan (Tu"'ey)sadogan@ataunLedu,tr
Wolfgang Von Engelhardt(Germany)
Qing Hai Fan (China)
. FOREIGN
Zaheer ud Din Khankhan_zaheerpk@yahoo,com
PAKISTAN
M. Anwar [email protected]
M. Sharif [email protected]
M. R. MirzaA.U. [email protected]
Shamshad [email protected]
BIOLOGICAL SOCIETY OF PAKISTANBiological Laboratories, GC University Lahore, Pakistan
/
PeiSheng-Ji (China)KazuoN.Watanabe (Japan)Jin lou (U. K.)Mary Tatnar (U. K.)William Bill Radke (U. S. A.)David B.Wilson (U. SA.)Lee A. Meserve (U..S. A.)Fabrizio Rueca (llaly)SilvanaDiverio (Italy)Giorgia Della Rocca (Italy)R. Pabst (Germany)
Editor-in-ChiejAzizullah
Department of Zoology,GC University, Lahore, Pakistan
AD VISOR Y BOARDMuhammadAshraf (Univ. of AgrL, Faisalabad)laheer Ahmed (GC University, Lahore)SaminaJalali (Quaid-e-AzamUniversity, Islamabad)SaeedAhmad Malik (B. l.U., Multan)'HadayatullahAbro (University of Sindh,'Jamshoro)JumaKhan KakarSulemankhel (University ofBalochistan,Quetta)MufakhiraJan DUffani(University of Balochistan,Quetta)Ajmal Khan (Universityof Karachi, Karachi)SanaUllah Khan Khattak (University of Peshawar,Peshawar)Ihsan.Elahi (Universily of Peshawar, Peshawar)lahid HussainMalik (Universityof AJK, Muzaffarabad)
\ 1-
ANNUAL MEMBERSHIP
PAKISTAN
BIOLOGIA(PAKISTAN) 2008, 54 (1), 1-15PK ISSN 0006 - 3096
Formulation of semi-purified diets for striped bass,Aforone saxatilis larvae
MUHAMMAD ASHRAF, DAVlIJ BENGSTON & K.L. SIMPSON
Complete Volume
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Deputy Director Fisheries, Fish Hatchery, Sationa Raad, Faisalahad (MA)Department of Food Science and Nutrition, University of
Rhode Island, US.A. (DB, KLS)
.ABSTRACT
One of the major problems that.limit Marone saxatilis mass culture is non-availability of seed in sufficient quantities due to unreliab.ilityof live feeds andtheir high cost. To completely replace it with artificial diets or at least reduce itsquantity in larval food without compromi_~ing growth and survival of fish,. variousiso-caloric and iso-nitrogenous semi-pudfied diets were formulated. Commercialstarter diets were also used for comparison. In two feeding trials, 8 and 12-day-old striped bass was used in trial 1 and 2, respectively.' Former had g dietarytreatments and was continued for two weeks. Instar I, live Anemia nauplii andzooplankton served as control while their encapsulated forms were secondarycontrols. Fish fed on live Artemia and zooplankton exhibited the highest growthand survival. Encapsulated live food was ranked second. Later trial had 11treatments and lasted for four weeks. Same age and weight larval fish were fedon live Anemia nauplii (control), Artemia capsules and casein based semi-purified diet. Tannic acid was incorporated in encapsulation material of some ofthe diets to enhance capsule stability. Similarly, betain was included in selectedinert diets to boost diet acceptability. Some inert di\its were also periodicallysupplemented with live Artemia nauplii. Fish reared on live Artemia maintained itssuperiority in growth and survival over its counterparts. Tannic acid containingdiets with periodic supplementation of live Artemia gave 40-50% survival. Itappears that striped bass larvae cannot digest microcapsules due to poorlydeveloped digestive system.
Key words: Striped bass, Artemia, Semipurified diets, Enzymes, Micro-encapsulation
•
All correspondence relating to membership and publication should be addressed to
Dr. M. Sharif MughalBusiness Manager,Department of Zoology,
GC University, Lahore, PakistanE-mail: [email protected]
BIOLOGICAL SOCIETY OF PAKISTAN,Biological Laboratories, GC University, Lahore, Pakistan
www.biosoc.org.pk
INTRODUCTION
Like other fish species, production of striped bass, Morone saxitilis, incommercial hatcheries still depends on supply of live food such as rotifers,Brachionus plicatilis and Artemia (Cahu & Infante, 2001). Pro-larvae and larvae,soon after yolk sac absorption, demand an exogenous and continuous source offood due to their rudimentary digestive system (Blaxter, 1969). Feeding fishlarvae from hatching to metamorphosis on live organisms such as algae andmicro crustaceans is technically difficult and may be impractical for large scaleoperations (Gabaudan, 1984). Persistent availability of sufficient food ofsustainable quality around the larval fish is and has been a major constraint
ANNUAL MEMBERSHIP
PAKISTAN
BIOLOGIA(PAKISTAN) 2008, 54 (1), 1-15PK ISSN 0006 - 3096
Formulation of semi-purified diets for striped bass,Aforone saxatilis larvae
MUHAMMAD ASHRAF, DAVlIJ BENGSTON & K.L. SIMPSON
Complete Volume
JOURNAL SUBSCRIPTION
Life Fellow
Fellow
Student Member(Will not receive free copyof journal)
FOREIGN
Fellow
,PAKISTAN
FOREIGN
• Surface mail
• Air Mail
RS.1000.00
US $100.00
US $120.00
RS.3000.00
RS.500.00(per annum)
RS.200.00(per annum)
US $ 30.00(per annum)
Single Copy
RS.500.00
US $ 60.00
US $ 70.00
Deputy Director Fisheries, Fish Hatchery, Sationa Raad, Faisalahad (MA)Department of Food Science and Nutrition, University of
Rhode Island, US.A. (DB, KLS)
.ABSTRACT
One of the major problems that.limit Marone saxatilis mass culture is non-availability of seed in sufficient quantities due to unreliab.ilityof live feeds andtheir high cost. To completely replace it with artificial diets or at least reduce itsquantity in larval food without compromi_~ing growth and survival of fish,. variousiso-caloric and iso-nitrogenous semi-pudfied diets were formulated. Commercialstarter diets were also used for comparison. In two feeding trials, 8 and 12-day-old striped bass was used in trial 1 and 2, respectively.' Former had g dietarytreatments and was continued for two weeks. Instar I, live Anemia nauplii andzooplankton served as control while their encapsulated forms were secondarycontrols. Fish fed on live Artemia and zooplankton exhibited the highest growthand survival. Encapsulated live food was ranked second. Later trial had 11treatments and lasted for four weeks. Same age and weight larval fish were fedon live Anemia nauplii (control), Artemia capsules and casein based semi-purified diet. Tannic acid was incorporated in encapsulation material of some ofthe diets to enhance capsule stability. Similarly, betain was included in selectedinert diets to boost diet acceptability. Some inert di\its were also periodicallysupplemented with live Artemia nauplii. Fish reared on live Artemia maintained itssuperiority in growth and survival over its counterparts. Tannic acid containingdiets with periodic supplementation of live Artemia gave 40-50% survival. Itappears that striped bass larvae cannot digest microcapsules due to poorlydeveloped digestive system.
Key words: Striped bass, Artemia, Semipurified diets, Enzymes, Micro-encapsulation
•
All correspondence relating to membership and publication should be addressed to
Dr. M. Sharif MughalBusiness Manager,Department of Zoology,
GC University, Lahore, PakistanE-mail: [email protected]
BIOLOGICAL SOCIETY OF PAKISTAN,Biological Laboratories, GC University, Lahore, Pakistan
www.biosoc.org.pk
INTRODUCTION
Like other fish species, production of striped bass, Morone saxitilis, incommercial hatcheries still depends on supply of live food such as rotifers,Brachionus plicatilis and Artemia (Cahu & Infante, 2001). Pro-larvae and larvae,soon after yolk sac absorption, demand an exogenous and continuous source offood due to their rudimentary digestive system (Blaxter, 1969). Feeding fishlarvae from hatching to metamorphosis on live organisms such as algae andmicro crustaceans is technically difficult and may be impractical for large scaleoperations (Gabaudan, 1984). Persistent availability of sufficient food ofsustainable quality around the larval fish is and has been a major constraint
2 M. ASHRAF ETAL. BIOLOGIAVOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 3
.'0
(Sorgeloos, 1980 and Watanabe et al., 1983). Collection of Daphnia. Artemiaand Brachionus from natural sources can import pathogens and parasites to thehatchery (Uys & Hecht, 1985). Pathogen free and cost effective compound dietsubstitution for live prey is therefore crucial for lowering production cost andensuring sustainable supply of quality fish seed (Uys & Hecht, 1985 qnd Bautistaet al., 1989).
le Ruyet et al. (1993) formulated a diet adequate to sustain good growthand survival in European sea bass, Dicentrarchus labrax. Similarly, Zambonino etal. (1997) obtained significant growth and good survival in sea bass when fed oncompound feed. Cahu et al. (1998) have achieved up to 35% survival when fedexclusively on compound feed from mouth opening with no cannibalism. The.survival rate can be attributed to the efficiency of the compound diet, since unfed.sea bass larvae do not survive after day fifteen. Concurrently some survival hasbeen obtained in sea bream, Sparus aurata (Fernandez-Diaz & Yufera, 1997)and in Pagrus major (Takeuchi et aI., 1998), but survival remained very poor inlarval Chitala when fed on boiled egg-yolk (Sarkar et al., 2006).
Formulation of an effective compound diet for fish larvae is not easy toachieve. Nutritional requirements of larval stages of most of the species are notknown and. are hard to know by traditional nutritional approaches and, further,they change with the development of larvae (Dabrowski, 1984). Further, the dietshould 'be palatable, nutritionally adequate and particle size should be compatiblewith the oral cavity of the target animal and must be the true representative of thewhole. These particles should resemble their natural counterparts in elasticityand collapsibility so that fish, chemically and visually, can recognize them.•..asfood' (Applebaum, 1980). Moreover, the individual particles must be fullyprotected from nutrient leaching. Poor diet stability leads to nutrient leaching.particle dissolution and subsequently bacterial fouling (langdon, 2003). Feedshould have a low moisture level to prolong the shelf life and inhibit the growth ofmicroorganisms.
Microencapsulated diets appear to be a good option to overcome theselimitations. They can combine the texture of moist diets and the high stability ofthe best dry pellets. Their high stability may protect the nutrients from leachingand make them available to fish (leibovitz et al., 1987). They are consumed byestuarine fish species e.g., Menidia beryllina and crustaceans (leibovitz et al.,1987 and Yufera et al.. 2002). The objective of present studies was to formulatenutritionally balanced and well protected water stable diets for striped bass. Dietformulae were based on the composition of natural zooplankton and particleswere carefully encapsulated. The prepared diets were evaluated against liveArtemia, live zooplankton and some commercially 'known starter diets.
MATERIALS AND METHODS
Experimental protocol and setupEight and 12-day old striped bass larvae were transported in oxygen
. permeated plastic bags, from the University of Maryland's Crane Aquaculture
Facility (Trial 1) and the Verplanck Hatchery Hudson River, New York (Trial 2),respectively. They were transferred to 190-l. aquaria and acclimated to thelaboratory conditions in 5% saline water for three days. The experimental systemconsisted of acrylic cylindrical fish egg hatching jars (150 x 46H cm; III capacity).On the 4'h, 11th and 15th day post hatching, fish were randomly placed into 28(Trial 1) and 31 (Trial 2) jars. A random sample of five fish Was taken from theoriginal stock for preliminary weight arid length measurements (Table 5). Fishwere raised in 5% saline water at 24-26'C.
Every morning before the first feeding, the jars were cleaned of uneaten feedand extraneous materials were removed from the jars. Any dead fish found wereremoved and counted. One-third of the total water was removed and replaced.with fresh saline water (5%). The fry were fed 100% of their wet weightthroughout the study period. Manually dispensed feeds were stirred in water for ashort time at 1000, 14.00 and 18.00 hours with 3% daily increase. At the end ofeach experiment, the water was completely drained off and the fish we'reharvested on a 150 ~m net suspended in 2-phenoxy ethanol. The euthanized'- .. animals were blotted dry, weighed by an electronic balance to the nearest 0.1 mgand measured with a caliper to the nearest 0.1 mm.Preparation of diet for trial 1Diet 1(lA): Artemia nauplii were hatched from a single batch of referenceArtemia cysts II (RAC II) (Bengtson et aI., 1985) in 500 ml separatorifunnelscontaining 30% saline water with pH range of 7.75-8.3 and at 26'C temperature.under constant light and aeration. After 36 hours of incubation, aeration wasstopped and the instar I nauplii were allowed to settle at the bcttom .of thehatching flask and then collected on a 150 ~m sieve. They were rinsed with'de-ionized water and blotted dry. The nauplii were weighed and hOrTlOgenouslydispersed in the tank once every day early in the morning.Diet 2. (lZ): Live zooplanktons, predominantly Brachionus plicatilis, collecteddaily from Pettaquamscutt River Narragansett, R. I., were concentrated atplankton net of 110 ~m mesh. Upto day three of the experiment, they were. treated exactly like Artemia nauplii and fed to fish but due to mortality of.zooplankton during the long handling time, the strategy was changed. They were'transferred to a graduated beaker that contained 50 ml sea water and examinedfor their total biomass. Repeated weighing and dissolution gave qualitative andquantitative estimation of zooplankton in a given volume of water. This practicewas repeated every 3'° day to make sure that fish were not under-fed.Diet 3 (EAN): RAC II Artemia were hatched as in diet 1. The instar I nauplii wereharvested on a 150 ~m sieve, rinsed with d.e-ionized water and blotted dry on apaper toWel. They were weighed and encapsulated according to the procedureexplain",d for diet 5 in this series except that the albumin-alginate ratio was 1:1.Diet 4 (EZ): Zooplanktons were collected as in treatment 2 and were sievedthrough several meshes to remove extraneous material and undesirableorganisms present in sample water. They were thoroughly rinsed witn de-ionizedwater, blotted dry, weighed and encapsulated following the procedure asexplained for diet 5.
2 M. ASHRAF ETAL. BIOLOGIAVOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 3
.'0
(Sorgeloos, 1980 and Watanabe et al., 1983). Collection of Daphnia. Artemiaand Brachionus from natural sources can import pathogens and parasites to thehatchery (Uys & Hecht, 1985). Pathogen free and cost effective compound dietsubstitution for live prey is therefore crucial for lowering production cost andensuring sustainable supply of quality fish seed (Uys & Hecht, 1985 qnd Bautistaet al., 1989).
le Ruyet et al. (1993) formulated a diet adequate to sustain good growthand survival in European sea bass, Dicentrarchus labrax. Similarly, Zambonino etal. (1997) obtained significant growth and good survival in sea bass when fed oncompound feed. Cahu et al. (1998) have achieved up to 35% survival when fedexclusively on compound feed from mouth opening with no cannibalism. The.survival rate can be attributed to the efficiency of the compound diet, since unfed.sea bass larvae do not survive after day fifteen. Concurrently some survival hasbeen obtained in sea bream, Sparus aurata (Fernandez-Diaz & Yufera, 1997)and in Pagrus major (Takeuchi et aI., 1998), but survival remained very poor inlarval Chitala when fed on boiled egg-yolk (Sarkar et al., 2006).
Formulation of an effective compound diet for fish larvae is not easy toachieve. Nutritional requirements of larval stages of most of the species are notknown and. are hard to know by traditional nutritional approaches and, further,they change with the development of larvae (Dabrowski, 1984). Further, the dietshould 'be palatable, nutritionally adequate and particle size should be compatiblewith the oral cavity of the target animal and must be the true representative of thewhole. These particles should resemble their natural counterparts in elasticityand collapsibility so that fish, chemically and visually, can recognize them.•..asfood' (Applebaum, 1980). Moreover, the individual particles must be fullyprotected from nutrient leaching. Poor diet stability leads to nutrient leaching.particle dissolution and subsequently bacterial fouling (langdon, 2003). Feedshould have a low moisture level to prolong the shelf life and inhibit the growth ofmicroorganisms.
Microencapsulated diets appear to be a good option to overcome theselimitations. They can combine the texture of moist diets and the high stability ofthe best dry pellets. Their high stability may protect the nutrients from leachingand make them available to fish (leibovitz et al., 1987). They are consumed byestuarine fish species e.g., Menidia beryllina and crustaceans (leibovitz et al.,1987 and Yufera et al.. 2002). The objective of present studies was to formulatenutritionally balanced and well protected water stable diets for striped bass. Dietformulae were based on the composition of natural zooplankton and particleswere carefully encapsulated. The prepared diets were evaluated against liveArtemia, live zooplankton and some commercially 'known starter diets.
MATERIALS AND METHODS
Experimental protocol and setupEight and 12-day old striped bass larvae were transported in oxygen
. permeated plastic bags, from the University of Maryland's Crane Aquaculture
Facility (Trial 1) and the Verplanck Hatchery Hudson River, New York (Trial 2),respectively. They were transferred to 190-l. aquaria and acclimated to thelaboratory conditions in 5% saline water for three days. The experimental systemconsisted of acrylic cylindrical fish egg hatching jars (150 x 46H cm; III capacity).On the 4'h, 11th and 15th day post hatching, fish were randomly placed into 28(Trial 1) and 31 (Trial 2) jars. A random sample of five fish Was taken from theoriginal stock for preliminary weight arid length measurements (Table 5). Fishwere raised in 5% saline water at 24-26'C.
Every morning before the first feeding, the jars were cleaned of uneaten feedand extraneous materials were removed from the jars. Any dead fish found wereremoved and counted. One-third of the total water was removed and replaced.with fresh saline water (5%). The fry were fed 100% of their wet weightthroughout the study period. Manually dispensed feeds were stirred in water for ashort time at 1000, 14.00 and 18.00 hours with 3% daily increase. At the end ofeach experiment, the water was completely drained off and the fish we'reharvested on a 150 ~m net suspended in 2-phenoxy ethanol. The euthanized'- .. animals were blotted dry, weighed by an electronic balance to the nearest 0.1 mgand measured with a caliper to the nearest 0.1 mm.Preparation of diet for trial 1Diet 1(lA): Artemia nauplii were hatched from a single batch of referenceArtemia cysts II (RAC II) (Bengtson et aI., 1985) in 500 ml separatorifunnelscontaining 30% saline water with pH range of 7.75-8.3 and at 26'C temperature.under constant light and aeration. After 36 hours of incubation, aeration wasstopped and the instar I nauplii were allowed to settle at the bcttom .of thehatching flask and then collected on a 150 ~m sieve. They were rinsed with'de-ionized water and blotted dry. The nauplii were weighed and hOrTlOgenouslydispersed in the tank once every day early in the morning.Diet 2. (lZ): Live zooplanktons, predominantly Brachionus plicatilis, collecteddaily from Pettaquamscutt River Narragansett, R. I., were concentrated atplankton net of 110 ~m mesh. Upto day three of the experiment, they were. treated exactly like Artemia nauplii and fed to fish but due to mortality of.zooplankton during the long handling time, the strategy was changed. They were'transferred to a graduated beaker that contained 50 ml sea water and examinedfor their total biomass. Repeated weighing and dissolution gave qualitative andquantitative estimation of zooplankton in a given volume of water. This practicewas repeated every 3'° day to make sure that fish were not under-fed.Diet 3 (EAN): RAC II Artemia were hatched as in diet 1. The instar I nauplii wereharvested on a 150 ~m sieve, rinsed with d.e-ionized water and blotted dry on apaper toWel. They were weighed and encapsulated according to the procedureexplain",d for diet 5 in this series except that the albumin-alginate ratio was 1:1.Diet 4 (EZ): Zooplanktons were collected as in treatment 2 and were sievedthrough several meshes to remove extraneous material and undesirableorganisms present in sample water. They were thoroughly rinsed witn de-ionizedwater, blotted dry, weighed and encapsulated following the procedure asexplained for diet 5.
4 M. ASHRAF ET AL BIOLOGIA VOL 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 5
"
Table 1: Ingredient composition (in percentages) of casein and yeast based. artificial diets for striped bass, Morone saxatilis larvae.
Ingredients Casein based Yeast based
Casein 60.0 -Yeast - 78.7Vitamin 5.0 2.6
Mineral mix 4.0 0.6Menhaden fish oil 13.0 10.9
Fatly acyl methyl esters 4.2 5.05Stay-C (Ascorbyl-PP) 0.1 005
Carboxymethyl cellulose 13.7 2.1
Table 2: Proximate analysis of live, live encapsulated, 'formulated andcommercial diets on dry weight basis.
Deitnumber 1 2 3 4 5 6 7 8 9~
NutrientsI
Protein 61 57 61 57 57 57 53 53 44Fats 11 17 11 17 17 17 10 10 11Ash 11 25 11 25 4 19 13 13 12Moisture - - 5 5 5 5 5 5 13Note: Diet follows the pattern as in feeding trial 1(Materials and Methods section)
Diet 5 (ECO): Casein (Erie Foods International, Inc.,ll) contained 94% protein,0% fats, 1% ash and 5% moisture (Table 1). Casein, vitamin, mineral mix andcarboxymethyl cellulose (Table 1), were individually powdered in a coffee 9rinderand sieved throu9h 106 J.1mmesh. The diet was formulated based on thechemical composition of zooplanktons (Table 2). The pre-sieved ingredients werethen thoroughly mixed in Sorvall Oman i-Mixer. Intermixing of dry ingredients wasfollowed by the addition of Menhadell-fish oil (Zapata Haynie Corporation,Virginia), Stay-C (VTI Vitamin Technologies International, 10) and fatly acid. methylesters (Ocean Organics, RI). The mixture obtained was then encapsulated(leibovitz, 1990) ,
I ,
"I
I;
I'
Ii'
j••
Table 3: Fatty acid composition (mg/gm dry weight) of live and inert diets .
Names I Diet numberI 1& 3 I 2 I 4 I 5 I 6 I 7&8 I 9,
14:0 1.1 1.9 19 15 1.6 26 10414:1 0.9 - - 01 0.1 - 0.815:0 0.6 - - 01 0.1 0.2 00415:1 0.5 - - - - - -16:0 125 7.5 7.5 4.3 4.5 12.9 10.916:1 - 1204 4.2 4.2 204 2.2 30417:0 3.0 - - 004 0.4 0.5 0.816:2 4.0 - - 004 004 I. 0.6 1318:0 4.0 1.0 1.0 104 104 304 2.918:1 33.5 33 3.3 404 r 4.4 10.7 180416:3 - - - '';'',J 0.2 0.2 - -18:2 10.0 - - 0.2 0.2 - -18:2 - 0.7 0.7 02 004 14.8 8620:0 - - - - - 0.2 -18:3 0.5 004 0.4 5 4.7 1.7 518:4 4.5 - - - 1.2 1.5 -21:5 19 1.9 0.7 0.6 0.7 1.020:2 - - - - - 0.2 -20:3 - - - 0.1 0.1 1.1 -22:0 - - - 2.2 22 10 -. 20:4 - - - 0.2 0.2 - -20:5 4.5 4.3 4.3 7.1 7.1 4.1 4.122:5 5.5 - - 0.1 0.1 - -22:6 - - - 0.3 0.3 - -22:5 - - - 09 0.8 0.5 -22:6 - 5 5 4.6 4.6 5.1 -
Diet 6 (EYO): Autolysed yeast extract (Rhone-Poulenc Food IngredientsDivision, PAl was finely ground (106 J.1m)and mixed with measured amount offish oil, fatty acid methylesters, vitamins and minerals and then encapsulated. (leibovitz, 1990). Protein, fat, ash and moisture contents of yeast were 77, 1, 13and 9 %, respectively. .Diet 7 (AP200): Closed formula commercial starter diet (AP200) (ZieglerBrothers Inc., PAl, was analyzed for its nutritional information and fed to fishwithout any further modification.Diet 8 (EAP200): Encapsulated diet 7.Diet 9 (ABO): Artemia meal-based commercial starter diet (PDM Associates,CT), was analyzed and sieved through 150 and 250 J.1mmesh and fed to fishwithout any modification.
4 M. ASHRAF ET AL BIOLOGIA VOL 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 5
"
Table 1: Ingredient composition (in percentages) of casein and yeast based. artificial diets for striped bass, Morone saxatilis larvae.
Ingredients Casein based Yeast based
Casein 60.0 -Yeast - 78.7Vitamin 5.0 2.6
Mineral mix 4.0 0.6Menhaden fish oil 13.0 10.9
Fatly acyl methyl esters 4.2 5.05Stay-C (Ascorbyl-PP) 0.1 005
Carboxymethyl cellulose 13.7 2.1
Table 2: Proximate analysis of live, live encapsulated, 'formulated andcommercial diets on dry weight basis.
Deitnumber 1 2 3 4 5 6 7 8 9~
NutrientsI
Protein 61 57 61 57 57 57 53 53 44Fats 11 17 11 17 17 17 10 10 11Ash 11 25 11 25 4 19 13 13 12Moisture - - 5 5 5 5 5 5 13Note: Diet follows the pattern as in feeding trial 1(Materials and Methods section)
Diet 5 (ECO): Casein (Erie Foods International, Inc.,ll) contained 94% protein,0% fats, 1% ash and 5% moisture (Table 1). Casein, vitamin, mineral mix andcarboxymethyl cellulose (Table 1), were individually powdered in a coffee 9rinderand sieved throu9h 106 J.1mmesh. The diet was formulated based on thechemical composition of zooplanktons (Table 2). The pre-sieved ingredients werethen thoroughly mixed in Sorvall Oman i-Mixer. Intermixing of dry ingredients wasfollowed by the addition of Menhadell-fish oil (Zapata Haynie Corporation,Virginia), Stay-C (VTI Vitamin Technologies International, 10) and fatly acid. methylesters (Ocean Organics, RI). The mixture obtained was then encapsulated(leibovitz, 1990) ,
I ,
"I
I;
I'
Ii'
j••
Table 3: Fatty acid composition (mg/gm dry weight) of live and inert diets .
Names I Diet numberI 1& 3 I 2 I 4 I 5 I 6 I 7&8 I 9,
14:0 1.1 1.9 19 15 1.6 26 10414:1 0.9 - - 01 0.1 - 0.815:0 0.6 - - 01 0.1 0.2 00415:1 0.5 - - - - - -16:0 125 7.5 7.5 4.3 4.5 12.9 10.916:1 - 1204 4.2 4.2 204 2.2 30417:0 3.0 - - 004 0.4 0.5 0.816:2 4.0 - - 004 004 I. 0.6 1318:0 4.0 1.0 1.0 104 104 304 2.918:1 33.5 33 3.3 404 r 4.4 10.7 180416:3 - - - '';'',J 0.2 0.2 - -18:2 10.0 - - 0.2 0.2 - -18:2 - 0.7 0.7 02 004 14.8 8620:0 - - - - - 0.2 -18:3 0.5 004 0.4 5 4.7 1.7 518:4 4.5 - - - 1.2 1.5 -21:5 19 1.9 0.7 0.6 0.7 1.020:2 - - - - - 0.2 -20:3 - - - 0.1 0.1 1.1 -22:0 - - - 2.2 22 10 -. 20:4 - - - 0.2 0.2 - -20:5 4.5 4.3 4.3 7.1 7.1 4.1 4.122:5 5.5 - - 0.1 0.1 - -22:6 - - - 0.3 0.3 - -22:5 - - - 09 0.8 0.5 -22:6 - 5 5 4.6 4.6 5.1 -
Diet 6 (EYO): Autolysed yeast extract (Rhone-Poulenc Food IngredientsDivision, PAl was finely ground (106 J.1m)and mixed with measured amount offish oil, fatty acid methylesters, vitamins and minerals and then encapsulated. (leibovitz, 1990). Protein, fat, ash and moisture contents of yeast were 77, 1, 13and 9 %, respectively. .Diet 7 (AP200): Closed formula commercial starter diet (AP200) (ZieglerBrothers Inc., PAl, was analyzed for its nutritional information and fed to fishwithout any further modification.Diet 8 (EAP200): Encapsulated diet 7.Diet 9 (ABO): Artemia meal-based commercial starter diet (PDM Associates,CT), was analyzed and sieved through 150 and 250 J.1mmesh and fed to fishwithout any modification.
6 M. ASHRAF ET AL. BIOLOGIA. VOL. 54 (1) FORMULATION OF SEMI.PURIFIED DIETS FOR STRIPED BASS LARVAE 7
RESULTS
Statistical AnalysisOne Way Analysis of Variance followed by Duncan's Multiple Range Test, wasused to evaluate the statistical significance of the treatment differences,Ditferences between the treatment means were considered significant at p<O,5,
Feeding Activity and BehaviorBoth live diets (Anemia nauplii and zooplankton) were eagerly taken up by fishand they consumed all the available live organisms within a short span of time.Response of larvae was slow towards inert diets, On the 3" day of theexperiment, fish larvae present in casein-based diet, Ap200 powder andencapsulated Anemia group, hesitantly started to swallow the capsules, Theresponse was comparatively better towards AP200 powder, Sometimes fish spitout the particles after devouring it. the food particles were visible throughtransparent larval gut by day five, During the next one and a half week, fishappeared healthy and active in those diet groups where they had shown somepositive response towards feed. The fish fed on encapsulated yeast-based dietand casein-based diet without tannic acid and unfed control, were totally dead atthe end of the first week,
Cannibalism was visually observed and was more prominent inencapsulated casein-based diet, yeast-based diet, encapsulated AP200, andencapsulated Anemia 'without tannic acid, It was observed many times duringfeeding trials that one fish running after the other and devouring it as a wholewhen felt convenient. At the end of the trial, nnortality was calculated bydifference, Number of live fish recovered was subtracted from the initial stock.The missing fish were considered the victims of cannibalism, Data showed that17-18% of the total stock was lost due to this problem.
--•..•.•. LA..••.• L2
-EAN---EZ--ECD~EYD-AP200
-EAP20015 I-+-ABD
-'-UF131197
Time(days)
531
~';f!. 150::::-.~ 100 ~~ 50 J ~'---:l l .,,'-f/J -, ,0. '#-::::: ~
o ,- . -,
'I
!I
Experimental DesignBoth feeding trials were designed as "One Way Analysis of Variance" (ANOVA),The treatment group fed on live Anemia nauplii served as control in bothexperiments, while "unfed" group was negative control, There were threereplicates in each treatment with 25 individuals in each replica, except unfedgroup that had single replica. All the jars were numbered and randomly allotted totreatment groups.
Chemical AnalysisCrude protein, ash and moisture were determined by the AOAC (1984) methods(Table 2), The amino acids were analyzed (Table 4) by the Pico-Tag method(Bennett & Solomon, 1986) that involves the protein hydrolysis (Ng et ai, 1987)and pre-eolumn derivatization of the amino acids followed by Reverse PhaseHigh Performance Liquid Chromatography (HPLC) (0' Hare et ai, 1987),Crude lipids were estimated (Table 2) by the Bligh & Dyer (1959) method asmodified by Kates (1986), Fatty acid profile (Table 3) was determined using theNOAA protocol (1988) of National Marine Fisheries for the analysis o! marine fishoil.
Table 4: Amino acid composition (mg/g of dry weight) of diets.
Preparation of diets for trial 2. Live, encapsulated and formulated diets were processed following the sameprocedures as described for different diets in trial 1. The differences were onlyaddition of tannic acid and attractant in some diets (Table 5).
Dietnumber-': 1&3 2 4 5 6 7&8 9Amino AcidslASP 31.2 40.4 40.4 29.4 60.9 44.6 30GLU 153 89.2 89.2 139 70.2 83.6 79.3SER 30.5 20 20 30.3 30.4 26.3 16.5GLY 22.7 39.7 39.7 19.6 29.1 38.5 22.4HIS 24.1 12.1 12.1 21.6 8.4 12.6 8.5ARG 31.8 54.7 54.7 31.2 49.7 50.7 29.9THR 20.8 21.6 21.6 18.2 33.8 25.2 13.9ALA 16.9 42.8 42.8 17 54.5 301 229PRO 85.7 39.6 39.6 78.6 30.4 352 288TYR 35.7 27.6 27.6 34.3 2.3 11.9 10.3VAL 28.6 32.9 32.1 27 34.6 2.1 23.7MET 12.3 12.6 12.6 10.8 55.3 11.9 7.8CYS - 0.8 0.8 0.2 0.4 2.1 -ISO 18.2 228 22.8 19.9 25.3 21.5 20.2 .LEU 29.2 38.1 38.1 30 33.7 332 31.1PHE 20.8 24.3 24.3 20.1 21.5 23.8 19LYS 46.7 37 37 41.8 47.2 34.5 27
,
I
I
, I! ';
6 M. ASHRAF ET AL. BIOLOGIA. VOL. 54 (1) FORMULATION OF SEMI.PURIFIED DIETS FOR STRIPED BASS LARVAE 7
RESULTS
Statistical AnalysisOne Way Analysis of Variance followed by Duncan's Multiple Range Test, wasused to evaluate the statistical significance of the treatment differences,Ditferences between the treatment means were considered significant at p<O,5,
Feeding Activity and BehaviorBoth live diets (Anemia nauplii and zooplankton) were eagerly taken up by fishand they consumed all the available live organisms within a short span of time.Response of larvae was slow towards inert diets, On the 3" day of theexperiment, fish larvae present in casein-based diet, Ap200 powder andencapsulated Anemia group, hesitantly started to swallow the capsules, Theresponse was comparatively better towards AP200 powder, Sometimes fish spitout the particles after devouring it. the food particles were visible throughtransparent larval gut by day five, During the next one and a half week, fishappeared healthy and active in those diet groups where they had shown somepositive response towards feed. The fish fed on encapsulated yeast-based dietand casein-based diet without tannic acid and unfed control, were totally dead atthe end of the first week,
Cannibalism was visually observed and was more prominent inencapsulated casein-based diet, yeast-based diet, encapsulated AP200, andencapsulated Anemia 'without tannic acid, It was observed many times duringfeeding trials that one fish running after the other and devouring it as a wholewhen felt convenient. At the end of the trial, nnortality was calculated bydifference, Number of live fish recovered was subtracted from the initial stock.The missing fish were considered the victims of cannibalism, Data showed that17-18% of the total stock was lost due to this problem.
--•..•.•. LA..••.• L2
-EAN---EZ--ECD~EYD-AP200
-EAP20015 I-+-ABD
-'-UF131197
Time(days)
531
~';f!. 150::::-.~ 100 ~~ 50 J ~'---:l l .,,'-f/J -, ,0. '#-::::: ~
o ,- . -,
'I
!I
Experimental DesignBoth feeding trials were designed as "One Way Analysis of Variance" (ANOVA),The treatment group fed on live Anemia nauplii served as control in bothexperiments, while "unfed" group was negative control, There were threereplicates in each treatment with 25 individuals in each replica, except unfedgroup that had single replica. All the jars were numbered and randomly allotted totreatment groups.
Chemical AnalysisCrude protein, ash and moisture were determined by the AOAC (1984) methods(Table 2), The amino acids were analyzed (Table 4) by the Pico-Tag method(Bennett & Solomon, 1986) that involves the protein hydrolysis (Ng et ai, 1987)and pre-eolumn derivatization of the amino acids followed by Reverse PhaseHigh Performance Liquid Chromatography (HPLC) (0' Hare et ai, 1987),Crude lipids were estimated (Table 2) by the Bligh & Dyer (1959) method asmodified by Kates (1986), Fatty acid profile (Table 3) was determined using theNOAA protocol (1988) of National Marine Fisheries for the analysis o! marine fishoil.
Table 4: Amino acid composition (mg/g of dry weight) of diets.
Preparation of diets for trial 2. Live, encapsulated and formulated diets were processed following the sameprocedures as described for different diets in trial 1. The differences were onlyaddition of tannic acid and attractant in some diets (Table 5).
Dietnumber-': 1&3 2 4 5 6 7&8 9Amino AcidslASP 31.2 40.4 40.4 29.4 60.9 44.6 30GLU 153 89.2 89.2 139 70.2 83.6 79.3SER 30.5 20 20 30.3 30.4 26.3 16.5GLY 22.7 39.7 39.7 19.6 29.1 38.5 22.4HIS 24.1 12.1 12.1 21.6 8.4 12.6 8.5ARG 31.8 54.7 54.7 31.2 49.7 50.7 29.9THR 20.8 21.6 21.6 18.2 33.8 25.2 13.9ALA 16.9 42.8 42.8 17 54.5 301 229PRO 85.7 39.6 39.6 78.6 30.4 352 288TYR 35.7 27.6 27.6 34.3 2.3 11.9 10.3VAL 28.6 32.9 32.1 27 34.6 2.1 23.7MET 12.3 12.6 12.6 10.8 55.3 11.9 7.8CYS - 0.8 0.8 0.2 0.4 2.1 -ISO 18.2 228 22.8 19.9 25.3 21.5 20.2 .LEU 29.2 38.1 38.1 30 33.7 332 31.1PHE 20.8 24.3 24.3 20.1 21.5 23.8 19LYS 46.7 37 37 41.8 47.2 34.5 27
,
I
I
, I! ';
8 M.ASHRAFET AL. BIOlOGIA VOL.54(1) FORMULATIONOFSEMI-PURIFIEDDIETSFORSTRIPEDBASSlARVAE 9
Figure 1: Survival percentage of striped bass larvae in trial 1
Survival and GrowthTrial 1; Survival: Survival was significantly higher in Artemia fed group than restof the treatment groups (Table 5). Consequential mortality started in treatment 6(yeast-based diet) and treatment 10 (unfed) on the second day of experimentand all the individuals were dead on or before day nine (Fig. 1). Althoughmortality had started in the other treatment groups, the rate of mortality, however,was comparatively low. Fish fed on live zooplankton was the second highest insurvival. Survival (%) was similar in encapsulated Artemia nauplii, encapsulatedzooplankton, encapsulated casein-based diet and encapsulated AP200. Survival,however, was comparatively lower in Artemia meal based diet (19%) but notstatistically different from aforementioned inert diets (Table 5).Growth: The fish fed on live. food (Artemia and zooplankton) showed asubstantial increase in weigh!. The final weight was 16 mg for fish fed onArtemia nauplii and 12 mg for live zooplankton.
The respective weight gain was 486% and 314% over the initial body weight inboth treatment groups. The weight gain in the rest of the treatment groupsranged from 0-139%. The fish fed on encapsulated zooplankton, AP200, caseinand Artemia meal based diet did not grow at all. The larvae fed on AP200capsules and Artemia meal based diet showed 14% reduction in their initialweigh!. Similar variations were noted when their specific growth rates werecompared (Table 5)
Trial 2; Survival: Significantly higher survival (78.7%) was observed in liveArtemia fed group than rest of the treatments, even after 30 days of rearing(Table 5). Like the previous experiment, mortality had started on the second dayArtemia group leveled off after 11 days. A sharp jump in mortality was observedin casein-based diet without tannic acid and unfed control and all the fish were. dead in the first half of the.2"' week of experiment (Fig. 2). Fish fed on casein-based diet with tannic acid and tannic acid attractant survived up to the beginningof the third week. Mortality then started and all the fish were dead within a shortperiod of time (Fig. 2). ;;;:)
Figure 2: Survival percentage of striped bass larvae in trial 2
Among the surviving treatments, casein-based diet containing tannic acidwith iive Artemia supplementation every 4th day, exhibited significantly highersurvival (51%) than those where live Artemia was omitted and capsules wereprepared without tannic acid. Survival in these groups ranged from 6% (caseinwithout Tannic Acid + live Artemia; Artemia capsules without tannic acid) to 40%(casein +Tannic Acid + Attractant+ live Artemia). Fish fed on diets with tannic
J
Table 5: Survival (%), final wet body weight (mg) and specific growth rate (SGR) ofstriped bass in feeding trials 1 and 2. Data are presented as mean :t SE. Initial meanwet weight of the larvae in feeding trials 1 and 2 Were 2.8fO.3 and 2.6f0.3 mg,respectively. Values in a column followed by the same superscript letters are notsignificantly different from each other (p>0.05).
Treatment' Survival _Weight SGR
(LA 80+43. 16:t:5i1 11.7lLA 79:t63. 64.17 10.7(lZ 67z14 12.5 9.5(EAN) 25+10'- ...., 7+2' 5.8IEAN+T)' 25.0 16.7 I 6(EAN+T+A)' 12+6' 15.5 6EANWIO Tl' 6>3' 11 :t3c 4.7EZ)' 27t8 ' 3.1' 0.2EYO) 0 -- --EGO) 29+7' 3.1' -0.1EGO+T)' 0 -- --IEGO+T+A)' 0 -- --(EGOWIO T)' 0 -- --(EGO+T+LA)' 51+13e 26.9 7.7(EGO+T+A+lA)' 40.24 26+7 7.7~(EGOWIO T+LA)' 6:!::1 26+4 7.7(AP200)' . 43+9 - 6:t2 c 4.6I (EAP200)' 28.17 ' 2.0.1 -0.6I (ABO) 19i:8e 3.1 0.2
A Superscript 1, 2 represents trials, T stands for tannic acid and A fOf attractant. Detail of other abbreviations canbe seen under heading, "Diet preparation" in -Materials and Methods' section.
S SGR (Average daily growth as percentage of the initial weight) :0 In (final weight) - In (initIalweight) ,. 100 + No. of days.
j.•
120-100
?i 80ji 60 '--\' ..- . '-\-'1: 40-- .1;, i:lIII 20
o1 5 9
,\.,.\ :,,","
1317212529
Days
..•..Days
..•...LA
-"-ECD+T--ECD+T+A...•..ECDw/o T..•...ECD+T+LA-+-- ECD+T+A+LA-ECDw/oT+LA-EAN+T-+-EAN+T+A..•...EANw/o T-"-Unfej
8 M.ASHRAFET AL. BIOlOGIA VOL.54(1) FORMULATIONOFSEMI-PURIFIEDDIETSFORSTRIPEDBASSlARVAE 9
Figure 1: Survival percentage of striped bass larvae in trial 1
Survival and GrowthTrial 1; Survival: Survival was significantly higher in Artemia fed group than restof the treatment groups (Table 5). Consequential mortality started in treatment 6(yeast-based diet) and treatment 10 (unfed) on the second day of experimentand all the individuals were dead on or before day nine (Fig. 1). Althoughmortality had started in the other treatment groups, the rate of mortality, however,was comparatively low. Fish fed on live zooplankton was the second highest insurvival. Survival (%) was similar in encapsulated Artemia nauplii, encapsulatedzooplankton, encapsulated casein-based diet and encapsulated AP200. Survival,however, was comparatively lower in Artemia meal based diet (19%) but notstatistically different from aforementioned inert diets (Table 5).Growth: The fish fed on live. food (Artemia and zooplankton) showed asubstantial increase in weigh!. The final weight was 16 mg for fish fed onArtemia nauplii and 12 mg for live zooplankton.
The respective weight gain was 486% and 314% over the initial body weight inboth treatment groups. The weight gain in the rest of the treatment groupsranged from 0-139%. The fish fed on encapsulated zooplankton, AP200, caseinand Artemia meal based diet did not grow at all. The larvae fed on AP200capsules and Artemia meal based diet showed 14% reduction in their initialweigh!. Similar variations were noted when their specific growth rates werecompared (Table 5)
Trial 2; Survival: Significantly higher survival (78.7%) was observed in liveArtemia fed group than rest of the treatments, even after 30 days of rearing(Table 5). Like the previous experiment, mortality had started on the second dayArtemia group leveled off after 11 days. A sharp jump in mortality was observedin casein-based diet without tannic acid and unfed control and all the fish were. dead in the first half of the.2"' week of experiment (Fig. 2). Fish fed on casein-based diet with tannic acid and tannic acid attractant survived up to the beginningof the third week. Mortality then started and all the fish were dead within a shortperiod of time (Fig. 2). ;;;:)
Figure 2: Survival percentage of striped bass larvae in trial 2
Among the surviving treatments, casein-based diet containing tannic acidwith iive Artemia supplementation every 4th day, exhibited significantly highersurvival (51%) than those where live Artemia was omitted and capsules wereprepared without tannic acid. Survival in these groups ranged from 6% (caseinwithout Tannic Acid + live Artemia; Artemia capsules without tannic acid) to 40%(casein +Tannic Acid + Attractant+ live Artemia). Fish fed on diets with tannic
J
Table 5: Survival (%), final wet body weight (mg) and specific growth rate (SGR) ofstriped bass in feeding trials 1 and 2. Data are presented as mean :t SE. Initial meanwet weight of the larvae in feeding trials 1 and 2 Were 2.8fO.3 and 2.6f0.3 mg,respectively. Values in a column followed by the same superscript letters are notsignificantly different from each other (p>0.05).
Treatment' Survival _Weight SGR
(LA 80+43. 16:t:5i1 11.7lLA 79:t63. 64.17 10.7(lZ 67z14 12.5 9.5(EAN) 25+10'- ...., 7+2' 5.8IEAN+T)' 25.0 16.7 I 6(EAN+T+A)' 12+6' 15.5 6EANWIO Tl' 6>3' 11 :t3c 4.7EZ)' 27t8 ' 3.1' 0.2EYO) 0 -- --EGO) 29+7' 3.1' -0.1EGO+T)' 0 -- --IEGO+T+A)' 0 -- --(EGOWIO T)' 0 -- --(EGO+T+LA)' 51+13e 26.9 7.7(EGO+T+A+lA)' 40.24 26+7 7.7~(EGOWIO T+LA)' 6:!::1 26+4 7.7(AP200)' . 43+9 - 6:t2 c 4.6I (EAP200)' 28.17 ' 2.0.1 -0.6I (ABO) 19i:8e 3.1 0.2
A Superscript 1, 2 represents trials, T stands for tannic acid and A fOf attractant. Detail of other abbreviations canbe seen under heading, "Diet preparation" in -Materials and Methods' section.
S SGR (Average daily growth as percentage of the initial weight) :0 In (final weight) - In (initIalweight) ,. 100 + No. of days.
j.•
120-100
?i 80ji 60 '--\' ..- . '-\-'1: 40-- .1;, i:lIII 20
o1 5 9
,\.,.\ :,,","
1317212529
Days
..•..Days
..•...LA
-"-ECD+T--ECD+T+A...•..ECDw/o T..•...ECD+T+LA-+-- ECD+T+A+LA-ECDw/oT+LA-EAN+T-+-EAN+T+A..•...EANw/o T-"-Unfej
10 M. ASHRAF ET AL. BIOLOGIA VOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 11
(I
J
acid-containing microcapsules showed overall better survival. Periodicsupplementation of live Artemia amplified this response (Fig. 2).
Growth: The fish fed on live Artemia nauplii yielded the highest weight (Table 5)while those fed on combined diets (live Artemia + artificial diets) were .second ingrowth. Artemia capsules prepared with and without tannic acid, showedsignificant growrh differences, more in tannic acid containing capsules and verypoor where tannic acid was excluded (Table 5).
DISCUSSION
During these studies larvae fed on experimental diets, ingested somediets but not others. The reasons for their total refusal to encapsulated yeast-based diet (Trial 1) and casein-based diet without tannic acid (Trial 2) might besmall mouth size or poor diet quality that failed to stimulate gustatory responses.Larval fish responded slowly towards casein-based diet with tannic acid incapsule material and/ or where allractants were also added. Unlike the former, inthe 'later, processing modifications, supported survival of larval fish up to 3" weekof experiment but sudden mortality then ensued and all the individuals perishedwithin a very short period of time, There can be multiple factors governing therearing process of this tiny creature and will be tried to explain in the following. text.
Nutrient level in the artificial diets or their chemical composition is thefirst step to consider in diet formulation. Since the nutrient requirements of thelarval striped bass like most of the other marine fish species (Park et aI., 2006)are not known, hence it is impossible to formulate a balanced diet and/or evenbalanced diet can be nutrient-deficient due to nutrient leaching (Medgyesy &Wieser, 1982; Yufera et aI., 2002 and Langdon, 2003) if diet particles are not fullyprotected. These problems were very carefully addressed in the present studies.The diets were based on the nutrient composition of natural zooplankton, thepresumed ideal diet for larval striped bass. The diet particles were well protectedin albumen-alginate capsules, whose ability to prevent leaching has beendocumented by Leibovitz (1990), Ozkizilicik & Chu (1996) and Yufera et al.(2002).
The encapsulated diet particles were though non-motile, yet their retentiontime in the water column was much longer than anticipated and they wereavailable to larvae providing ample opportunity for sufficient intake. Larvae wereobserved schooling both at the top and bottom striking the capsules and theywere visible inside the transparent gut of the larvae soon after feeding. It means. that like other species such as blue spotted goby (Clack, 2006) and Chitalachitala larvae (Sarkar et al., 2006), larval striped bass was also capable ofcapturing and ingesting non-living and non-motile.prey.
Stout outer albumen-alginate tannic acid bonded coating can be a barrierto the proper dissolution of capsules and release of nutrients. Walford et al.(1991) found that protein membrane capsules did not break down even after 2hour retention in the gut of sea bass larvae and the capsules were expelledintact. Bengtson et al. (1993) in their:detailed histological studies discovered that
digestion of albumen-alginate capsules in striped bass larvae differed from that oflive Artemia. Although the capsules were broken down in the passage throughthe digestive .tract, very little of capsule contents were actually digested andabsorbed. This indicates that there was no proper mechanical or physiologicalmechanisms in larval gut which could breakdown the outer coating of the capsuleand let the nutrients go in the gut. It was obvious from the superiority in growthand survival of fish fed on AP200 powder (Trial 1) to that of its counterpart. Might_be the nutrients in mesh were comparatively more available (Tonheim et al.,2005) to digestive set-up and they supported growth and survival of fish.
Dabrowski (1979), Dabrowski et al. (2000), Yufera et al. (2000) and Cahu &Infante (2001) demonstrated the importance of exogenous enzyme~ tor larvae,. available in li\'e foods. Live foods transferred 70% of total protease activity inwhitefish larvae (Hofer, 1985) and 89-94% ot the total esterase, 79-88%exonuclease, 43-60% protease and 15-27% amylase in turbot larvae (Munilla-Moran et al., 1990). Kurokawa et al. (1998), on the other hand, observed thatonly 0.6% protease of the total enzyme present in 2-day-old Japanese sa(dinelarval gut, was due to rolifers. Our observations support some but contradictothers and affirm that some enzymes were present in larval gut at the onset offirst feeding but might not be in sufficient quantities to support ongoing digestiveprocesses. Sirvas-Cornejo et al. (2007) have recently observed beller growth in.post larval Fenneropenaeus indicus when they supplemented micro-encapsulated diet with genetically modified enzyme (protease) producingbacteria. Similarly, Keys;3mi et al. (2007) have reported higher survival and fasterrate of metamorphosis in those Macrobrachium rosenbergii larvae which werefed on Artemia treated with Bacillus subtilis bacterium than those which werehatched in the absence of bacteria. Hence, it can be deduced from recent andprevious studies that addition of an appropriate exogenous enzyme source ismust for successful weaning of larvae on artificial diets. Similarly when we fedlarvae on combined diets (artificial diets + live Artemia nauplii on every 4th day),growth and survival remained in between to those fed on sole artificial diets or. live Artemia With no cannibalism. Battaglene & Cobcroft (2007), observedreduced rate of malformations and beller growth in striped bass trumpeter larvaewhen fed on mixed (artificial + live food) feeding schedule. Investigations of theseworkers strongly support and confirm our findings. It seems that inert dietsoffered, maintained proper nutrient balance and motility, collapsibility andcharacteristic colour/odor of live food, not only instigated larvae to take morefeeds but also transferred some digestive factors into the larval gut. Thecombined effect of two dietary sources helped the larvae to nearly approachcontrol group in growth and survival. Tonheim et al. (2007) and Conceicao et al.(2007) had similar findings in their investigative work. Therefore, total eliminationof live food for successful larval rearing (Fletcher et aI., 2007), at least at thistime, without reducing growth potential, needs further extensive research work.
Addition of attractants did not make any difference on the performance oflarval fish though quite encouraging results have been obtained previously inlarval yellow perch (Dabrowski et a'., 2000). Fatty acid and amino acidcomposition was almost uniform in all the diets applied because the formulationwas based on the composition of the natural food. Concentration of fatty andamino acids was even higher in some inert diets, Unlike previous studies, this
10 M. ASHRAF ET AL. BIOLOGIA VOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 11
(I
J
acid-containing microcapsules showed overall better survival. Periodicsupplementation of live Artemia amplified this response (Fig. 2).
Growth: The fish fed on live Artemia nauplii yielded the highest weight (Table 5)while those fed on combined diets (live Artemia + artificial diets) were .second ingrowth. Artemia capsules prepared with and without tannic acid, showedsignificant growrh differences, more in tannic acid containing capsules and verypoor where tannic acid was excluded (Table 5).
DISCUSSION
During these studies larvae fed on experimental diets, ingested somediets but not others. The reasons for their total refusal to encapsulated yeast-based diet (Trial 1) and casein-based diet without tannic acid (Trial 2) might besmall mouth size or poor diet quality that failed to stimulate gustatory responses.Larval fish responded slowly towards casein-based diet with tannic acid incapsule material and/ or where allractants were also added. Unlike the former, inthe 'later, processing modifications, supported survival of larval fish up to 3" weekof experiment but sudden mortality then ensued and all the individuals perishedwithin a very short period of time, There can be multiple factors governing therearing process of this tiny creature and will be tried to explain in the following. text.
Nutrient level in the artificial diets or their chemical composition is thefirst step to consider in diet formulation. Since the nutrient requirements of thelarval striped bass like most of the other marine fish species (Park et aI., 2006)are not known, hence it is impossible to formulate a balanced diet and/or evenbalanced diet can be nutrient-deficient due to nutrient leaching (Medgyesy &Wieser, 1982; Yufera et aI., 2002 and Langdon, 2003) if diet particles are not fullyprotected. These problems were very carefully addressed in the present studies.The diets were based on the nutrient composition of natural zooplankton, thepresumed ideal diet for larval striped bass. The diet particles were well protectedin albumen-alginate capsules, whose ability to prevent leaching has beendocumented by Leibovitz (1990), Ozkizilicik & Chu (1996) and Yufera et al.(2002).
The encapsulated diet particles were though non-motile, yet their retentiontime in the water column was much longer than anticipated and they wereavailable to larvae providing ample opportunity for sufficient intake. Larvae wereobserved schooling both at the top and bottom striking the capsules and theywere visible inside the transparent gut of the larvae soon after feeding. It means. that like other species such as blue spotted goby (Clack, 2006) and Chitalachitala larvae (Sarkar et al., 2006), larval striped bass was also capable ofcapturing and ingesting non-living and non-motile.prey.
Stout outer albumen-alginate tannic acid bonded coating can be a barrierto the proper dissolution of capsules and release of nutrients. Walford et al.(1991) found that protein membrane capsules did not break down even after 2hour retention in the gut of sea bass larvae and the capsules were expelledintact. Bengtson et al. (1993) in their:detailed histological studies discovered that
digestion of albumen-alginate capsules in striped bass larvae differed from that oflive Artemia. Although the capsules were broken down in the passage throughthe digestive .tract, very little of capsule contents were actually digested andabsorbed. This indicates that there was no proper mechanical or physiologicalmechanisms in larval gut which could breakdown the outer coating of the capsuleand let the nutrients go in the gut. It was obvious from the superiority in growthand survival of fish fed on AP200 powder (Trial 1) to that of its counterpart. Might_be the nutrients in mesh were comparatively more available (Tonheim et al.,2005) to digestive set-up and they supported growth and survival of fish.
Dabrowski (1979), Dabrowski et al. (2000), Yufera et al. (2000) and Cahu &Infante (2001) demonstrated the importance of exogenous enzyme~ tor larvae,. available in li\'e foods. Live foods transferred 70% of total protease activity inwhitefish larvae (Hofer, 1985) and 89-94% ot the total esterase, 79-88%exonuclease, 43-60% protease and 15-27% amylase in turbot larvae (Munilla-Moran et al., 1990). Kurokawa et al. (1998), on the other hand, observed thatonly 0.6% protease of the total enzyme present in 2-day-old Japanese sa(dinelarval gut, was due to rolifers. Our observations support some but contradictothers and affirm that some enzymes were present in larval gut at the onset offirst feeding but might not be in sufficient quantities to support ongoing digestiveprocesses. Sirvas-Cornejo et al. (2007) have recently observed beller growth in.post larval Fenneropenaeus indicus when they supplemented micro-encapsulated diet with genetically modified enzyme (protease) producingbacteria. Similarly, Keys;3mi et al. (2007) have reported higher survival and fasterrate of metamorphosis in those Macrobrachium rosenbergii larvae which werefed on Artemia treated with Bacillus subtilis bacterium than those which werehatched in the absence of bacteria. Hence, it can be deduced from recent andprevious studies that addition of an appropriate exogenous enzyme source ismust for successful weaning of larvae on artificial diets. Similarly when we fedlarvae on combined diets (artificial diets + live Artemia nauplii on every 4th day),growth and survival remained in between to those fed on sole artificial diets or. live Artemia With no cannibalism. Battaglene & Cobcroft (2007), observedreduced rate of malformations and beller growth in striped bass trumpeter larvaewhen fed on mixed (artificial + live food) feeding schedule. Investigations of theseworkers strongly support and confirm our findings. It seems that inert dietsoffered, maintained proper nutrient balance and motility, collapsibility andcharacteristic colour/odor of live food, not only instigated larvae to take morefeeds but also transferred some digestive factors into the larval gut. Thecombined effect of two dietary sources helped the larvae to nearly approachcontrol group in growth and survival. Tonheim et al. (2007) and Conceicao et al.(2007) had similar findings in their investigative work. Therefore, total eliminationof live food for successful larval rearing (Fletcher et aI., 2007), at least at thistime, without reducing growth potential, needs further extensive research work.
Addition of attractants did not make any difference on the performance oflarval fish though quite encouraging results have been obtained previously inlarval yellow perch (Dabrowski et a'., 2000). Fatty acid and amino acidcomposition was almost uniform in all the diets applied because the formulationwas based on the composition of the natural food. Concentration of fatty andamino acids was even higher in some inert diets, Unlike previous studies, this
12 M. ASHRAF ET AL BIOlOGIA VOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 13
,rI
. slight variation in the present situation apparently did not play any differential rolein larval growth and survival, hence does not warrant further explanation.
From these studies it can be concluded that not a single factor iscontributing in rearing of larval fish but a series of factors are involved. Digestivecapabilities and dietary requirements of the larvae need to be determinedthrough the combination of biochemical, physiological and morphological studies.Feed formulation then needs to be based on the integration of. informationobtained from these studies which is attractive to larvae and can stimulate therequired enzymes as and when required and is digestible eliminating thedependence on costly and unreliable natural foods. Specific sites for digestionand absorption of nutrients in the larval gut need to be thoroughly studied by theuse of radioisotopes and fluorescence dyes. Not less important is to learn theinherent attractant in natural food that motivates the larvae and stimulates theingestion of live foods. Further refinement in encapsulation technology for thesafe delivery of nutrients and their release at suitable sites and at appropriatetime is of great significance in preparation of effective weaning diets which will bea major breakthrough in larval rearing technology.
REFERENCESAOAC (Association of Official Analytical Chemists). 1984. Official Methods of
Analysis. 14th ed. AOAC, Arlington Virginia, U. S AAppelbaum, S. 1980. Versuche zur geschmacksperzeption einiger susswasserfische
in larvalen und adulten stadium. Archives Fischereiwiss., 31 (2): 105-114.Battaglene, S. C. & Cobcroft, J. M., 2007. Advances in the culture of striped trumpeter
larvae: A review. Aquaculture, 268: 195-208.Bautista, N. M., Millamena, O. M. & Kanazawa, A., 1989. Use of Kappa-carragenan
microbound diet (C-MBD) as feed for Penaeus monodon larvae. Journal ofMarine Biology, 103169-173.
Bengtson, D. A, Beck, A D. & Simpson, K. L., 1985. Standardization of the nutritionof fish in aquatic toxicological testing. (eds Cowey, C. B., Mackie, A M. & Bell, J.G.). Nutrition and feeding in fish. Academic Press, London. pp. 431-446.
Bengtson, D. A .. Borrus, D., Leibovitz, H. E. & Simpson, K. L., 1993. Studies ondigestive structure and function in larvae of inland silvers ides. Menidia beryl/ina.In: Physiological and Biological Aspects of Fish Development (eds Walther, B. &Fyhn. H. J.), Un;' Bergen, Bergen, Norway. 199pp.
Bennet, H. P. J. & Solomon, S., 1986. Use of Pico-Tag methodology in the chemicalanalysis of peptides with carboxyl terminal amides. Journal of Chromatography.359:221-230.
Blaxter, J. H. S. 1969. Development: eggs and larvae. (eds Hoar, H. S. & Randall, D.J.). Fish physiology, volume 3. Academic press, New York, U. S. A .. pp. 152-178.
Bligh, E. G., & Dyer, W. J., 1959. A rapid method of total lipid extraction andpurification. Canadian Journal of Biochemistry and Physiology, 37: 911-937.
Cahu, CL. Zambonino-Infante, J. L., Escaffre, AM., Bergot, P. & Kaushik, S., 1998.Preliminary results on sea bass, Dicentrarchus labrax larvae rearing with
compound diet from first feeding. Comparison with carp Cyprinus carpio larvae .Aquaculture, 169:1-7.
Cahu, C. & Infante, J. Z., 2001. Substitution of live food by formulated diets in marinefish larvae. Aquaculture, 200:161-180.
Clack, B. W., 2006. Development of microparticulate feeds and methods to improveacceptability of artificial diets by blue spotted goby (Asterropteryx semipunctata).Oregon State University (HMSC) Publications, 121-The Valley Library, Corvallis,U.SA
Conceicao, L. E. C., Reibero, L., Engrola, S., Aragao, C., Morais, S., Lacuisse, M.,. Soares, M. & Dinis, M. T., 2007. Nutritional physiology during development otSenegalese sole (Solea senegalensis). Aquaculture, 268: 64-81.
Dabrowski, K., 1979. Role of proteolytic enzymes in fish digestion. In: Cultivation offish fry and its live food. (eds Styczynskjurewicz, E., Backiel, E., Jaspers, T. &Persoone, E.). European Mariculture Society Special Publication 4, Bredene,Belgium. pp.107-126.
Dabrowski, K. 1984. The feeding of fish larvae: present "state of the art" andperspectives Reprod. Nutr. Dev., 24i:a07-833.
Dabrowski, K., Kolkovski, S. & Theis, C., 2000. Application of New Biotechnology ofMicro-encapsulation. Department of Natural Resources, Project Number: R/A-12, The Ohio State University, U. S. A pp. 1-3.
Fernanandez-Diaz, C. & Yutera, M., 1997. Detecting growth in gilthead sea breamSparus aurata L. larvae fed microcapsules. Aquaculture, 153:93-102.
Fletcher, Jr. R.C. Roy, w., Davie, A, Taylor, J., Robertson, D. & Migaud, H, 2007.Evaluation of new particulate diets for early weaning of Atlantic cod (Gadusmorhua): Implications on larval performance and tank hygiene. Aquaculture, 263:35-51.
Gabaudan, J., 1984. Posthatching morphogenesis of the digestive system of stripedbass. Doctoral dissertation. Auburn University, Alabama, U. S. A
Hofer, R., 1985. Effects of artificial diets on the digestive process of larvae. In:Nutrition and Feeding in Fish, (eds Cowey, C. B., Mackie, A M. & Bell, J. B).Institute of Marine Biochemistry, Aberdeen, Scotland. pp. 213-216.
Kates, M., 1986. Lipid extraction procedures. In: Techniques of Lipido/ogy: Isolation.Analysis and Identification of Lipids. Elsevier Press, Amesterdam. 347pp.
Keysami, M. A, Saad, C. R., Sijam, K, Daud, H. M. & Alimon, A R, 2007. Effect ofBacil/us subtilis on growth and survival of larvae Macrobrachium rosenbergii (deMan). Aquaculture Nutrition, 13:131-136.
Kurokawa, T., Shirashi, M. & Suzuki, T., 1998. Quantification of exogenous proteasederived from zooplankton in the intestine of Japanese sardine (Sardinopsmelnoticus) larvae. Aquaculture, 161 :491-499.
Langdon, C., 2003. Microparticle types for delivering nutrients to marine fish larvae.Aquaculture. 227: 259-275.
Leibovitz, H. E., Bengtson, D. A, Maugle, P. D. & Simpson, K L., 1987. Effects ofdietary Artemia lipid fractions on growth of larval inland silversides, Menidiaberyl/ina. In: Artemia Research and its Applications, (eds Decleir. W .. Moens, I.,Sorgeloos, P. & Jaspers. E.). Volume 3. Universal Press Wettern. Belgium. pp469-478.
12 M. ASHRAF ET AL BIOlOGIA VOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 13
,rI
. slight variation in the present situation apparently did not play any differential rolein larval growth and survival, hence does not warrant further explanation.
From these studies it can be concluded that not a single factor iscontributing in rearing of larval fish but a series of factors are involved. Digestivecapabilities and dietary requirements of the larvae need to be determinedthrough the combination of biochemical, physiological and morphological studies.Feed formulation then needs to be based on the integration of. informationobtained from these studies which is attractive to larvae and can stimulate therequired enzymes as and when required and is digestible eliminating thedependence on costly and unreliable natural foods. Specific sites for digestionand absorption of nutrients in the larval gut need to be thoroughly studied by theuse of radioisotopes and fluorescence dyes. Not less important is to learn theinherent attractant in natural food that motivates the larvae and stimulates theingestion of live foods. Further refinement in encapsulation technology for thesafe delivery of nutrients and their release at suitable sites and at appropriatetime is of great significance in preparation of effective weaning diets which will bea major breakthrough in larval rearing technology.
REFERENCESAOAC (Association of Official Analytical Chemists). 1984. Official Methods of
Analysis. 14th ed. AOAC, Arlington Virginia, U. S AAppelbaum, S. 1980. Versuche zur geschmacksperzeption einiger susswasserfische
in larvalen und adulten stadium. Archives Fischereiwiss., 31 (2): 105-114.Battaglene, S. C. & Cobcroft, J. M., 2007. Advances in the culture of striped trumpeter
larvae: A review. Aquaculture, 268: 195-208.Bautista, N. M., Millamena, O. M. & Kanazawa, A., 1989. Use of Kappa-carragenan
microbound diet (C-MBD) as feed for Penaeus monodon larvae. Journal ofMarine Biology, 103169-173.
Bengtson, D. A, Beck, A D. & Simpson, K. L., 1985. Standardization of the nutritionof fish in aquatic toxicological testing. (eds Cowey, C. B., Mackie, A M. & Bell, J.G.). Nutrition and feeding in fish. Academic Press, London. pp. 431-446.
Bengtson, D. A .. Borrus, D., Leibovitz, H. E. & Simpson, K. L., 1993. Studies ondigestive structure and function in larvae of inland silvers ides. Menidia beryl/ina.In: Physiological and Biological Aspects of Fish Development (eds Walther, B. &Fyhn. H. J.), Un;' Bergen, Bergen, Norway. 199pp.
Bennet, H. P. J. & Solomon, S., 1986. Use of Pico-Tag methodology in the chemicalanalysis of peptides with carboxyl terminal amides. Journal of Chromatography.359:221-230.
Blaxter, J. H. S. 1969. Development: eggs and larvae. (eds Hoar, H. S. & Randall, D.J.). Fish physiology, volume 3. Academic press, New York, U. S. A .. pp. 152-178.
Bligh, E. G., & Dyer, W. J., 1959. A rapid method of total lipid extraction andpurification. Canadian Journal of Biochemistry and Physiology, 37: 911-937.
Cahu, CL. Zambonino-Infante, J. L., Escaffre, AM., Bergot, P. & Kaushik, S., 1998.Preliminary results on sea bass, Dicentrarchus labrax larvae rearing with
compound diet from first feeding. Comparison with carp Cyprinus carpio larvae .Aquaculture, 169:1-7.
Cahu, C. & Infante, J. Z., 2001. Substitution of live food by formulated diets in marinefish larvae. Aquaculture, 200:161-180.
Clack, B. W., 2006. Development of microparticulate feeds and methods to improveacceptability of artificial diets by blue spotted goby (Asterropteryx semipunctata).Oregon State University (HMSC) Publications, 121-The Valley Library, Corvallis,U.SA
Conceicao, L. E. C., Reibero, L., Engrola, S., Aragao, C., Morais, S., Lacuisse, M.,. Soares, M. & Dinis, M. T., 2007. Nutritional physiology during development otSenegalese sole (Solea senegalensis). Aquaculture, 268: 64-81.
Dabrowski, K., 1979. Role of proteolytic enzymes in fish digestion. In: Cultivation offish fry and its live food. (eds Styczynskjurewicz, E., Backiel, E., Jaspers, T. &Persoone, E.). European Mariculture Society Special Publication 4, Bredene,Belgium. pp.107-126.
Dabrowski, K. 1984. The feeding of fish larvae: present "state of the art" andperspectives Reprod. Nutr. Dev., 24i:a07-833.
Dabrowski, K., Kolkovski, S. & Theis, C., 2000. Application of New Biotechnology ofMicro-encapsulation. Department of Natural Resources, Project Number: R/A-12, The Ohio State University, U. S. A pp. 1-3.
Fernanandez-Diaz, C. & Yutera, M., 1997. Detecting growth in gilthead sea breamSparus aurata L. larvae fed microcapsules. Aquaculture, 153:93-102.
Fletcher, Jr. R.C. Roy, w., Davie, A, Taylor, J., Robertson, D. & Migaud, H, 2007.Evaluation of new particulate diets for early weaning of Atlantic cod (Gadusmorhua): Implications on larval performance and tank hygiene. Aquaculture, 263:35-51.
Gabaudan, J., 1984. Posthatching morphogenesis of the digestive system of stripedbass. Doctoral dissertation. Auburn University, Alabama, U. S. A
Hofer, R., 1985. Effects of artificial diets on the digestive process of larvae. In:Nutrition and Feeding in Fish, (eds Cowey, C. B., Mackie, A M. & Bell, J. B).Institute of Marine Biochemistry, Aberdeen, Scotland. pp. 213-216.
Kates, M., 1986. Lipid extraction procedures. In: Techniques of Lipido/ogy: Isolation.Analysis and Identification of Lipids. Elsevier Press, Amesterdam. 347pp.
Keysami, M. A, Saad, C. R., Sijam, K, Daud, H. M. & Alimon, A R, 2007. Effect ofBacil/us subtilis on growth and survival of larvae Macrobrachium rosenbergii (deMan). Aquaculture Nutrition, 13:131-136.
Kurokawa, T., Shirashi, M. & Suzuki, T., 1998. Quantification of exogenous proteasederived from zooplankton in the intestine of Japanese sardine (Sardinopsmelnoticus) larvae. Aquaculture, 161 :491-499.
Langdon, C., 2003. Microparticle types for delivering nutrients to marine fish larvae.Aquaculture. 227: 259-275.
Leibovitz, H. E., Bengtson, D. A, Maugle, P. D. & Simpson, K L., 1987. Effects ofdietary Artemia lipid fractions on growth of larval inland silversides, Menidiaberyl/ina. In: Artemia Research and its Applications, (eds Decleir. W .. Moens, I.,Sorgeloos, P. & Jaspers. E.). Volume 3. Universal Press Wettern. Belgium. pp469-478.
14 M. ASHRAF ET AL. BIOLOGIA VOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 15
Leibovitz, H. E. 1990. Albumen-alginate microcapsules for delivering food to larvalinland silversides, Menidia berylfina. Doctrol dissertation. University of RhodeIsl.and, U. S. A
Le Ruyet, P. L. J., Alexandre, J. C., Thebaud, L. & Mugnier, C., 1993. Marine fish... ,!arvae feeding: formulated diets or iive preys? J. World Aquae. Soc., 24:211-224.
MedgYeSY, N. & Wieser, W., 1982. Rearing of white fish, Coregonus lavaretus, with. frozen zooplankton by means of new feeding apparatus. Aquaculture, 28:327-337. .
Munilla-Moran, R, Stark, J. R & Barbour, A, 1990. The role of exogenous enzymesin digestion, of cultured turbot larvae, Scophthalmus maximus. Aquaculture,88:337-350.
Ng, L. T., Pascaud, A & Pasvaud, M., 1987. Hydrochloric acid hydrolysis of proteinand determination of tryptophan by Reverse Phase High Performance LiquidChromatography. Analytical Biochemistry, 167: 47-52.
NOAA., 1988. Fatty acid composition. In: Biomedical Test Materials Program:Analytical Methods for the Quality Assurance of Fish Oil. (eds VanDolah, F. M. &Galloway, S. B.). Charleston, SC: U. D. Department of Commerce, U. S. A pp. 6-11.
O'Hare, M, Tortora, M. T. 0., Gether, U, Nielson, H. V. & Schwartz, T. W,1987.High Performance Liquid Chromatography of phenylthiocarbamyl derivatives of
.,' amino acids and side chain derivatized amino acids. Journal of Chromatography,389: 379-388.
OZKiziiicik,' S. & Chu, F. L., 1996. Preparation and Characterization of a complexmicroencapsulated diet for striped bass, Morone saxatilis, larvae. J.
f~Microencapsul., 13: 331-343.Park, G. H, Puvanendran, V, Kellett, A., Christopher, C. P. & Brown, A. J., 2006.
Effect of enriched rotifers on growth, survival and composition of 12rval Atlanticcod (Gadus morhua). Journal of Marine Science, 63: 285-295.
Sarkar, U. K., Lakra, W. S., Deepak, P. K., Nagi, R S., Paul, S. K. & Srivastava, A,2006.-Performance of different types of diets on experimental larval rearing ofendangered Chitala chitala (Hamilton) in re-circulatory system. Aquaculture, 261:141c150.
Sirvas-Cornejo, S., Latchford, J. W. & Jones, D. A, 2007. Effect of microencapsulated., diets supplemented with genetically modified bacteria on the growth and survival.. of Penneropenaeus indicus postlarvae. Aquaculture Nutrition, 13: 10-16.
Sorgeloos. P., 1980. The use of brine shrimp Arlemia, in aquaculture. In: Ecology,. culturing and use in aquaculture. (eds Persoone, G., Sorgeloos, P., Roels, O. &Jaspers, E.). Volume 3. Universal Press, Western Belgium. pp. 25-26.
Takeuchi, T., Ohkuma, N., Ishida, S., Ishizuka, W., Tomota, M., Hayasawa, H. &Miyakawa, H, 1998. Development of micro-particle diet for marine fish larvae.VIII Int. Symp Nutrition and Feeding of Fish. Las Palmas, Spain, June 1-4. 193pp.
Tonheim, K. S., Espe, M., Hamre, K. & Ronnestad, I., 2005. Pre-hydrolysis improvesutilization of dietary protein in the larval teleost Atlantic halibut (Hippoglossushippoglossus L.). Journal of Experimental Marine Biology and Ecology, 321: 19-34.
Tonheim, K. S., Nordgreen, A, Hogoy, I., Hamre, K. & Ronnestad, I., 2007. In vitrodigestibility of water-soluble and water-insoluble protein fractions of somecommon fish larval feeds and feed ingredients. Aquaculture, 262: 426-435.
Uys, W. & Hecht, T., 1985. Evaluation and preparation of an optimal dry feed for theprimary nursing of Clarias gariepinus larvae. Aquaculture, 47: 173-183.
Walford, J., Lim, T. M. & Lam, T. J., 1991. Replacing live foods withmicroencapsulated diets in the rearing of sea bass, Lates calcarifer, larvae. Do. the larvae ingest and digest protein membrane microcapsules. Aquaculture, 92:225-235.
Watanabe, T., Kitajima, C. & Fujita, S., 1983. Nutritional value of live organisms usedin Japan for mass propagation of fish: A review. Aquaculture, 34: 115-143.
Yufera, F. D., Pasuial, S., Moyano, D., Alercon, G. & Parra, G. G., 2000. Towards aninert diet for first feeding gilthead seabream, Sparus aurata L. larvae.Aquaculture Nutrition, 6: 143pp.
Yufera, M., Kolkovski, S., Fernandez, C., Diaz & Dabrowski, K., 2002. Free aminoacid leaching from protein walled (llicroencapsulated diet for fish larvae.Aquaculture, 214: 273-287.
Zambonino, J. L., Cahu, C. L. & Peres. A, 1997. Partial substitution of di- and tri-peptides for native proteins in sea bass diet improves Dicentrarchus labrax larvaldevelopment. J. Nutr., 127: 608-614 .
14 M. ASHRAF ET AL. BIOLOGIA VOL. 54 (1) FORMULATION OF SEMI-PURIFIED DIETS FOR STRIPED BASS LARVAE 15
Leibovitz, H. E. 1990. Albumen-alginate microcapsules for delivering food to larvalinland silversides, Menidia berylfina. Doctrol dissertation. University of RhodeIsl.and, U. S. A
Le Ruyet, P. L. J., Alexandre, J. C., Thebaud, L. & Mugnier, C., 1993. Marine fish... ,!arvae feeding: formulated diets or iive preys? J. World Aquae. Soc., 24:211-224.
MedgYeSY, N. & Wieser, W., 1982. Rearing of white fish, Coregonus lavaretus, with. frozen zooplankton by means of new feeding apparatus. Aquaculture, 28:327-337. .
Munilla-Moran, R, Stark, J. R & Barbour, A, 1990. The role of exogenous enzymesin digestion, of cultured turbot larvae, Scophthalmus maximus. Aquaculture,88:337-350.
Ng, L. T., Pascaud, A & Pasvaud, M., 1987. Hydrochloric acid hydrolysis of proteinand determination of tryptophan by Reverse Phase High Performance LiquidChromatography. Analytical Biochemistry, 167: 47-52.
NOAA., 1988. Fatty acid composition. In: Biomedical Test Materials Program:Analytical Methods for the Quality Assurance of Fish Oil. (eds VanDolah, F. M. &Galloway, S. B.). Charleston, SC: U. D. Department of Commerce, U. S. A pp. 6-11.
O'Hare, M, Tortora, M. T. 0., Gether, U, Nielson, H. V. & Schwartz, T. W,1987.High Performance Liquid Chromatography of phenylthiocarbamyl derivatives of
.,' amino acids and side chain derivatized amino acids. Journal of Chromatography,389: 379-388.
OZKiziiicik,' S. & Chu, F. L., 1996. Preparation and Characterization of a complexmicroencapsulated diet for striped bass, Morone saxatilis, larvae. J.
f~Microencapsul., 13: 331-343.Park, G. H, Puvanendran, V, Kellett, A., Christopher, C. P. & Brown, A. J., 2006.
Effect of enriched rotifers on growth, survival and composition of 12rval Atlanticcod (Gadus morhua). Journal of Marine Science, 63: 285-295.
Sarkar, U. K., Lakra, W. S., Deepak, P. K., Nagi, R S., Paul, S. K. & Srivastava, A,2006.-Performance of different types of diets on experimental larval rearing ofendangered Chitala chitala (Hamilton) in re-circulatory system. Aquaculture, 261:141c150.
Sirvas-Cornejo, S., Latchford, J. W. & Jones, D. A, 2007. Effect of microencapsulated., diets supplemented with genetically modified bacteria on the growth and survival.. of Penneropenaeus indicus postlarvae. Aquaculture Nutrition, 13: 10-16.
Sorgeloos. P., 1980. The use of brine shrimp Arlemia, in aquaculture. In: Ecology,. culturing and use in aquaculture. (eds Persoone, G., Sorgeloos, P., Roels, O. &Jaspers, E.). Volume 3. Universal Press, Western Belgium. pp. 25-26.
Takeuchi, T., Ohkuma, N., Ishida, S., Ishizuka, W., Tomota, M., Hayasawa, H. &Miyakawa, H, 1998. Development of micro-particle diet for marine fish larvae.VIII Int. Symp Nutrition and Feeding of Fish. Las Palmas, Spain, June 1-4. 193pp.
Tonheim, K. S., Espe, M., Hamre, K. & Ronnestad, I., 2005. Pre-hydrolysis improvesutilization of dietary protein in the larval teleost Atlantic halibut (Hippoglossushippoglossus L.). Journal of Experimental Marine Biology and Ecology, 321: 19-34.
Tonheim, K. S., Nordgreen, A, Hogoy, I., Hamre, K. & Ronnestad, I., 2007. In vitrodigestibility of water-soluble and water-insoluble protein fractions of somecommon fish larval feeds and feed ingredients. Aquaculture, 262: 426-435.
Uys, W. & Hecht, T., 1985. Evaluation and preparation of an optimal dry feed for theprimary nursing of Clarias gariepinus larvae. Aquaculture, 47: 173-183.
Walford, J., Lim, T. M. & Lam, T. J., 1991. Replacing live foods withmicroencapsulated diets in the rearing of sea bass, Lates calcarifer, larvae. Do. the larvae ingest and digest protein membrane microcapsules. Aquaculture, 92:225-235.
Watanabe, T., Kitajima, C. & Fujita, S., 1983. Nutritional value of live organisms usedin Japan for mass propagation of fish: A review. Aquaculture, 34: 115-143.
Yufera, F. D., Pasuial, S., Moyano, D., Alercon, G. & Parra, G. G., 2000. Towards aninert diet for first feeding gilthead seabream, Sparus aurata L. larvae.Aquaculture Nutrition, 6: 143pp.
Yufera, M., Kolkovski, S., Fernandez, C., Diaz & Dabrowski, K., 2002. Free aminoacid leaching from protein walled (llicroencapsulated diet for fish larvae.Aquaculture, 214: 273-287.
Zambonino, J. L., Cahu, C. L. & Peres. A, 1997. Partial substitution of di- and tri-peptides for native proteins in sea bass diet improves Dicentrarchus labrax larvaldevelopment. J. Nutr., 127: 608-614 .
BIOLOGIA(PAKISTAN) 2008,54 (1), 17.32PK ISSN 0006 - 3096
The occurrence of the genus PropotamocllOerus in Dhokpathanformation ofthe Middle Siwaliks of Pakistan
ZAHEER AHMED
Department alZoology, GC University. Lahore, Pakistan
ABSTRACT
The genus Propotamochoerus has been recorded for the first time from theNagri formation of the Middle Siwaliks of Pakistan. Among the subfamily suinae,the genus Propotamochoerus was abundantly present in the Dhokpathanformation. The early Propotamochoeroides were very close to the Palaeochoerusand were considered to be the ancestors of other suinae. Three major groupsevolved from early Propotamochoeroid stock, one leading to generaPotamochoerus and Sus, and the other to the genera Hippohyus, Sivahyus andstill other to the genera Microstonyx, Hippopotamodon and Jhelumia. In the firstline, Ihe cheek teeth remained bunodont with the development of accessorytubercles due to which the crown became much complicated; in the second line,there was a trend towards elongations of the cusps while in the third line, themajor trend was the attainment of gigantic size. Propotamochoerus hysudricusdiffers from the other species due to the dental morphology of premolars. It showsadmixture of primitive and advanced characters of Propolamochoerus andPotamochoerus. There are several trends to be discerned in the material such asvariation in size, complexity and hypsodonty of the premolars, and change inenamel thickness of the molars. It is suggested that Propotamochoerus evolvedfrom Hyotherium during Chinji times about 11 million years ago.
Key words: Propotamochoerus, Artiodactyla, Suinae, Potamochoerus,Hippopotamodon, Saggital cusplets.
INTRODUCTION
The genus Propotamochoerus was erected by Pilgrim (1926).On thebasis of detailed study, he transferred the species hysudricus from thegenus Potamochoerus to Propotamochoerus as Propotamochoerushysudricus(Stehlin) and designated it as type species of the. genus. Headded three species I.e., Propotamochoerus salinus, P.uliginosus andP.ingens to this genus. Pickford (1986, 1988) studied the genus and he wasof the view that genus Korynochoerus and Palaeochoerus are so close toPropotamochoerus that these may be declared as congeneric. The P. salinusis a small suid with rugose molar enamel and a small heal in the third molar(Pilgrim, 1926). The species P. hysudricus is a large form with complexmolars (SIehlin, 1899-1900) characterized by the large canine flanges,elongated premolars with thick enamel on p2-3 and anterior ling"al fossa, p3with lingual vertical ridges. The M' is elongated with accessory su!d grooves.
18 Z.AHMED BIOLOGIA VOL 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 19
Pilgrim (1926) founded the species P. hysudricus upon a mandibularramus (Ind. Mus. B-30) described and figured as Sub hysudricus by Lydekker(1884). It was later on assigned to the genus Potamochoerus by Stehlins(1899-1900). The type mandibular .ramus bears P,--M,. The molars areelongated and transversely thick. The species P. uliginosus is similar to P.salin us and they show minor difference in their dental morphology. Accordingto Colbert (1935), the species is likely synonymous with P. safinus. Thespecies P. ingens is very large, resembling the genus Hippopotamadon incanine and premolar morphology. The other type specimen included in thisspecies by Pil~rim (1926) is a maxillary fragment bearing P'-M', the crownstructure of P -M' indicates that it is closely related to genus Sivachoerus.therefore, it is bett.er to refer the specimen to the genus Sivachoerus. Thespecies P. salinus"resembles P. hysudricus and it differs only in size; the P.salin us is probably variant species of P. hysudricus.
The skull of Potamochoerus is of about the same size and resembleswith P.hysudricus in its morphology. The difference between the two is of theposition and size of the orbit that is more centrally placed and smaller in P.hysudricus (Pilgrim 1926). The 1st upper premolar is present in P. hysudricusbut absent in Potamochoerus (Colbert, 1935). The species P. hysudricus is alarge suid with complex molars, smooth enamel and vertical ridges, thegrooves are present on the crown surface. From the present studies itappears that there exist only three species of the genus Propotamochoerusin Hie Siwaliks. These are the following:
The material comprises fossil specimens of mandibular ramus, maxillaryfragments and isolated upperllower molars from Lehri, Padhri, Dhokgaal, districtJhelum, Dhokpathan, district Chakwal and Mirkhal, district Attock, the MiddleSiwaliks; Punjab, Pakistan. The specimens were partially exposed; theembedded material was carefully procured with the help of the chisel,., pick-headhammer, and various types of needles. The material was carefully prepared forstudy by using different types of gums. The unwanted embedded sediments inthe specimens were removed by using various types of Acids such asHydrochloric acid, Phosphoric acid, and Acetic acid. The specimens werecatalogued, showing the collection year and serial number of the year e.g.P.U.P.C. No 69/277, the upper figure denotes the collection year and lower one,. the serial number of the respective year. The photographs were taken with thehelp of Minolta Camera; Accessory lenses were used for smaller specimens. Thecontrast photo paper-and various photo~~emicals were used for photo printing, inorder to obtain maximum contrast of photograph, ratio of potassium bromide waschanged during the preparation of developer.
Systematic Account
Order ARTIODACTYLA Owen R. 1844- 1846.Suborder SUINAE Gray, 1821.Super family SUOIDEA Cope, 1888.Family SUIDAE Gray, 1821.Subfamily SUINAE, Zittel, 1893.
Genus Propotamochoerus, Pilgrim, 1926.Species Propotamochoerus hysudricus (Stehlin) 1899-1900.1828 Sus sp Clift, XL, Fig.5.1836 Sus Baker & Durand, pI. XLIV, Fig. 6.1876 Sus hysudricus FALCONER, CAUTLEY & LYDEKKER, 901884. Sus hysudricus, FALCONER, CAUTLEY & LYDEKKER, 13-48, pI. VIIIFigs. 2,3,5,6,8,10,111899-1900 Potamochoerus hysudricus sp. nov. (non F and C) STEHLIN, 18, 1641926 Dicoryhochoerus vagus sp. Nov. PILGRIM, 43, PI.XI Figs. 1-2, 7, PI. XXFig.8 (non PI. XV fig8=Hippohyus. PI. XX fig. 7=Sus)
MATERIALS AND METHODS
in Zoology Department,Palaeontological collection storedPunjab University, Lahore, PakistanIndian Museum, Calcutta.Upper Incisor one to fourone to four upper premolarsone to four lower premolarsone to three upper molarsone to three lower molarsPropotamochoerus
Ind. Mus.I '.4p'-4P, .•M'"M'_3P
AbbreviationsPUPC
Propotamochoerus hysudricus (Stehlin). A large species of thegenus Propotamochoerus is characterized by large canine flanges,elongated premolars, thick p'" with anterior lingual fossa, p' withlingual ridges, M' is elongated with additional accessory conules andgrooves.Propotamochoerus khani (Ahmed, 2003 a). It is a transitionalspecies between Propotamochoerus hysudricus andPropotamochoerus chinjiensis. It is characterized by the transverselythin p', the antero-lingual fossa of p'-' is absent, p' is transverselynarrow, and the vertical ridges on p' are absent. The premolars arequite small antero-posteriorly, canine is also very small with flan~esand without cement, M' is mostly squared. The shape of M inPropotamochoerus khani and Propotamochoerus hysudrincus ismore or less similar but conspicuously differs in size. The molarmorphology of Propotamochoerus khani is relatively simpler thanPropotamochoerus hysudricus. The distribution of the two speciesindicates that former occurs in older strata i.e Nagri and Dhokpathanformation. The latter is only found in Dhokpathan formation.Propotamochoerus chinjiensis (Ahmed, 2003 b). It is very smalland primitive species of the genus Propotamochoerus and is similarto the species Conohyus sindiensis but is differentiated in having awell defined multituberculated post talon of M3, which ischaracteristic of the species Propotamochoerus khani.
1.
2.
3.
18 Z.AHMED BIOLOGIA VOL 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 19
Pilgrim (1926) founded the species P. hysudricus upon a mandibularramus (Ind. Mus. B-30) described and figured as Sub hysudricus by Lydekker(1884). It was later on assigned to the genus Potamochoerus by Stehlins(1899-1900). The type mandibular .ramus bears P,--M,. The molars areelongated and transversely thick. The species P. uliginosus is similar to P.salin us and they show minor difference in their dental morphology. Accordingto Colbert (1935), the species is likely synonymous with P. safinus. Thespecies P. ingens is very large, resembling the genus Hippopotamadon incanine and premolar morphology. The other type specimen included in thisspecies by Pil~rim (1926) is a maxillary fragment bearing P'-M', the crownstructure of P -M' indicates that it is closely related to genus Sivachoerus.therefore, it is bett.er to refer the specimen to the genus Sivachoerus. Thespecies P. salinus"resembles P. hysudricus and it differs only in size; the P.salin us is probably variant species of P. hysudricus.
The skull of Potamochoerus is of about the same size and resembleswith P.hysudricus in its morphology. The difference between the two is of theposition and size of the orbit that is more centrally placed and smaller in P.hysudricus (Pilgrim 1926). The 1st upper premolar is present in P. hysudricusbut absent in Potamochoerus (Colbert, 1935). The species P. hysudricus is alarge suid with complex molars, smooth enamel and vertical ridges, thegrooves are present on the crown surface. From the present studies itappears that there exist only three species of the genus Propotamochoerusin Hie Siwaliks. These are the following:
The material comprises fossil specimens of mandibular ramus, maxillaryfragments and isolated upperllower molars from Lehri, Padhri, Dhokgaal, districtJhelum, Dhokpathan, district Chakwal and Mirkhal, district Attock, the MiddleSiwaliks; Punjab, Pakistan. The specimens were partially exposed; theembedded material was carefully procured with the help of the chisel,., pick-headhammer, and various types of needles. The material was carefully prepared forstudy by using different types of gums. The unwanted embedded sediments inthe specimens were removed by using various types of Acids such asHydrochloric acid, Phosphoric acid, and Acetic acid. The specimens werecatalogued, showing the collection year and serial number of the year e.g.P.U.P.C. No 69/277, the upper figure denotes the collection year and lower one,. the serial number of the respective year. The photographs were taken with thehelp of Minolta Camera; Accessory lenses were used for smaller specimens. Thecontrast photo paper-and various photo~~emicals were used for photo printing, inorder to obtain maximum contrast of photograph, ratio of potassium bromide waschanged during the preparation of developer.
Systematic Account
Order ARTIODACTYLA Owen R. 1844- 1846.Suborder SUINAE Gray, 1821.Super family SUOIDEA Cope, 1888.Family SUIDAE Gray, 1821.Subfamily SUINAE, Zittel, 1893.
Genus Propotamochoerus, Pilgrim, 1926.Species Propotamochoerus hysudricus (Stehlin) 1899-1900.1828 Sus sp Clift, XL, Fig.5.1836 Sus Baker & Durand, pI. XLIV, Fig. 6.1876 Sus hysudricus FALCONER, CAUTLEY & LYDEKKER, 901884. Sus hysudricus, FALCONER, CAUTLEY & LYDEKKER, 13-48, pI. VIIIFigs. 2,3,5,6,8,10,111899-1900 Potamochoerus hysudricus sp. nov. (non F and C) STEHLIN, 18, 1641926 Dicoryhochoerus vagus sp. Nov. PILGRIM, 43, PI.XI Figs. 1-2, 7, PI. XXFig.8 (non PI. XV fig8=Hippohyus. PI. XX fig. 7=Sus)
MATERIALS AND METHODS
in Zoology Department,Palaeontological collection storedPunjab University, Lahore, PakistanIndian Museum, Calcutta.Upper Incisor one to fourone to four upper premolarsone to four lower premolarsone to three upper molarsone to three lower molarsPropotamochoerus
Ind. Mus.I '.4p'-4P, .•M'"M'_3P
AbbreviationsPUPC
Propotamochoerus hysudricus (Stehlin). A large species of thegenus Propotamochoerus is characterized by large canine flanges,elongated premolars, thick p'" with anterior lingual fossa, p' withlingual ridges, M' is elongated with additional accessory conules andgrooves.Propotamochoerus khani (Ahmed, 2003 a). It is a transitionalspecies between Propotamochoerus hysudricus andPropotamochoerus chinjiensis. It is characterized by the transverselythin p', the antero-lingual fossa of p'-' is absent, p' is transverselynarrow, and the vertical ridges on p' are absent. The premolars arequite small antero-posteriorly, canine is also very small with flan~esand without cement, M' is mostly squared. The shape of M inPropotamochoerus khani and Propotamochoerus hysudrincus ismore or less similar but conspicuously differs in size. The molarmorphology of Propotamochoerus khani is relatively simpler thanPropotamochoerus hysudricus. The distribution of the two speciesindicates that former occurs in older strata i.e Nagri and Dhokpathanformation. The latter is only found in Dhokpathan formation.Propotamochoerus chinjiensis (Ahmed, 2003 b). It is very smalland primitive species of the genus Propotamochoerus and is similarto the species Conohyus sindiensis but is differentiated in having awell defined multituberculated post talon of M3, which ischaracteristic of the species Propotamochoerus khani.
1.
2.
3.
20 Z. AHMED BIOLOGIA VOL. 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 21
1926 Sus comes sp.nov. PILGRIM, 60, PI. XIX Fig.81970 Dicoryhochoerus vagus var nagrii nov. var. PRASAD, 33 PI. IX Fig.8
DiagnosisA large species of the genus characterized by the large cement covering canine,the large canine flanges, elongated premolars, large transversely thick p"', p'"with antero-lingual fossa, p' with vertical lingual ridge/s and elongated M'.
HolotypeG.S,1. No. B.30, a right mandibular ramus (illustrated by Lydekker, 1884, pI. VIII,Fig.3)
7
8
9.
10
P.U.P.C. No. 94/2, a left mandibular fragment bearing M2 and rootsof M1 from middle Dhokpathan formation of Mirkhal, district Attock,Punjab, Pakistan.P.U.P.C. No. 69/228, a left mandibular fragment bearing anteriorlydamaged M1 and anterior part of M3 from middle Dhokpathanformation Dhokpathan Rest House area, district Attock, Punjab,Pakistan.PU.P.C. No. 68/94, a left mandibular fragment bearing M2-3 frommiddle Dhokpathan formation of Dhokpathan Rest House area,district Attock, Punjab, Pakistan.P.U.P.C. No. 68/211, a right mandibular fragment bearing erupting.M3 from upper Dhokpathan formation of Dudial, district Mirpur, AJK.
HorizonDhokpathan formation of Middle Siwaliks.
LocalityNo specific locality mentioned.
Distribution'The species is known from Ava, Burma (Clift, 1828); Nagri, district Chakwal
. (Pilgrim, 1926); Hasnot, Mithrala and Bahita, district Jhelum (Pilgrim, 1926),Punjab, Pakistan. .
The present collection is from Lehri, Padhri, Dhokgaal, district Jhelum andDhokpathan and Mirkhal, district Attock, Punjab, Pakistan.
Specimens Examined (Figs.1-4)
Fig. 1: Propotamochoerus hysudricusTop (A), labial (B) and lingual (C) views of a left maxillabearing I P'-M' (P.UP.C No. 69/227) from Lehri,district Jhelum, Punjab, Pakistan.Scale bar: 5 mm.
P.U.P.C. No. 69/227, two mandibular fragments and two maxillaryfragments of a skull bearing right P'-M', left P'-M' ,right P2, rightP4-M2, left M1-3, roots of right P3 and anterior part of erupting rightM3 . from lower Dhokpathan formation of Lehri, district Jhelum,Punjab, Pakistan (Last molars have been exposed by removingbone).P.U.P.C. No. 67/158, a right maxillary fragment bearing p' frommiddle Dhokpathan formation of Padhri, district Jhelum, Punjab,Pakistan.P.U.P.C. No. 94/4, a left maxillary fragment bearing p'" from middleDhokpathan formation of Padhri, district Jhelum, Punjab, Pakistan.P.U.P.C. No. 94/3, a left mandibular ramus bearing P2 and roots ofP1 from middle Dhokpathan formation of Dhokpathan Rest Housearea, district Attock, Punjab, Pakistan.P.U.P.C. No. 91/1, a right mandibular ramus bearing P3-M2, roots ofP2 and erupting M3 from middle Dhokpathan formatioll of Padhri,district Jhelum, Punjab, Pakistan.P.U.P.C. No. 93/1, an isolated left M1 from lower Dhokpathanformation of Dhokgaal, district Jhelum, Punjab, Pakistan.
1.
2.
3.
4.
5.
6.
20 Z. AHMED BIOLOGIA VOL. 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 21
1926 Sus comes sp.nov. PILGRIM, 60, PI. XIX Fig.81970 Dicoryhochoerus vagus var nagrii nov. var. PRASAD, 33 PI. IX Fig.8
DiagnosisA large species of the genus characterized by the large cement covering canine,the large canine flanges, elongated premolars, large transversely thick p"', p'"with antero-lingual fossa, p' with vertical lingual ridge/s and elongated M'.
HolotypeG.S,1. No. B.30, a right mandibular ramus (illustrated by Lydekker, 1884, pI. VIII,Fig.3)
7
8
9.
10
P.U.P.C. No. 94/2, a left mandibular fragment bearing M2 and rootsof M1 from middle Dhokpathan formation of Mirkhal, district Attock,Punjab, Pakistan.P.U.P.C. No. 69/228, a left mandibular fragment bearing anteriorlydamaged M1 and anterior part of M3 from middle Dhokpathanformation Dhokpathan Rest House area, district Attock, Punjab,Pakistan.PU.P.C. No. 68/94, a left mandibular fragment bearing M2-3 frommiddle Dhokpathan formation of Dhokpathan Rest House area,district Attock, Punjab, Pakistan.P.U.P.C. No. 68/211, a right mandibular fragment bearing erupting.M3 from upper Dhokpathan formation of Dudial, district Mirpur, AJK.
HorizonDhokpathan formation of Middle Siwaliks.
LocalityNo specific locality mentioned.
Distribution'The species is known from Ava, Burma (Clift, 1828); Nagri, district Chakwal
. (Pilgrim, 1926); Hasnot, Mithrala and Bahita, district Jhelum (Pilgrim, 1926),Punjab, Pakistan. .
The present collection is from Lehri, Padhri, Dhokgaal, district Jhelum andDhokpathan and Mirkhal, district Attock, Punjab, Pakistan.
Specimens Examined (Figs.1-4)
Fig. 1: Propotamochoerus hysudricusTop (A), labial (B) and lingual (C) views of a left maxillabearing I P'-M' (P.UP.C No. 69/227) from Lehri,district Jhelum, Punjab, Pakistan.Scale bar: 5 mm.
P.U.P.C. No. 69/227, two mandibular fragments and two maxillaryfragments of a skull bearing right P'-M', left P'-M' ,right P2, rightP4-M2, left M1-3, roots of right P3 and anterior part of erupting rightM3 . from lower Dhokpathan formation of Lehri, district Jhelum,Punjab, Pakistan (Last molars have been exposed by removingbone).P.U.P.C. No. 67/158, a right maxillary fragment bearing p' frommiddle Dhokpathan formation of Padhri, district Jhelum, Punjab,Pakistan.P.U.P.C. No. 94/4, a left maxillary fragment bearing p'" from middleDhokpathan formation of Padhri, district Jhelum, Punjab, Pakistan.P.U.P.C. No. 94/3, a left mandibular ramus bearing P2 and roots ofP1 from middle Dhokpathan formation of Dhokpathan Rest Housearea, district Attock, Punjab, Pakistan.P.U.P.C. No. 91/1, a right mandibular ramus bearing P3-M2, roots ofP2 and erupting M3 from middle Dhokpathan formatioll of Padhri,district Jhelum, Punjab, Pakistan.P.U.P.C. No. 93/1, an isolated left M1 from lower Dhokpathanformation of Dhokgaal, district Jhelum, Punjab, Pakistan.
1.
2.
3.
4.
5.
6.
22 Z. AHMED BIOLOGIA VOL. 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 23
F
A
E
c _,~~L
._.~~,.~
.' ..•.
>::,,;';'::II
~
'IE
Fig. 2: Propotamochoerus hysudricus
Top (A) lingual (8) labial views of a right maxilla and top (C)crown(O) and labial (E) views of a right mandibular ramusbearing rP2, P4-M2, (P.U.P.C No. 69/227) from Lehri, districtJhelum, Punjab, Pakistan.Scale bar 7mm.
Fig. 3: Propotamochoerus hysudricus
Top (A), and labial (8) views of a left mandibular ramusbearing left M'.3, (P.U.P.C. No. 69/227) from Lehri,district Jhelum, Punjab, Pakistan
Crown (C) and lingual (0) views of right maxillaryfragments bearing p3 I PUPC No. 67/158) fromPadhri, district Jhelum, Punjab, Pakistan
Crown (E) and labial IF) views of a left maxillaryfragments bearing p3.' ( P.U.P.C. No. 94/4) from Padhri,district Jhelum, Punjab, Pakistan
Crown (G) and lingual (H) views of a left mandibularfragment bearing P, and roots of P, (P.U.P.C. No. 94/3)from near Ohokpalhan Resl House area, districl Attock,Punjab, Pakistan.Scale bar 7 mm.
;0.-4-
22 Z. AHMED BIOLOGIA VOL. 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 23
F
A
E
c _,~~L
._.~~,.~
.' ..•.
>::,,;';'::II
~
'IE
Fig. 2: Propotamochoerus hysudricus
Top (A) lingual (8) labial views of a right maxilla and top (C)crown(O) and labial (E) views of a right mandibular ramusbearing rP2, P4-M2, (P.U.P.C No. 69/227) from Lehri, districtJhelum, Punjab, Pakistan.Scale bar 7mm.
Fig. 3: Propotamochoerus hysudricus
Top (A), and labial (8) views of a left mandibular ramusbearing left M'.3, (P.U.P.C. No. 69/227) from Lehri,district Jhelum, Punjab, Pakistan
Crown (C) and lingual (0) views of right maxillaryfragments bearing p3 I PUPC No. 67/158) fromPadhri, district Jhelum, Punjab, Pakistan
Crown (E) and labial IF) views of a left maxillaryfragments bearing p3.' ( P.U.P.C. No. 94/4) from Padhri,district Jhelum, Punjab, Pakistan
Crown (G) and lingual (H) views of a left mandibularfragment bearing P, and roots of P, (P.U.P.C. No. 94/3)from near Ohokpalhan Resl House area, districl Attock,Punjab, Pakistan.Scale bar 7 mm.
;0.-4-
24 Z. AHMED BIOLOGIA VOL 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 25
j
Description
p'Third premolar is very similar to the p
2except that it is transversely much broad
(Table 1) and more highly crowned. The three main cusps forming summit of thetooth are almost equally high and very closely applied. Anterior cingulum isrelatively stronger in PUPC. Nos. 69/227 and 94/4 than in P.UP.C. No. 67/158(Fig.1A; 3C&E)
p2The tooth is moderately elongated, anteriorly narrow and posteriorly broad.Anterior cingulum is well developed and covers the antero-lingual part of thetooth to enclose a small anterior fossette. The postero-Iingual cusp of hypoconeis very low and along with the cingulum forms a large posterior fossette. At themedian lingual side, there is an elongated] vertical ridge (protocone) rising to theapex of the tooth. A relatively weak cingulum covers the postero-Iabial side of thetooth. The extreme anterior end of the tooth comprising a very small portion ofthe crown is missing. The apex of the tooth is made by proto-and para-cones .. Metacone is a relatively low cusp present at some distance posterior to the maincusp Whole of the crown surface is rugose (Fig.1A).
SkullThe preserved parts of the skull do not throw much light upon the morphology ofthe skull except that it was moderately elongated.
Upper dentition
p'It is almost molarised with squared crown. Anterior and posterior cingula are welldeveloped, crenulated and fairly high, There are two labial cusps of equal heightThese are anteroposteriorly elongated and are well separated by a deep groovewhich may be seen running down to the crown base in labial view. Of the twolabial cusps, the anterior one is larger than the other. Between the anterior cuspand the anterior cingulum, a small supplementary cusplet is present which ismore visible in PU.PC No. 94/4 than in PU.P.C. No, 69/227, The two sagittal. cusplets are anteroposteriorly much elongated and transversaly much narrower,The anterior sagittal cusplet is actually a combination of the two, The lingualtubercle is made by the two closely appressed cusps cif unequal size, Thesmaller one is anteriorly located and the larger one posteriorly, The two cuspsare marked by a shallow groove seen in the lingual view (Fig.1A; 3E).
M'The tooth is longer than broad (Table 1). Anterior and posterior cingula arestrong. Cingulum is absent at the lateral sides except for a small, very low basalpillar at the labial entrance of the transverse valley. Anterior accessory conule is
Fig. 4: Propotamochoerus hysudricus
Top (A), and labial (B) views of a right mandibular ramusbearing p'" M, roots of P, and erupting M,( P.UP.C. No. 91/1)from Padhri, district Jhelum, Punjab, Pakistan ..Crown(C) and lingual (0) views of an isolated 1M' (P.UPC No.93/1) from Ohokgaal, district Jhelum, Punjab, PakistanCrown(E) and labial (F) views of a left maxillary fragmentsbearing M' and roots of M' ( P,U,PC. No, 94/1) from Mirkhal,district Attock, Punjab, PakistanCrown(G)and lingual (H) ) views of a left mandibular fragment
, bearing anteriorly damaged M' and anterior part of M' ( P.U.P.C.No. 69/228)Crown (I) and lingual (J) views of a left mandibular fragmentbearing M"3 ( P,U,PC, No, 68/94) from near Ohokpathan RestHouse, district Attock, Punjab, PakistanCrown (K) and labial (L) views of a right mandibular fragmentbearing erupting M, (P,U.P.C, No. 68/211) from Oudial, districtMirpur, Azad Jammu and Kashmir.Scale bar; 7 mm
•
24 Z. AHMED BIOLOGIA VOL 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 25
j
Description
p'Third premolar is very similar to the p
2except that it is transversely much broad
(Table 1) and more highly crowned. The three main cusps forming summit of thetooth are almost equally high and very closely applied. Anterior cingulum isrelatively stronger in PUPC. Nos. 69/227 and 94/4 than in P.UP.C. No. 67/158(Fig.1A; 3C&E)
p2The tooth is moderately elongated, anteriorly narrow and posteriorly broad.Anterior cingulum is well developed and covers the antero-lingual part of thetooth to enclose a small anterior fossette. The postero-Iingual cusp of hypoconeis very low and along with the cingulum forms a large posterior fossette. At themedian lingual side, there is an elongated] vertical ridge (protocone) rising to theapex of the tooth. A relatively weak cingulum covers the postero-Iabial side of thetooth. The extreme anterior end of the tooth comprising a very small portion ofthe crown is missing. The apex of the tooth is made by proto-and para-cones .. Metacone is a relatively low cusp present at some distance posterior to the maincusp Whole of the crown surface is rugose (Fig.1A).
SkullThe preserved parts of the skull do not throw much light upon the morphology ofthe skull except that it was moderately elongated.
Upper dentition
p'It is almost molarised with squared crown. Anterior and posterior cingula are welldeveloped, crenulated and fairly high, There are two labial cusps of equal heightThese are anteroposteriorly elongated and are well separated by a deep groovewhich may be seen running down to the crown base in labial view. Of the twolabial cusps, the anterior one is larger than the other. Between the anterior cuspand the anterior cingulum, a small supplementary cusplet is present which ismore visible in PU.PC No. 94/4 than in PU.P.C. No, 69/227, The two sagittal. cusplets are anteroposteriorly much elongated and transversaly much narrower,The anterior sagittal cusplet is actually a combination of the two, The lingualtubercle is made by the two closely appressed cusps cif unequal size, Thesmaller one is anteriorly located and the larger one posteriorly, The two cuspsare marked by a shallow groove seen in the lingual view (Fig.1A; 3E).
M'The tooth is longer than broad (Table 1). Anterior and posterior cingula arestrong. Cingulum is absent at the lateral sides except for a small, very low basalpillar at the labial entrance of the transverse valley. Anterior accessory conule is
Fig. 4: Propotamochoerus hysudricus
Top (A), and labial (B) views of a right mandibular ramusbearing p'" M, roots of P, and erupting M,( P.UP.C. No. 91/1)from Padhri, district Jhelum, Punjab, Pakistan ..Crown(C) and lingual (0) views of an isolated 1M' (P.UPC No.93/1) from Ohokgaal, district Jhelum, Punjab, PakistanCrown(E) and labial (F) views of a left maxillary fragmentsbearing M' and roots of M' ( P,U,PC. No, 94/1) from Mirkhal,district Attock, Punjab, PakistanCrown(G)and lingual (H) ) views of a left mandibular fragment
, bearing anteriorly damaged M' and anterior part of M' ( P.U.P.C.No. 69/228)Crown (I) and lingual (J) views of a left mandibular fragmentbearing M"3 ( P,U,PC, No, 68/94) from near Ohokpathan RestHouse, district Attock, Punjab, PakistanCrown (K) and labial (L) views of a right mandibular fragmentbearing erupting M, (P,U.P.C, No. 68/211) from Oudial, districtMirpur, Azad Jammu and Kashmir.Scale bar; 7 mm
•
26 Z. AHMED 810LOGIAVOL 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 27
very small. Posterior acc",ssory conule is larger than the anterior one and themedian one is the largest. All the accessory conules are somewhat rounded andlow. All the four principal cusps are rounded with some additional grooves. Thereare 6 grooves on protocone, 3 on paracone, 4 on metacone and 6 on hypocone(Fig.1, 4C, E, G).
Table 1: Measurement of the referred upper dentition of Propotamochoerus. hysudricus (Stehlin)
Lower Dentition
P,It is represented by the roots only in PU.P.C. No. 94/3. The roots Indicate thatthe tooth was much smaller anteroposteriorly as well as transversely than the P2(Fi9.3G) .
NumericalSerration
p'p'p'p'p'p'p'p'p'M'M'M'M'M'M'
Catalogue No.
P.U.P.C No. 691227Ind. Mus B.688*P.U.P.C. No. 691227P.U.P.C. No. 9414P.U.PC. No. 671158Ind. Mus 8688"P.U.P.C. No. 69/227P.U.P.C. No. 69/227Ind. Mus B.688*P.U.PC. No. 691227Ind. Mus B.688*Ind. Mus B.688*PU.P.C No. 691227P.U.P.C. No. 691227Ind. Mus 8.688"
Lengthmm
15.614.616.0t6.817.614.714.815.01.3.018.915.319.625.235.230.0
Widthmm
10.87.014815.015.713.216.916.714.817.214.518.9
23.422.4
"Measurements taken from illustration made by Pilgrim (1926)
.M'
The second molar is just a larger version of the preceding tooth except that avery small basal pillar is also present 2t the lingual entrance of the transversevalley. Also, it is relatively longer than the M1(Fig.1 AAE)
M'The last molar is similar to the M
2except for the shape, cingulum and the talon.
The tooth is triangular with maximum transverse width in the anterior half whichgradually decreases towards the posterior end. The anterior cingulum is high andstrong with quite distinct anterior accessory conule. The median transverse valleybears one small basal pillar at its each entrance. The talon is unituberculated.The tubercle is small 'and low with a small cingular tubercle anterior to its lingualedge. A small tuberculated cingular ridge is present labially between themetacone and the talon. A small, very low additional tubercle is present betweenthe talon and the posterior accessory conule (Fig.1A, 4G).
l l
P,The tooth is moderately elongated and thick. Cingula .are high and are confinedto the anterior and posterior sides. The posterior cingulum is slightly higher inPUPC No. 69/227 than in P.U.P.C. NO.91/1. The tooth is almost equally broadanteriorly as well as posteriorly. The summit of the tooth is bifid and the twotubercles (proto- and ento-conids) are very closely appressed, separated by ashallow groove The anterior cusp (metaconid) is also high but mu.ch less thanthe summit of the tooth. The posterior cusp (hypoconid) is also low but IScomparatively very thick. It is separated from the anterior part of the tooth by adeep transverse groove (Fig.2A, 4A).
M,Like other molars, the first molar is a low-crowned and anteroposteriorlyelongated tooth (Tables 1 & 2). The crown in PU.P.C No. 69/227 is partiallydamaged but IS well preserved in other specimens. Ml is very small as comparedto the M2. Anterior cingulum is weak and the posterior one is moderatelydeveloped. Anterior accessory conule is very small and difficult to identify. Themedian and the posterior accessory conules are somewhat anteroposteriorlycompressed and are conspicuous. Apart from 3 suid grooves, an additionalgroove may also be seen in each of the principal cusp. The labial entrance of themedian transverse valley is blocked at the base by a weak cingular ridge. Thespecimen P.U.P.C. No. 93/01 shows abnormal wear and tear 'In this tooth, theposterior half is more worn out than the anterior one (Fig.2A, 3A, 4A).
26 Z. AHMED 810LOGIAVOL 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 27
very small. Posterior acc",ssory conule is larger than the anterior one and themedian one is the largest. All the accessory conules are somewhat rounded andlow. All the four principal cusps are rounded with some additional grooves. Thereare 6 grooves on protocone, 3 on paracone, 4 on metacone and 6 on hypocone(Fig.1, 4C, E, G).
Table 1: Measurement of the referred upper dentition of Propotamochoerus. hysudricus (Stehlin)
Lower Dentition
P,It is represented by the roots only in PU.P.C. No. 94/3. The roots Indicate thatthe tooth was much smaller anteroposteriorly as well as transversely than the P2(Fi9.3G) .
NumericalSerration
p'p'p'p'p'p'p'p'p'M'M'M'M'M'M'
Catalogue No.
P.U.P.C No. 691227Ind. Mus B.688*P.U.P.C. No. 691227P.U.P.C. No. 9414P.U.PC. No. 671158Ind. Mus 8688"P.U.P.C. No. 69/227P.U.P.C. No. 69/227Ind. Mus B.688*P.U.PC. No. 691227Ind. Mus B.688*Ind. Mus B.688*PU.P.C No. 691227P.U.P.C. No. 691227Ind. Mus 8.688"
Lengthmm
15.614.616.0t6.817.614.714.815.01.3.018.915.319.625.235.230.0
Widthmm
10.87.014815.015.713.216.916.714.817.214.518.9
23.422.4
"Measurements taken from illustration made by Pilgrim (1926)
.M'
The second molar is just a larger version of the preceding tooth except that avery small basal pillar is also present 2t the lingual entrance of the transversevalley. Also, it is relatively longer than the M1(Fig.1 AAE)
M'The last molar is similar to the M
2except for the shape, cingulum and the talon.
The tooth is triangular with maximum transverse width in the anterior half whichgradually decreases towards the posterior end. The anterior cingulum is high andstrong with quite distinct anterior accessory conule. The median transverse valleybears one small basal pillar at its each entrance. The talon is unituberculated.The tubercle is small 'and low with a small cingular tubercle anterior to its lingualedge. A small tuberculated cingular ridge is present labially between themetacone and the talon. A small, very low additional tubercle is present betweenthe talon and the posterior accessory conule (Fig.1A, 4G).
l l
P,The tooth is moderately elongated and thick. Cingula .are high and are confinedto the anterior and posterior sides. The posterior cingulum is slightly higher inPUPC No. 69/227 than in P.U.P.C. NO.91/1. The tooth is almost equally broadanteriorly as well as posteriorly. The summit of the tooth is bifid and the twotubercles (proto- and ento-conids) are very closely appressed, separated by ashallow groove The anterior cusp (metaconid) is also high but mu.ch less thanthe summit of the tooth. The posterior cusp (hypoconid) is also low but IScomparatively very thick. It is separated from the anterior part of the tooth by adeep transverse groove (Fig.2A, 4A).
M,Like other molars, the first molar is a low-crowned and anteroposteriorlyelongated tooth (Tables 1 & 2). The crown in PU.P.C No. 69/227 is partiallydamaged but IS well preserved in other specimens. Ml is very small as comparedto the M2. Anterior cingulum is weak and the posterior one is moderatelydeveloped. Anterior accessory conule is very small and difficult to identify. Themedian and the posterior accessory conules are somewhat anteroposteriorlycompressed and are conspicuous. Apart from 3 suid grooves, an additionalgroove may also be seen in each of the principal cusp. The labial entrance of themedian transverse valley is blocked at the base by a weak cingular ridge. Thespecimen P.U.P.C. No. 93/01 shows abnormal wear and tear 'In this tooth, theposterior half is more worn out than the anterior one (Fig.2A, 3A, 4A).
I 28 z. AHMED BIOLOGIA . VOl. 54 ill OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 29
'MeasuremenlstakenIromillustralionmadeby Pilgrim(1926)M, Table 3: Dental variation limits in P. hysudricus, P.chinjiensis and P.khaniIt is represented by six teeth. Like Ml, the M2 is also anteroposteriorly elongated (length and width in mm').but it is also thick transverselly (Table 2). Anterior cingulum is weaker than theposterior one. Lateral cingula are lacking in all the teeth except P.U.P.C. No. Numerical Propotamochoerus P.chinjiensis Pkhani
Serration hysudn'cus(Stehlin) Ahmed AhmedTable 2: Measurement of the referred lower dentition of Propotamochoerus
from Table from Pickford from Biologia,49 from Biologia,49. hysudricus (Stehlin)1 2 (1988) (2003) (2003)
Numerical CalalogueNo. Length Wldlh L W L W L W L WSerration mm mmP' 14.6 7.0 no 8.7 13.7 64
15.6 10.8 14.0P, P.UP.C.No.691227 14.4' 6.0'(Fromroot) P' 14.7 n2 13.0 11.0 12.8 95P, .P.UP.C No.9413 13.8' 5.5' 17.6 15.7 14.7 no 9.8(Fromrool)
(:jP, Ind.MusB.687' 153 58 . P' no 148 12.5 150 11.4 12.5P, PUP.C. No.691227 16.7 150 17.0 146 17.0 126 13.2P, P.UPC. No.94/4 16.2 8.2 M' 15.3 145 16.0 15.1 12.5 13.1P, PUP.C. No.91/1 16.8' 8.4
189 17.3 175 17.0 14.4 142(Fromrool) (Fromrool)P, Ind.MusB.687' 202 108 M' 19.6 18.9 20.8 183 17.0 13.2 20.4 17.7P, PUPC. No.691227 20.6 12.4 ' 25.4 21.9 25.0 220 18.0(Fromrool (Fromrool)P, P.UP.C No.91/1 182 10.8 M' 300 22.4 265 17.8 21.7 13.3 27.5 17.7P, P.UP.C.No.9311 18.5 139 35.2 23.4 31.4 226 312 208P, Ind.MusB.715' 17.4 12.4P, 15.3 5.8 141 76 120 5.9
P; Ind.MusB.687' 17.5 13.016.8 8.4P, P.UP.C. No.691227 17.4 13.0P, P.U.PC. No.9111 17.8 129 P:1 182 10.8 14.7 7.6 n9 82M, Ind.MusB.715' 17.6 13.0 20.6 13.9 16.7 10.0M, Ind.MusB687' 158 14.0M, PUP.C. No.69/227 18.4 13.2 P4 17.4 124 13.6 9.2 13.5 11.1M, P.UPC. No.9412 18.5 ' 112' 178 13.0 18.0 12.6 144
(Fromroot) (Fromroot) M, 158 13.0 14.5 108 140 11.0M, PUPC. No.91/1 183 13.2
18.5 14.0 18.8 no 148 11.2~b PUP.C. No.69/227 222 16.5M, P.UPC No.68194 22.1 154 M., 21.0 15.4 19.0 13.9 16.8 125 183 13.5M, P.UP.C.No.94/2 210 15.4 242 17.1 24.0 18.0 207 15.0M, P.UP.C No.69/228 225 16.2M2 P.UP.C. No.9111 242 17.1M, Ind.MusB715' 23.0 16.0 M, 336 17.1 25.5 15.0 268 13.7M, Ind.MusB687' 216 16.0 35.6 18.6 360 19.5 29.1 156M, Ind.Mus8.687' 35.6 171M, Ind.MusB71S' 35.4 18.8 'Measurements taken from illustration made by Pilgrim (1926)M) PU.P.C.No.691227 34.2Mj P.UPC. No.68/94 33.6 17.5M, PU.P.C No.68/211 17.4M.l P.U.P.C.No.691228 180M, P.UP.C No.9111 erupting
T
I 28 z. AHMED BIOLOGIA . VOl. 54 ill OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 29
'MeasuremenlstakenIromillustralionmadeby Pilgrim(1926)M, Table 3: Dental variation limits in P. hysudricus, P.chinjiensis and P.khaniIt is represented by six teeth. Like Ml, the M2 is also anteroposteriorly elongated (length and width in mm').but it is also thick transverselly (Table 2). Anterior cingulum is weaker than theposterior one. Lateral cingula are lacking in all the teeth except P.U.P.C. No. Numerical Propotamochoerus P.chinjiensis Pkhani
Serration hysudn'cus(Stehlin) Ahmed AhmedTable 2: Measurement of the referred lower dentition of Propotamochoerus
from Table from Pickford from Biologia,49 from Biologia,49. hysudricus (Stehlin)1 2 (1988) (2003) (2003)
Numerical CalalogueNo. Length Wldlh L W L W L W L WSerration mm mmP' 14.6 7.0 no 8.7 13.7 64
15.6 10.8 14.0P, P.UP.C.No.691227 14.4' 6.0'(Fromroot) P' 14.7 n2 13.0 11.0 12.8 95P, .P.UP.C No.9413 13.8' 5.5' 17.6 15.7 14.7 no 9.8(Fromrool)
(:jP, Ind.MusB.687' 153 58 . P' no 148 12.5 150 11.4 12.5P, PUP.C. No.691227 16.7 150 17.0 146 17.0 126 13.2P, P.UPC. No.94/4 16.2 8.2 M' 15.3 145 16.0 15.1 12.5 13.1P, PUP.C. No.91/1 16.8' 8.4
189 17.3 175 17.0 14.4 142(Fromrool) (Fromrool)P, Ind.MusB.687' 202 108 M' 19.6 18.9 20.8 183 17.0 13.2 20.4 17.7P, PUPC. No.691227 20.6 12.4 ' 25.4 21.9 25.0 220 18.0(Fromrool (Fromrool)P, P.UP.C No.91/1 182 10.8 M' 300 22.4 265 17.8 21.7 13.3 27.5 17.7P, P.UP.C.No.9311 18.5 139 35.2 23.4 31.4 226 312 208P, Ind.MusB.715' 17.4 12.4P, 15.3 5.8 141 76 120 5.9
P; Ind.MusB.687' 17.5 13.016.8 8.4P, P.UP.C. No.691227 17.4 13.0P, P.U.PC. No.9111 17.8 129 P:1 182 10.8 14.7 7.6 n9 82M, Ind.MusB.715' 17.6 13.0 20.6 13.9 16.7 10.0M, Ind.MusB687' 158 14.0M, PUP.C. No.69/227 18.4 13.2 P4 17.4 124 13.6 9.2 13.5 11.1M, P.UPC. No.9412 18.5 ' 112' 178 13.0 18.0 12.6 144
(Fromroot) (Fromroot) M, 158 13.0 14.5 108 140 11.0M, PUPC. No.91/1 183 13.2
18.5 14.0 18.8 no 148 11.2~b PUP.C. No.69/227 222 16.5M, P.UPC No.68194 22.1 154 M., 21.0 15.4 19.0 13.9 16.8 125 183 13.5M, P.UP.C.No.94/2 210 15.4 242 17.1 24.0 18.0 207 15.0M, P.UP.C No.69/228 225 16.2M2 P.UP.C. No.9111 242 17.1M, Ind.MusB715' 23.0 16.0 M, 336 17.1 25.5 15.0 268 13.7M, Ind.MusB687' 216 16.0 35.6 18.6 360 19.5 29.1 156M, Ind.Mus8.687' 35.6 171M, Ind.MusB71S' 35.4 18.8 'Measurements taken from illustration made by Pilgrim (1926)M) PU.P.C.No.691227 34.2Mj P.UPC. No.68/94 33.6 17.5M, PU.P.C No.68/211 17.4M.l P.U.P.C.No.691228 180M, P.UP.C No.9111 erupting
T
30 Z. AHMED BIOLOGIAVOL. 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 31
91/01. In this tooth, there is a weak labial cingulum mostly confined to theentrance of the median transverse valley. Anterior accessory conule is very smallin all the teeth. Median accessory conule is anteroposteriorly compressed andblocks the median transverse valley at the base. Posterior accessory conule is. somewhat larger than the median accessory conule in all the teeth exceptP.U.P.C. No. 91/01 in which the two are of equal size. The posterior accessoryconule, together with the cingulum, forms a significant talonid. Suid grooves varyin number due to the varying degree of wear. These are very well seen in thetooth P.U.P.C. NO.94/2 which is unworn. There are 5 suid grooves on protoconid,5 on metaconid, 4 on entoconid and 5 on hypoconid. The postero-median limb ofthe metaconid is quite thick distally and looks like an additional cusplet anterior tothe median accessory conule. This additional cusplet may be traced more or lessin all the teeth (Fig.2A, 3A, 4A). •
M,Except for the shape and size, the last molar is just a larger version of thesecond molar. It is comparatively much elongated due to the presence of talonid(Table 3) Its talonid is complex as compared to that found in the M3 of thespecies Propotamochoerus khani. Apart from the major posterior tubercle, thereis a transverse row of three tubercles anterior to it, a labial, a lingual and amedian one. Anterior to the lingual and labial tubercles, variable number ofcingular tubercles may also be seen. Posterior median accessory conule is lesscompressed and comparatively larger than the anterior median accessory conule. (Fig3A; 4A)
DISCUSSION
The specimens under study have been collected from Lehri, Padhri,Dhokgaal, district Jhelum; Dhokpathan, district Chakwal and Mirkhal, districtAttock, Punjab, Middle Siwaliks of Pakistan. The Molars and Premolars aretetracuspid which means that they belong to some Eutherian mammals. Inherbivores it resembles best with Artiodactyles particularly the pigs belonging tothe family Suidae. This is based on the reasons that the specimens have morethan one accessory conule 1 conulids, possessing bunodont dentition withcrowned conelets which also favour its inclusion in the family Suidae of Gray(1821). Simpson (1945) was the firsCto classify Family Suidae into fivesubfamilies, namely: Hyotheriinae, Listridontinae, Tetraconodontinae,Sanitheriinae and Suinae. In the subfamily Hyotheriinae and Sanitheriinae, theteeth are very much complicated, whereas, in Listriodontinae the teeth areLophodont (Meyer, 1866 and Cope, 1888). In the subfamily Tetraconodontinae,the middle cusps of lower fourth premolar are not bifurcated whereas these are. bifurcated in Suinae (Pilgrim, 1926); similar is the case in the specimens understudy.
Pilgrim (1926) founded the species Propotamochoerus hysudricus upon amandibular ramus (Ind. Mus. B.30) which was described and figured as Sushysudricus by Lydekker (1884). It was later on transferred to the genus
Ii ,...:::.
Potamochoerus by Stehlin (1899-1900). The type mandibular ramus bore P3-M3.The premolars were elongated and transversely thick. The M' was elongated.Molar's enamel was moderately thick. Talonid of M3 was simple. Teeth withsimilar characters were described by Clift (1828) as Sus sp., by Baker & Durand(1836) as Sus, by Pilgrim (1926) as Dicoryphochoerus vinayaki, and by Prasad(1970) as Dicoryphochoerus vagus var. nagrii.
The skulls of Propotamochoerus hysudricus and the modern AfricanPotamochoerus are of about the same size and are very similar to each other.The difference between the two lies in the position and size of the orbit which ismore centrally placed and smaller in Propotamo<;hoerus hysudricus (Pilgrim,1926). The upper premolar is present in P. hysudricus but absent in the genusPotamochoerus (Colbert, 1935)
Propotamochoerus hysudricus occurs in relatively younger horizon thanthe other two species. Consequently, it shows advancement over the species. P.chinjiensis and P. khani both in size (Table 3), cusp morphology and enamelfolding. (:5
The major difference between the species Propotamochoerus hysudricusand P. khani lies in the size of the canine flanges and the nature of the upperpremolars. The canine flanges are much more expanded in the species P.hysudricus but very small in P. khani, Upper premolars, especially the p', ismuch narrower transversely than P'. The lingual vertical ridges which are seen inp' of P. hysudricus are absent in P. khani. A very wide range exists in the dentalvariation of the species Propotamochoerus hysudricus (Table 3). It is justpossible that some samples of P. khani may have been erroneously referred tothe species P. hysudricus by the workers, possibly due to the reason that themajor difference between the two species lies in the structure and size of the p'It may be mentioned that the material of P. hysudricus contains a lot of isolatedcheek teeth and a large number of samples are without P'.
ACKNOWLEDGEMENTS
I am deeply indebted to and acknowledge the valuable guidance of my. Research supervisor Dr. Muhammad Sarwar, Zoology Department, University ofthe Punjab, Lahore, Pakistan. I would like to express my deep gratitude to Dr.Muhammad Akhtar, Chairman, Zoology Department, University of the Punjab.Lahore, Pakistan, for his help in photography and field work. I have deep senseof gratitude for my Ph.D. Research students Kiran Aftab and Muhammad AkramJan and M..Phil. students Samia Azad, Saima Jameel and Aasia Khaliq for typingthis manuscript
REFERENCES
Ahmed, Z., 2003 a. A new suid species Propotamochoerus khani from Siwalik ofPakistan. Biologia, 49( 1& 2): 9-20.
Ahmed, Z, 2003 b. A new species of the genus Propotamochuerus from lowerChinji formation of lower Siwalik, District Chakwal, Punjab, Pakistan.Biologia, 49(1 &2) 71 - 76.
30 Z. AHMED BIOLOGIAVOL. 54 (1) OCCURENCE OF GENUS PROPOTAMOCHOERUS IN PAKISTAN 31
91/01. In this tooth, there is a weak labial cingulum mostly confined to theentrance of the median transverse valley. Anterior accessory conule is very smallin all the teeth. Median accessory conule is anteroposteriorly compressed andblocks the median transverse valley at the base. Posterior accessory conule is. somewhat larger than the median accessory conule in all the teeth exceptP.U.P.C. No. 91/01 in which the two are of equal size. The posterior accessoryconule, together with the cingulum, forms a significant talonid. Suid grooves varyin number due to the varying degree of wear. These are very well seen in thetooth P.U.P.C. NO.94/2 which is unworn. There are 5 suid grooves on protoconid,5 on metaconid, 4 on entoconid and 5 on hypoconid. The postero-median limb ofthe metaconid is quite thick distally and looks like an additional cusplet anterior tothe median accessory conule. This additional cusplet may be traced more or lessin all the teeth (Fig.2A, 3A, 4A). •
M,Except for the shape and size, the last molar is just a larger version of thesecond molar. It is comparatively much elongated due to the presence of talonid(Table 3) Its talonid is complex as compared to that found in the M3 of thespecies Propotamochoerus khani. Apart from the major posterior tubercle, thereis a transverse row of three tubercles anterior to it, a labial, a lingual and amedian one. Anterior to the lingual and labial tubercles, variable number ofcingular tubercles may also be seen. Posterior median accessory conule is lesscompressed and comparatively larger than the anterior median accessory conule. (Fig3A; 4A)
DISCUSSION
The specimens under study have been collected from Lehri, Padhri,Dhokgaal, district Jhelum; Dhokpathan, district Chakwal and Mirkhal, districtAttock, Punjab, Middle Siwaliks of Pakistan. The Molars and Premolars aretetracuspid which means that they belong to some Eutherian mammals. Inherbivores it resembles best with Artiodactyles particularly the pigs belonging tothe family Suidae. This is based on the reasons that the specimens have morethan one accessory conule 1 conulids, possessing bunodont dentition withcrowned conelets which also favour its inclusion in the family Suidae of Gray(1821). Simpson (1945) was the firsCto classify Family Suidae into fivesubfamilies, namely: Hyotheriinae, Listridontinae, Tetraconodontinae,Sanitheriinae and Suinae. In the subfamily Hyotheriinae and Sanitheriinae, theteeth are very much complicated, whereas, in Listriodontinae the teeth areLophodont (Meyer, 1866 and Cope, 1888). In the subfamily Tetraconodontinae,the middle cusps of lower fourth premolar are not bifurcated whereas these are. bifurcated in Suinae (Pilgrim, 1926); similar is the case in the specimens understudy.
Pilgrim (1926) founded the species Propotamochoerus hysudricus upon amandibular ramus (Ind. Mus. B.30) which was described and figured as Sushysudricus by Lydekker (1884). It was later on transferred to the genus
Ii ,...:::.
Potamochoerus by Stehlin (1899-1900). The type mandibular ramus bore P3-M3.The premolars were elongated and transversely thick. The M' was elongated.Molar's enamel was moderately thick. Talonid of M3 was simple. Teeth withsimilar characters were described by Clift (1828) as Sus sp., by Baker & Durand(1836) as Sus, by Pilgrim (1926) as Dicoryphochoerus vinayaki, and by Prasad(1970) as Dicoryphochoerus vagus var. nagrii.
The skulls of Propotamochoerus hysudricus and the modern AfricanPotamochoerus are of about the same size and are very similar to each other.The difference between the two lies in the position and size of the orbit which ismore centrally placed and smaller in Propotamo<;hoerus hysudricus (Pilgrim,1926). The upper premolar is present in P. hysudricus but absent in the genusPotamochoerus (Colbert, 1935)
Propotamochoerus hysudricus occurs in relatively younger horizon thanthe other two species. Consequently, it shows advancement over the species. P.chinjiensis and P. khani both in size (Table 3), cusp morphology and enamelfolding. (:5
The major difference between the species Propotamochoerus hysudricusand P. khani lies in the size of the canine flanges and the nature of the upperpremolars. The canine flanges are much more expanded in the species P.hysudricus but very small in P. khani, Upper premolars, especially the p', ismuch narrower transversely than P'. The lingual vertical ridges which are seen inp' of P. hysudricus are absent in P. khani. A very wide range exists in the dentalvariation of the species Propotamochoerus hysudricus (Table 3). It is justpossible that some samples of P. khani may have been erroneously referred tothe species P. hysudricus by the workers, possibly due to the reason that themajor difference between the two species lies in the structure and size of the p'It may be mentioned that the material of P. hysudricus contains a lot of isolatedcheek teeth and a large number of samples are without P'.
ACKNOWLEDGEMENTS
I am deeply indebted to and acknowledge the valuable guidance of my. Research supervisor Dr. Muhammad Sarwar, Zoology Department, University ofthe Punjab, Lahore, Pakistan. I would like to express my deep gratitude to Dr.Muhammad Akhtar, Chairman, Zoology Department, University of the Punjab.Lahore, Pakistan, for his help in photography and field work. I have deep senseof gratitude for my Ph.D. Research students Kiran Aftab and Muhammad AkramJan and M..Phil. students Samia Azad, Saima Jameel and Aasia Khaliq for typingthis manuscript
REFERENCES
Ahmed, Z., 2003 a. A new suid species Propotamochoerus khani from Siwalik ofPakistan. Biologia, 49( 1& 2): 9-20.
Ahmed, Z, 2003 b. A new species of the genus Propotamochuerus from lowerChinji formation of lower Siwalik, District Chakwal, Punjab, Pakistan.Biologia, 49(1 &2) 71 - 76.
32 Z. AHMED BIOLOGIA BIOLOGI(PAKISTAN} 2008,54 (1), 33.42PK ISSN 0006 - 3096
Baker, W. E. & Durand, 1836. Table of sub-Himalayan fossil remains of thecollection. Journal Asiatic. Soc. Bengal, 5: 661-669.PI XLIV; XXXIV.
Clift, W., 1828. On the fossil remains of two new species of mastodon and othervertebrated animals found on the left bank of the Irawadi. Tran Geol. Soc.Lond. , Ser, 2, 11(3): 369-376.
Colbert, E. H.~ 1935. Distribution and phylogenetic studies on Indian fossilmammals. V. The phylogeny of the Indian suidae and the origin of theHippopotamidae. Amer. Mus. Novit., 799: 1-24.
Cope, E. D., 1888. Lydekker's catalogue of fossil mammalian in the BritishMuseum, Part-5. Amer. Nat., 22: 164-165.
Gray, J. E., 1821. On the natural arrangement of vertebrate animals. LondonMed. Reposit., 15(1): 296-310.
Lydekker, R, 1884. Indian Tertiary and Post-Tertiary vertebrata. Siwalik andNarbada Bunodont Suina. Mem. Geol'Surv. India, Pal. Indica, (10), 3(2):35-104.
Meyer, H. M., 1866. Uber die fossilen reste von Wirbelthieren ,welche die Herrenvon Schlagen tweit von ihren Reisen in Indien and Hochasien mitgebrachthaben. Pal. Habsen. Palaeontographica, Stuttgart, 15: 1-40.
Owen, R., 1848. Description of teeth portions of jaws of two 'extinctanthracotheriod quadrupeds discovered in the Eocene deposits on the N.W.coast of the Isle of Wight Quart. Jour. Geol.Soc. London, 4 103-141, pis7-8.
. Pickford, M., 1986. A revision of the Miocene Suidae and Tayassuidae(Artiodactyla Mammalia) of Africa. Tertiary Research, 7 1-83.
Pickford, M., 1988 .Revision of the Miocene suidae. of the Indian subcontinentMunchner Geowiss. Ab/:J.,4(12): 1-92.
Pilgrim, G. E., 1926. The fossil Suidae of India. Pal. Indica, ns, 8 (4):1-65, Pls,-IXX.
Prasad, K. N., 1970.The vertebrate fauna .from Siwalik beds of HaritalyangarHimachal Pradesh, India. Mem. Geol. Surv. India. Palaeont. Indica, ns., 39:1-54.
Simpson, G. G., 1945. The principles of classification of Mammals. Bull. Amer.Mus, Nat Hist, 85: 1-350.
Stehlin, H. G., 1899-1900. Uber die Geschichte des suiden. Gebissesabh.Schweiz, Palaeont. Ges, 26: 1-527.
Zitlel, K. A, 1893. Handbunch der Palaeontologie Abteilung I. Palaeozoologie.Band IV Vertebrata (Mammalia). Munich, R Oldenbourg, XI 799PP.
?
l' '~"
Effect of agrochemicals inducing genetic diversity among earthwormspecies, as revealed by RAPD, in sugarcane fields
SHAHNAZ AKHTAR RANA, NAUREEN RANA, TAHIRA RUBY, ZAHIDMUSTAFA, FAISAL SAEED ~ SAJIDA SABAHAT
Deporlmen{ C!fZoolol!Y and Fisheries, University (!/AgriclI!fllre,Faisalahad, Pakistan (.~AR, NR, TR, SS)
CABB DepartmenJ, Universily QfAgriculture, Faisalahad, PakisJan (FS)Center.f{Jr Advanced Studies in Vaccinology & Biotechnology, Universityal
Balachistan, Quella, Pakistan (ZM)
ABSTRACT
Genetic diversity of six earthworm species was assessed using Random.Amplified Polymorphic DNA (RAPD) matk1rs. A total of 123 loci were amplified by15 polymorphic RAPD primers with an average of 8.2 loci per primer. A total of 89fragments were found polymorphic. The genetic relationship among nineor9anisms was identified with the help of cluster analysis constructed on the basisof similarity matrix. Two main cluster groups were depicted. One group includingsix species, namely, Pheretima morrisi, P. hawayana, P. posthuma, P. elongata,P. suctoria and P. houl/eli, collected from low input fields. The other groupconsisted of three species, P. posthuma, P. suctoria and P. hawayana, collectedfrom high input fields. These three later species seemed highly adaptable as theywere found in both types of fields, but, these species tolerated agrochemicalstreated soils at the expense of some changes, probably in their DNAs.
Key words: Random Amplified Polymorphic DNA (RAPD), Earthworms, Geneticdistinction, Genetic relationship
INTRODUCTION
Earthworms are perhaps the most important soil organisms in terms of theirinfluence on the organic matler breakdown, soil structure development, andnutrient cycling, especially in productive ecosystems. During the last twodecades, there has been a great increase in scientific literature on earthworms,especially on rearing of culturable species and their competence to improving soilenvironment (Curry, 1987; Lee & Foster, 1991 and Edwards et al., 1995). Stillmuch remains to be known of their complete species inventory, abundance andtheir specific roles in the functioning of different crop systems. It is lamentablethat very few studies on the identification and abundance of the earthworms ofPunjab are available (Qureshi et aI., 1999; Rana et aI., 2000 and Rafique &Rana, 2001). The changed scenario of agriculture due to the intensive farmingprompted many researchers to know the effects of chemicals on earthworms intheir countries (Edwards & Lofty, 1972; Stenerson, 1979; Hans et aI., 1990;Edwards & Bohlen, 1992 and Panda et aI., 2000). Accordingly, many sub-lethalchronic toxicity symptoms in the earthworms exposed to different pesticides were
32 Z. AHMED BIOLOGIA BIOLOGI(PAKISTAN} 2008,54 (1), 33.42PK ISSN 0006 - 3096
Baker, W. E. & Durand, 1836. Table of sub-Himalayan fossil remains of thecollection. Journal Asiatic. Soc. Bengal, 5: 661-669.PI XLIV; XXXIV.
Clift, W., 1828. On the fossil remains of two new species of mastodon and othervertebrated animals found on the left bank of the Irawadi. Tran Geol. Soc.Lond. , Ser, 2, 11(3): 369-376.
Colbert, E. H.~ 1935. Distribution and phylogenetic studies on Indian fossilmammals. V. The phylogeny of the Indian suidae and the origin of theHippopotamidae. Amer. Mus. Novit., 799: 1-24.
Cope, E. D., 1888. Lydekker's catalogue of fossil mammalian in the BritishMuseum, Part-5. Amer. Nat., 22: 164-165.
Gray, J. E., 1821. On the natural arrangement of vertebrate animals. LondonMed. Reposit., 15(1): 296-310.
Lydekker, R, 1884. Indian Tertiary and Post-Tertiary vertebrata. Siwalik andNarbada Bunodont Suina. Mem. Geol'Surv. India, Pal. Indica, (10), 3(2):35-104.
Meyer, H. M., 1866. Uber die fossilen reste von Wirbelthieren ,welche die Herrenvon Schlagen tweit von ihren Reisen in Indien and Hochasien mitgebrachthaben. Pal. Habsen. Palaeontographica, Stuttgart, 15: 1-40.
Owen, R., 1848. Description of teeth portions of jaws of two 'extinctanthracotheriod quadrupeds discovered in the Eocene deposits on the N.W.coast of the Isle of Wight Quart. Jour. Geol.Soc. London, 4 103-141, pis7-8.
. Pickford, M., 1986. A revision of the Miocene Suidae and Tayassuidae(Artiodactyla Mammalia) of Africa. Tertiary Research, 7 1-83.
Pickford, M., 1988 .Revision of the Miocene suidae. of the Indian subcontinentMunchner Geowiss. Ab/:J.,4(12): 1-92.
Pilgrim, G. E., 1926. The fossil Suidae of India. Pal. Indica, ns, 8 (4):1-65, Pls,-IXX.
Prasad, K. N., 1970.The vertebrate fauna .from Siwalik beds of HaritalyangarHimachal Pradesh, India. Mem. Geol. Surv. India. Palaeont. Indica, ns., 39:1-54.
Simpson, G. G., 1945. The principles of classification of Mammals. Bull. Amer.Mus, Nat Hist, 85: 1-350.
Stehlin, H. G., 1899-1900. Uber die Geschichte des suiden. Gebissesabh.Schweiz, Palaeont. Ges, 26: 1-527.
Zitlel, K. A, 1893. Handbunch der Palaeontologie Abteilung I. Palaeozoologie.Band IV Vertebrata (Mammalia). Munich, R Oldenbourg, XI 799PP.
?
l' '~"
Effect of agrochemicals inducing genetic diversity among earthwormspecies, as revealed by RAPD, in sugarcane fields
SHAHNAZ AKHTAR RANA, NAUREEN RANA, TAHIRA RUBY, ZAHIDMUSTAFA, FAISAL SAEED ~ SAJIDA SABAHAT
Deporlmen{ C!fZoolol!Y and Fisheries, University (!/AgriclI!fllre,Faisalahad, Pakistan (.~AR, NR, TR, SS)
CABB DepartmenJ, Universily QfAgriculture, Faisalahad, PakisJan (FS)Center.f{Jr Advanced Studies in Vaccinology & Biotechnology, Universityal
Balachistan, Quella, Pakistan (ZM)
ABSTRACT
Genetic diversity of six earthworm species was assessed using Random.Amplified Polymorphic DNA (RAPD) matk1rs. A total of 123 loci were amplified by15 polymorphic RAPD primers with an average of 8.2 loci per primer. A total of 89fragments were found polymorphic. The genetic relationship among nineor9anisms was identified with the help of cluster analysis constructed on the basisof similarity matrix. Two main cluster groups were depicted. One group includingsix species, namely, Pheretima morrisi, P. hawayana, P. posthuma, P. elongata,P. suctoria and P. houl/eli, collected from low input fields. The other groupconsisted of three species, P. posthuma, P. suctoria and P. hawayana, collectedfrom high input fields. These three later species seemed highly adaptable as theywere found in both types of fields, but, these species tolerated agrochemicalstreated soils at the expense of some changes, probably in their DNAs.
Key words: Random Amplified Polymorphic DNA (RAPD), Earthworms, Geneticdistinction, Genetic relationship
INTRODUCTION
Earthworms are perhaps the most important soil organisms in terms of theirinfluence on the organic matler breakdown, soil structure development, andnutrient cycling, especially in productive ecosystems. During the last twodecades, there has been a great increase in scientific literature on earthworms,especially on rearing of culturable species and their competence to improving soilenvironment (Curry, 1987; Lee & Foster, 1991 and Edwards et al., 1995). Stillmuch remains to be known of their complete species inventory, abundance andtheir specific roles in the functioning of different crop systems. It is lamentablethat very few studies on the identification and abundance of the earthworms ofPunjab are available (Qureshi et aI., 1999; Rana et aI., 2000 and Rafique &Rana, 2001). The changed scenario of agriculture due to the intensive farmingprompted many researchers to know the effects of chemicals on earthworms intheir countries (Edwards & Lofty, 1972; Stenerson, 1979; Hans et aI., 1990;Edwards & Bohlen, 1992 and Panda et aI., 2000). Accordingly, many sub-lethalchronic toxicity symptoms in the earthworms exposed to different pesticides were
II
34 S. A. RANA ET AL BIOLOGIA VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 35
Conditions optimizing for RAPD-PCR Analysis
For RAPD analysis, protocol by Williams et aJ. (1990) was used. Ten baseoligonucleotide primers obtained from Genelink Company were used for
. amplification of the genomic DNA
PCR was carried out in 25~1 reactior, ,~lixture containing 3mM MgCI" atotal of 20 primers were used for the analysis. The thermal cycler wasprogrammed for 5 minutes. Initial 1 minute denaturation at 95°C was followed by1 minute primer annealing at 3rC, and 2 minutes extension at 72°C for 40cycles and then final extension occurred at 72°C for 10 minutes.
Amplified fragments were scored by starting from top of the lane to itsbottom. All visible and unambiguously scorable fragments amplified by theprimers were scored under the heading of total scorable fragments. Amplificationprofile of six earthworm species were compared with each other and :0 molecularsize marker and bands of DNA fragments were scored 1 as present and 0 asabent. ~
The data of primers was used to estimate genetic similarity on the basis ofnumber of shared amplification products (Nei & Li, 1979). The coefficients werecalculated by the following statistical equation:
F = 2N,y I (N, + Ny)
where, "F" is the similarity coefficient in which N, and Ny are the numbers offragments in population x and y, respectively, whereas N,y is the fragment sharedby the two populations.
Similarity coefficients were utilized to generate a dendrogram by using un-weighted pair group method of arithmetic means (UPGMA) (Sneath & Sokal,1973).
recorded. Some of which were serious and short term effects, while others wereminor, but involved long term effects on the earthworms and their functions.Genetic diversity assessment of the earthworm species can facilitate anunderstanding of their basic biology and ecology, but little effort has been madeto assess the genetic characterization of earthworm species. Among severaltechnologies, Random Amplified Polymorphic DNA (RAPD) (Welsh &McClelland, 1990) gained importance due to its simplicity, efficiency and non-requirement of sequence information (Karp et a/., 1997). RAPD markers havesuccessfully been used for the determination of genetic similarity andphylogenetic analysis (Wells & Sperling, 1999; Cognato & Sperling, 2000;Caterino et al., 2001 and Krzywinski & Besanki, 2003).
Earthworms are important for top soil fertility. Their abundance anddiversity in the field cannot be ruled out because of diversified vegetation in thecrop field which can give rise to diversified micro-fragments, organic matter andother soil biota. The conventional farming methods using various agrochemicalsin our croplands are damaging most of the top soil biota and macro-organismslike earthworms. The phenotypic anomalies as reported earlier (Rana et aI.,2000) may have some genetic basis. The hypothesis for present study was thedeCline in diversity and induction of genetic changes in earthworm speciesstriving under pesticidal stress.,
MATERIALS AND METHODS
High input crop fields (cultivations with intensive farming usingconventional doses of pesticides and synthetic fertilizers) and low input cropfields (cultivations using relatively minimal doses of pesticides, syntheticfertilizers and frequent use of organic manures) of sugarcane were selected atrandom, each from an area of 10 acres, for the collection of earthworms.
Extraction of earthworms was made from the soil by simple digging andhand sorting. An iron rectangle of one ft. sq. was used to dig out the earthwormsfrom the soil up to one ft deep at various locations in the fields. The suspectedinduction of changes in the genetic make-up of earthworms due to agrochemicalswas determined in terms of genetic diversity by RAPD analysis having thefollowing procedure.
DNA extraction
Prior to DNA extraction, the earthworms were starved for 48 hours to. allow soil to pass out from their guts. DNA extraction was done using alkalinelysis method with minor alteration in the procedure (Rocke et aI., 2002; Jonas etal., 2001 and Gerardo et al., 2001). Extracted DNA was quantified by usingspectrophotometer (Cecil, CE 2021 2000 series) at 260 nm wavelength Qualityof DNA was assessed by running 5~1 DNA on 1.0% agarose gel prepared in 0.5X TBE buffer. The samples giving smear in the gel were rejected.
l ~
. Abbreviations used
AFLP
Bp
DNA
DNTPs
EDTA
Kb
ml
~IPCR
RAPD
RFLP
Rpm
Amplified fragment length polymorphism
base pair
Deoxyribonucleic acid
Deoxyribonuleotide triphosphate
Ethylene diamine tetraacetic acidkilo bases
milli liter
micro liter
Polymerase Chain Reaction
Random amplified polymorphic DNA
Restriction fragment length polymorphism
revolutions per minute
II
34 S. A. RANA ET AL BIOLOGIA VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 35
Conditions optimizing for RAPD-PCR Analysis
For RAPD analysis, protocol by Williams et aJ. (1990) was used. Ten baseoligonucleotide primers obtained from Genelink Company were used for
. amplification of the genomic DNA
PCR was carried out in 25~1 reactior, ,~lixture containing 3mM MgCI" atotal of 20 primers were used for the analysis. The thermal cycler wasprogrammed for 5 minutes. Initial 1 minute denaturation at 95°C was followed by1 minute primer annealing at 3rC, and 2 minutes extension at 72°C for 40cycles and then final extension occurred at 72°C for 10 minutes.
Amplified fragments were scored by starting from top of the lane to itsbottom. All visible and unambiguously scorable fragments amplified by theprimers were scored under the heading of total scorable fragments. Amplificationprofile of six earthworm species were compared with each other and :0 molecularsize marker and bands of DNA fragments were scored 1 as present and 0 asabent. ~
The data of primers was used to estimate genetic similarity on the basis ofnumber of shared amplification products (Nei & Li, 1979). The coefficients werecalculated by the following statistical equation:
F = 2N,y I (N, + Ny)
where, "F" is the similarity coefficient in which N, and Ny are the numbers offragments in population x and y, respectively, whereas N,y is the fragment sharedby the two populations.
Similarity coefficients were utilized to generate a dendrogram by using un-weighted pair group method of arithmetic means (UPGMA) (Sneath & Sokal,1973).
recorded. Some of which were serious and short term effects, while others wereminor, but involved long term effects on the earthworms and their functions.Genetic diversity assessment of the earthworm species can facilitate anunderstanding of their basic biology and ecology, but little effort has been madeto assess the genetic characterization of earthworm species. Among severaltechnologies, Random Amplified Polymorphic DNA (RAPD) (Welsh &McClelland, 1990) gained importance due to its simplicity, efficiency and non-requirement of sequence information (Karp et a/., 1997). RAPD markers havesuccessfully been used for the determination of genetic similarity andphylogenetic analysis (Wells & Sperling, 1999; Cognato & Sperling, 2000;Caterino et al., 2001 and Krzywinski & Besanki, 2003).
Earthworms are important for top soil fertility. Their abundance anddiversity in the field cannot be ruled out because of diversified vegetation in thecrop field which can give rise to diversified micro-fragments, organic matter andother soil biota. The conventional farming methods using various agrochemicalsin our croplands are damaging most of the top soil biota and macro-organismslike earthworms. The phenotypic anomalies as reported earlier (Rana et aI.,2000) may have some genetic basis. The hypothesis for present study was thedeCline in diversity and induction of genetic changes in earthworm speciesstriving under pesticidal stress.,
MATERIALS AND METHODS
High input crop fields (cultivations with intensive farming usingconventional doses of pesticides and synthetic fertilizers) and low input cropfields (cultivations using relatively minimal doses of pesticides, syntheticfertilizers and frequent use of organic manures) of sugarcane were selected atrandom, each from an area of 10 acres, for the collection of earthworms.
Extraction of earthworms was made from the soil by simple digging andhand sorting. An iron rectangle of one ft. sq. was used to dig out the earthwormsfrom the soil up to one ft deep at various locations in the fields. The suspectedinduction of changes in the genetic make-up of earthworms due to agrochemicalswas determined in terms of genetic diversity by RAPD analysis having thefollowing procedure.
DNA extraction
Prior to DNA extraction, the earthworms were starved for 48 hours to. allow soil to pass out from their guts. DNA extraction was done using alkalinelysis method with minor alteration in the procedure (Rocke et aI., 2002; Jonas etal., 2001 and Gerardo et al., 2001). Extracted DNA was quantified by usingspectrophotometer (Cecil, CE 2021 2000 series) at 260 nm wavelength Qualityof DNA was assessed by running 5~1 DNA on 1.0% agarose gel prepared in 0.5X TBE buffer. The samples giving smear in the gel were rejected.
l ~
. Abbreviations used
AFLP
Bp
DNA
DNTPs
EDTA
Kb
ml
~IPCR
RAPD
RFLP
Rpm
Amplified fragment length polymorphism
base pair
Deoxyribonucleic acid
Deoxyribonuleotide triphosphate
Ethylene diamine tetraacetic acidkilo bases
milli liter
micro liter
Polymerase Chain Reaction
Random amplified polymorphic DNA
Restriction fragment length polymorphism
revolutions per minute
In RAPD-PCR amplification. the conditions were optimized for MgCI,. TaqDNA polymerase and template DNA concentration. Template concentration of 15ng/lJl was found to be optimum. The reagents (phenol, EDTA, Proteinase-K etc.)used in DNA purification procedures were potent inhibitor of Taq DNApolymerase activity (Newton & Graham, 1997). Nearly in all routine methodsethanc;>1precipitation of DNA and repetitive treatments of DNA pellets with 70%ethanol was effective in removing traces of these inhibitors. Annealingtemperature of 34°C was found optimum in the study. It was also noted that Ic;>w. annealing temperature e.g. 30°C produced less number of banes and highannealing temperature produced non-specific bands. Similarly, 3mM MgCl,concentration and one unit of Taq DNA polymerase per 25 IJI reaction were foundoptimum (Newton & Graham, 1997).
36
Taq
TBE
TNE
Tris
S. A. RANA ET AL.
Thermophilus aquaticus DNA polymerase.
Tris boric acid EDTA (buffer)
Tris-NaCI EDTA (buffer)
Trizma base
RESULTS AND DISCUSSION
BIOLOGIA
VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 37
Table 1: RAPD primers amplified and total number of fragments scored for. each primer
Sr. No. Primer Code No. of Polymorphic Totalamplified loci amplified
loci loci1 A-05 7. 5 462 A-09 8 7 463 A-13 10 7 664 B-02 8 4 505 B-13 9 3 766 B-16 8 7 567 8-19 10 9 42.8 C-09 9 8 629 C-13 8,""'1 7 32 .10 0-18 6 5 4411 0-05 10 10 5212 0-07 8 4 5113 0-11 8 8 2914 0-12 5 2 4115 0-17 8 4 43
DNA of nine organisms was amplified using different primers of arbitrarysequence. Of these isolates, a total of 123 loci were amplified by 15 primers withan average of 8.2 loci per primer; 724 fragments were amplified, out of which 89fragments were found polymorphic. In the initial study, primers were screened fortheir polymorphic amplification. A total of 25 primers of random sequence wereselected, four primers resulted in low amplification profile, while six (C-19, C-11,0-13. A-12, A-08 and A-07) were found non-polymorphic and were excluded(Table 1). Reactions were repeated three times to check the consistency ofamplified products. Only easily resolved bright DNA bands were considered andscored. All the nine organisms showed diversity with each other on theiramplification profile bases. The level of polymorphism was found different withdifferent primers among these organisms.
When isolated DNA was electrophoresed on 1% agarose gel, no protein. as well as RNA contamination was observed (no smear). Absorbance valueswere taken at rando'm 260nm and 280nm to obtain 260/280 ratio. Value of morethan 1.8 for each isolate was obtained that further confirmed the purity of DNA(Table 2).
l - b
Table 2: DNA estimationl quantification extracted from earthworm speciesusing alkaline lysis method as determined by UV spectrophotometry
Sr. Species UV absorbance value at DNA yieldNo. wavelen!lth 270 nm (1J!l/ml)1 Pheretima morrisi (UP) 0.045 2252 P. hawayana (UP) 0005 2553 P. posthuma (UP) 0.043 2154 Phou/leti (UP) 0061 3055 P.e/onaate (LIP) 0045 2256 P.suctoria (UP) 0.050 2507 P posthuma (HIP) 0.041 2058 P. suctoria (HIP) 0053 2659 P. hawayana (HIP) 0061 305
The reproducibility of the RAPD technique could be influenced by manyfactors, such as sequence of a primer, template quality and quantity, the type ofthermal cycler and polymerase employed (Schierwater & Ender, 1993). The useof a standardized RAPD protocol could ensure the reproducible results in RAPD. methodology. Variant concentrations of MgCl" Taq DNA polymerase and
In RAPD-PCR amplification. the conditions were optimized for MgCI,. TaqDNA polymerase and template DNA concentration. Template concentration of 15ng/lJl was found to be optimum. The reagents (phenol, EDTA, Proteinase-K etc.)used in DNA purification procedures were potent inhibitor of Taq DNApolymerase activity (Newton & Graham, 1997). Nearly in all routine methodsethanc;>1precipitation of DNA and repetitive treatments of DNA pellets with 70%ethanol was effective in removing traces of these inhibitors. Annealingtemperature of 34°C was found optimum in the study. It was also noted that Ic;>w. annealing temperature e.g. 30°C produced less number of banes and highannealing temperature produced non-specific bands. Similarly, 3mM MgCl,concentration and one unit of Taq DNA polymerase per 25 IJI reaction were foundoptimum (Newton & Graham, 1997).
36
Taq
TBE
TNE
Tris
S. A. RANA ET AL.
Thermophilus aquaticus DNA polymerase.
Tris boric acid EDTA (buffer)
Tris-NaCI EDTA (buffer)
Trizma base
RESULTS AND DISCUSSION
BIOLOGIA
VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 37
Table 1: RAPD primers amplified and total number of fragments scored for. each primer
Sr. No. Primer Code No. of Polymorphic Totalamplified loci amplified
loci loci1 A-05 7. 5 462 A-09 8 7 463 A-13 10 7 664 B-02 8 4 505 B-13 9 3 766 B-16 8 7 567 8-19 10 9 42.8 C-09 9 8 629 C-13 8,""'1 7 32 .10 0-18 6 5 4411 0-05 10 10 5212 0-07 8 4 5113 0-11 8 8 2914 0-12 5 2 4115 0-17 8 4 43
DNA of nine organisms was amplified using different primers of arbitrarysequence. Of these isolates, a total of 123 loci were amplified by 15 primers withan average of 8.2 loci per primer; 724 fragments were amplified, out of which 89fragments were found polymorphic. In the initial study, primers were screened fortheir polymorphic amplification. A total of 25 primers of random sequence wereselected, four primers resulted in low amplification profile, while six (C-19, C-11,0-13. A-12, A-08 and A-07) were found non-polymorphic and were excluded(Table 1). Reactions were repeated three times to check the consistency ofamplified products. Only easily resolved bright DNA bands were considered andscored. All the nine organisms showed diversity with each other on theiramplification profile bases. The level of polymorphism was found different withdifferent primers among these organisms.
When isolated DNA was electrophoresed on 1% agarose gel, no protein. as well as RNA contamination was observed (no smear). Absorbance valueswere taken at rando'm 260nm and 280nm to obtain 260/280 ratio. Value of morethan 1.8 for each isolate was obtained that further confirmed the purity of DNA(Table 2).
l - b
Table 2: DNA estimationl quantification extracted from earthworm speciesusing alkaline lysis method as determined by UV spectrophotometry
Sr. Species UV absorbance value at DNA yieldNo. wavelen!lth 270 nm (1J!l/ml)1 Pheretima morrisi (UP) 0.045 2252 P. hawayana (UP) 0005 2553 P. posthuma (UP) 0.043 2154 Phou/leti (UP) 0061 3055 P.e/onaate (LIP) 0045 2256 P.suctoria (UP) 0.050 2507 P posthuma (HIP) 0.041 2058 P. suctoria (HIP) 0053 2659 P. hawayana (HIP) 0061 305
The reproducibility of the RAPD technique could be influenced by manyfactors, such as sequence of a primer, template quality and quantity, the type ofthermal cycler and polymerase employed (Schierwater & Ender, 1993). The useof a standardized RAPD protocol could ensure the reproducible results in RAPD. methodology. Variant concentrations of MgCl" Taq DNA polymerase and
38 s. A. RANA ET AL BIOLOGIA VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 39
T<!ble 3: Similarity matrix of nine earthworm species obt<!ined fromRAPD m<!rkers,
concentration of template DNA were optimized for PCR conditions. DNAconcentrations of 5,7,10, 12 and 15ng/ 25~1 in each reaction were studied. Theconcentration of 10 ng/ 25 ~I was found to produce the most consistent andreproducible results. Of all the concentrations of MgCl, studied (1.0, 1.5, 2.0, 2.5,3.0 mM), 3mM concentration was found optimum for consistency. Similarly, Taq. DNA polymerase concentrations were studied (0.5, 1.0 and 1.5 unitsl 25~1reaction). One unit concentration of Taq was found optimum for betteramplification of genomic DNA. Other reaction conditions were also kept constantand results were found consistant and reproducible.
P./llIlrri.'
P.f",,,.,,.,",,,,P. h""v~I'",,,r 0.8056
UP 1IfPP.hl/lt'll.I'llnn P.p"",hu",n ".h"ulli'ri P.dOI/KIIIII P.,"I"',,,ria P.p'''''!'''''''' p'.,""I",i" 1',1I(1"'''.I"UII
1~1""tl,um" 0.8762 0737
E p,suc/oria
0.833
f J!.hou/ll!li
k P.lwlI-"ayall
r Il.e/OJ/gil/II
r J1.po,l'r!lIlJIIl7
_I! 1-'.Il1llrayww
r I'.sue/or/a
0,715
O.J54
- f 1'.11(1]1'0.1"(1//(1
f VpOSI/W/I/(/
lJ.74
0./48
0.7740,703
0.615
0.6
0.871o.mO,67~
0.746
0.189.....,0,7)
0.614
0.7[6
0.711
0,705
0.645
0.787
0,691
0.807
0,739
0684
0,1617
0.7049P,'I"ltx"r" 0,7148
P./""H"",,,, 0.749
P."''''lori" 0.718
P.h",.lf",; 0.7888
p..,,,•.,,,,i,, 0,86]
P.",'"'III'UIIIIL - 0,743
Identified from their phenotypic characters, six earthworm species, namely,P morrisi, P. hawayana, P. posthuma, P. e/ongata, P. suctoria and P. hOll/leti,from LIP sugarcane fields were confirmed as a genetically different group. Butthe individuals of the three species collected from HIP fields i.e. P. posthllma, P.suetoria and P. hawayana formed another genetically diverse group (Figure 2). Itis assumed that the pesticidal impact on the genetic structure of the earthwormscould have the ability to cause mutation changing the path of evolution. Sublethaldoses of different chemicals have been found to cause varied effects onearthworm populations (Bharathi & Rao, 1984; Drewes & Callahan, 1988; Antonet al., 1990 and Hans et al., 1990). Present study is the first report, using RAPDanalysis, to understand the genetic diversity in earthworms. Based on the results,it is suggested that RAPD can be used for the identification of different species.Producing much polymorphic information of target genomes with more primerscan compensate the shortcoming of this method. These results can be taken asa starting point for future researches aimed at defining the level of intra- andinter-species genetic diversity.
The genetic similarity matrix was obtained through RAPD-PCR based onNei & Li (1979) coefficient of similarity (Table 3). The genetic similarities wereranging from 60.0% to 87.62%. The maximum. similarity 87.62% was foundbetween P. morrisi an,d P. posthuma and minimum similarity was observedbetween P. e/ongata and P. hawayana which was 60.0%. The genetic similaritymatrix of RAPD data for the nine organisms was constructed based on coefficientof similarity. It was evidenced from the cluster analysis that there was high levelof genetic similarity between P. prosthuma and P. morrisi and they were closelyclustered with P. hawayana at 84%. The same genetic similarity was observed. between P. e/ongata and P. suetoria. Dendrogram was constructed on the basisof similarity matrix. Interestingly, the two genetically diverse groups were found,which were rightly correlated with their collection sites (sugarcane) receivingagrochemicals (Figure 1).
06 0.05 0.70 0.75 O.RO 0.85 0.90 0.95 1.0
..y.,Fig. 1: Dendrogr<!m showing lhe clustering of (HIP <!nd LIP) s<!mple ofearthworms 'IS genetically mut<!ted P. posthuma, P. suetoria <!nd P.hawayana suspected to be tolerant 'It the cost of their genetic modification.
I
38 s. A. RANA ET AL BIOLOGIA VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 39
T<!ble 3: Similarity matrix of nine earthworm species obt<!ined fromRAPD m<!rkers,
concentration of template DNA were optimized for PCR conditions. DNAconcentrations of 5,7,10, 12 and 15ng/ 25~1 in each reaction were studied. Theconcentration of 10 ng/ 25 ~I was found to produce the most consistent andreproducible results. Of all the concentrations of MgCl, studied (1.0, 1.5, 2.0, 2.5,3.0 mM), 3mM concentration was found optimum for consistency. Similarly, Taq. DNA polymerase concentrations were studied (0.5, 1.0 and 1.5 unitsl 25~1reaction). One unit concentration of Taq was found optimum for betteramplification of genomic DNA. Other reaction conditions were also kept constantand results were found consistant and reproducible.
P./llIlrri.'
P.f",,,.,,.,",,,,P. h""v~I'",,,r 0.8056
UP 1IfPP.hl/lt'll.I'llnn P.p"",hu",n ".h"ulli'ri P.dOI/KIIIII P.,"I"',,,ria P.p'''''!'''''''' p'.,""I",i" 1',1I(1"'''.I"UII
1~1""tl,um" 0.8762 0737
E p,suc/oria
0.833
f J!.hou/ll!li
k P.lwlI-"ayall
r Il.e/OJ/gil/II
r J1.po,l'r!lIlJIIl7
_I! 1-'.Il1llrayww
r I'.sue/or/a
0,715
O.J54
- f 1'.11(1]1'0.1"(1//(1
f VpOSI/W/I/(/
lJ.74
0./48
0.7740,703
0.615
0.6
0.871o.mO,67~
0.746
0.189.....,0,7)
0.614
0.7[6
0.711
0,705
0.645
0.787
0,691
0.807
0,739
0684
0,1617
0.7049P,'I"ltx"r" 0,7148
P./""H"",,,, 0.749
P."''''lori" 0.718
P.h",.lf",; 0.7888
p..,,,•.,,,,i,, 0,86]
P.",'"'III'UIIIIL - 0,743
Identified from their phenotypic characters, six earthworm species, namely,P morrisi, P. hawayana, P. posthuma, P. e/ongata, P. suctoria and P. hOll/leti,from LIP sugarcane fields were confirmed as a genetically different group. Butthe individuals of the three species collected from HIP fields i.e. P. posthllma, P.suetoria and P. hawayana formed another genetically diverse group (Figure 2). Itis assumed that the pesticidal impact on the genetic structure of the earthwormscould have the ability to cause mutation changing the path of evolution. Sublethaldoses of different chemicals have been found to cause varied effects onearthworm populations (Bharathi & Rao, 1984; Drewes & Callahan, 1988; Antonet al., 1990 and Hans et al., 1990). Present study is the first report, using RAPDanalysis, to understand the genetic diversity in earthworms. Based on the results,it is suggested that RAPD can be used for the identification of different species.Producing much polymorphic information of target genomes with more primerscan compensate the shortcoming of this method. These results can be taken asa starting point for future researches aimed at defining the level of intra- andinter-species genetic diversity.
The genetic similarity matrix was obtained through RAPD-PCR based onNei & Li (1979) coefficient of similarity (Table 3). The genetic similarities wereranging from 60.0% to 87.62%. The maximum. similarity 87.62% was foundbetween P. morrisi an,d P. posthuma and minimum similarity was observedbetween P. e/ongata and P. hawayana which was 60.0%. The genetic similaritymatrix of RAPD data for the nine organisms was constructed based on coefficientof similarity. It was evidenced from the cluster analysis that there was high levelof genetic similarity between P. prosthuma and P. morrisi and they were closelyclustered with P. hawayana at 84%. The same genetic similarity was observed. between P. e/ongata and P. suetoria. Dendrogram was constructed on the basisof similarity matrix. Interestingly, the two genetically diverse groups were found,which were rightly correlated with their collection sites (sugarcane) receivingagrochemicals (Figure 1).
06 0.05 0.70 0.75 O.RO 0.85 0.90 0.95 1.0
..y.,Fig. 1: Dendrogr<!m showing lhe clustering of (HIP <!nd LIP) s<!mple ofearthworms 'IS genetically mut<!ted P. posthuma, P. suetoria <!nd P.hawayana suspected to be tolerant 'It the cost of their genetic modification.
I
40 S. A. RANA ET AL BIOLOGIA VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 41
J-• ~ n- 0
ci='e;,C=S:=~' , ! •
",'it
=.. '.'-
Figure 2: Amplification profile of nine earthworm species by using primerGLB-13
From left to right
RAPD Marker
Lane 1.representing P. morrisi
Lane 2 representing P. hawayana
Lane 3 representing P, posthuma
Lane 4 representing P. houletti
Lane 5 representing P elongata
Lane 6 representing P. suctoria
. Lane 7 representing P, posthuma
Lane 8 represer,ting P. suctoriaLane 9 representing Phawayana
RAPD Marker
'\,
-
REFERENCESAnton, F, Laborda, E. & Laborda, P., 1990. Acute toxicity of the fungicide captan
to the earthworm Eisenia fetida (Savigny). Bull. Environ Contam. Toxicol.,45 82-87.
Bharathi, C. & Rao, S., 1984. Toxicity of phosphamidon to the common SouthIndian earthworm Lampito mauritti. Bull. Environ. Contam. Toxicol., 32: 295-300.
Caterino, M. S, Reed, RD., Kuo, M. M. & Sperling, F. A H, 2001. A partitionedlikelihood analysis of swallowtail butterfly phylogeny (Lepidoptera:Paplionidae). Syst Bioi., 50: 106-127.
Cognato, A I. & Sperling, F. A H., 2000. Phylogeny of Ips DeGeer species(Coleoptera Scolytidae) inferred fr<;'111Mitochondrial Cytochrome OxidaseDNA Sequence. Mol Phylog. Evol, 14J 445-460. .
Curry, J. P., 1987. The invertebrate fauna of grassland and its influence onproductivity. III. Effect on soil fertility and plant growth. Grass Forage Sci.,42: 325-341. . .
Drewes, C. D. & Callahan, C. A, 1988. Electrophysiological .detection ofsublethal neurotoxic effects in intact earthworm, In: Earthworms In WasteAnd Environment Management (eds Edwards, C. A. & Neuhauser, E.F.),SPB Acad. Pub!. The Hague, The Netherlands. pp 355-366.
Edwards, C. A & Bohlen, P. J., 1992. The effects of toxic chemicals onearthworms. Rev. Environ. Cotam. Toxicol, 125: 23-29.
Edwards, C. A, Bohlen, P. J., Linden, D. R & Subler, S., 199'5. Earthworms inagroecosystems, In: Earthworm Ecology and Biography in North America.(ed P F. Hendrix), Lewis Publishers, Boca Raton, FL, pp 185-213.
Edwards, C. A & Lofty, J. R, 1972. Biology of Earthworms. Chapman and Hall,London. 283pp.
Gerardo, S. H, Erardo, M. C., Diane, C. C, Marina, C C., Helenf, 1. H. & Ellie,C. G., 2001. Pasteurells multocida subsp. Multicoda and subsp. Septicadifferentiation by PCR fingerprinting and a-glucosidase activity. J ClinMicrobiol., 39: 2558-2564.
Hans, K. R, Gupta, R C. & Big, M. U., 1990. Toxicity assessment of fourinsecticides on earthworm, Pheretima posthuma. Bull. Environ. ContamToxicol., 45: 358-364. .
Jonas, M., Morishota, 1. Y" Angirk, E. J..& Jahja, B., 2001. Characterization ofnine Pasteurells multocida isolates from avian cholera outbreaks inIndonesia, Avian Dis., 45: 34-42.
Karp. A, Krevoich, S., Bhat, K. V. & Ayad, W. G" 1997. Molecular tools in plantgenetic resources conservation: a guide to the technologies. In: InternationalPlant Genetic Resource Institute. Rome, Italy. Tech. Bull. NO.2: 165pp.
40 S. A. RANA ET AL BIOLOGIA VOL 54 (1) EFFECT OF AGROCHEMICALS IN EARTHWORMS AS REVEALED BY RAPD 41
J-• ~ n- 0
ci='e;,C=S:=~' , ! •
",'it
=.. '.'-
Figure 2: Amplification profile of nine earthworm species by using primerGLB-13
From left to right
RAPD Marker
Lane 1.representing P. morrisi
Lane 2 representing P. hawayana
Lane 3 representing P, posthuma
Lane 4 representing P. houletti
Lane 5 representing P elongata
Lane 6 representing P. suctoria
. Lane 7 representing P, posthuma
Lane 8 represer,ting P. suctoriaLane 9 representing Phawayana
RAPD Marker
'\,
-
REFERENCESAnton, F, Laborda, E. & Laborda, P., 1990. Acute toxicity of the fungicide captan
to the earthworm Eisenia fetida (Savigny). Bull. Environ Contam. Toxicol.,45 82-87.
Bharathi, C. & Rao, S., 1984. Toxicity of phosphamidon to the common SouthIndian earthworm Lampito mauritti. Bull. Environ. Contam. Toxicol., 32: 295-300.
Caterino, M. S, Reed, RD., Kuo, M. M. & Sperling, F. A H, 2001. A partitionedlikelihood analysis of swallowtail butterfly phylogeny (Lepidoptera:Paplionidae). Syst Bioi., 50: 106-127.
Cognato, A I. & Sperling, F. A H., 2000. Phylogeny of Ips DeGeer species(Coleoptera Scolytidae) inferred fr<;'111Mitochondrial Cytochrome OxidaseDNA Sequence. Mol Phylog. Evol, 14J 445-460. .
Curry, J. P., 1987. The invertebrate fauna of grassland and its influence onproductivity. III. Effect on soil fertility and plant growth. Grass Forage Sci.,42: 325-341. . .
Drewes, C. D. & Callahan, C. A, 1988. Electrophysiological .detection ofsublethal neurotoxic effects in intact earthworm, In: Earthworms In WasteAnd Environment Management (eds Edwards, C. A. & Neuhauser, E.F.),SPB Acad. Pub!. The Hague, The Netherlands. pp 355-366.
Edwards, C. A & Bohlen, P. J., 1992. The effects of toxic chemicals onearthworms. Rev. Environ. Cotam. Toxicol, 125: 23-29.
Edwards, C. A, Bohlen, P. J., Linden, D. R & Subler, S., 199'5. Earthworms inagroecosystems, In: Earthworm Ecology and Biography in North America.(ed P F. Hendrix), Lewis Publishers, Boca Raton, FL, pp 185-213.
Edwards, C. A & Lofty, J. R, 1972. Biology of Earthworms. Chapman and Hall,London. 283pp.
Gerardo, S. H, Erardo, M. C., Diane, C. C, Marina, C C., Helenf, 1. H. & Ellie,C. G., 2001. Pasteurells multocida subsp. Multicoda and subsp. Septicadifferentiation by PCR fingerprinting and a-glucosidase activity. J ClinMicrobiol., 39: 2558-2564.
Hans, K. R, Gupta, R C. & Big, M. U., 1990. Toxicity assessment of fourinsecticides on earthworm, Pheretima posthuma. Bull. Environ. ContamToxicol., 45: 358-364. .
Jonas, M., Morishota, 1. Y" Angirk, E. J..& Jahja, B., 2001. Characterization ofnine Pasteurells multocida isolates from avian cholera outbreaks inIndonesia, Avian Dis., 45: 34-42.
Karp. A, Krevoich, S., Bhat, K. V. & Ayad, W. G" 1997. Molecular tools in plantgenetic resources conservation: a guide to the technologies. In: InternationalPlant Genetic Resource Institute. Rome, Italy. Tech. Bull. NO.2: 165pp.
42 S. A. RANA ET AL. BIOLOGIA BIOLOGIA(PAKISTAN) 2008,54 (1), 43-48PK ISSN 0006 - 3096
Krzywinski, J. & Besanki, N. K., 2003. Molecular systemics of Anopheles: FromSubgenera to sub populations ..Am. Rev. En/omol., 48: 111-139.
Lee, K. & Foster, R. C., 1991. Soil fauna and soil structure. Aus/. J. Soil Res., 29:745-776.
Nei, N. & Li, w., 1979. Mathematical model for studying genetic variation in termsof restriction endonucleases. Proc. Nail. Acad. Sci., 76: 5269-5273.
Newton, C. R & Graham, A., 1997. PCR, BIOS Scientific Pub, Ltd. U.K. pp. 18-22.
Panda, S., Sahuo, S. & Panda, S., 2000. Assessment of recovery o'i population,biomass and reproduction of the earthworm Drawida willsi following theapplication of'Malathion under field conditions. Bioi. And Fer!. of Soil .. 32:82-86.
Qureshi, J. I., Rana, S. A & Ghafoor, A., 1999. Habitat preference of earthwormspecies in Faisalabad. J.A.P.S., 9: 3-4.
Rafique, A & Rana, S. A, 2001. Species association of some earthworms in theagro-ecosystem of Faisalabad and Sargodha. Pak. J. Agri. Sci., 38: 27-28.
Rana, S. A, Rafique, A & Qureshi, J. I., 2000. Ecological distribution ofearthworm species along some water channels, water ditches, canal andriver in the agro-system of Faisalabad Division. J.A.P.S., 10: 126-130.
Rocke, T. E, Susan, R S., Amy, M. & Daniel, J. S., 2002. A serotype specificpolymerase chain reaction for identification of Pasteurella mul/ocidaserotype 1. Avian Dis., 46: 370-377,
Scheirwater, B. & Ender, A, 1993. Different thermostable DNA polymerase mayamplify different RAPD products. Nucl. Acids Res., 21: 4647-4648.
Sneath, P. H. A & Sokal, R R 1973. Numerical Taxonomy. Freeman, SanFransisco ..359pp.
Stenerson, J., 1979. Action of pesticides on earthworms. Part I. The toxicity 0/cholinesterase inhibiting insecticides to earthworm as evaluated bylaboratory test. Pes/ic. Sci., 10: 66-74.
Wells, J. D. & Sperling, F. A H., 1999. Molecular phylogeny of Chysoma albicepsand C. rufifacies ( Diptera: Calliphoridae). J. Med. Entomol., 34: 222-226.
Welsh, J. & McClieland, M., 1990. Fingerprinting genomes using PCR witharbitrary primers. Nucl. Acids Res., 18: 7213-7219.
Williams, J. G., Kubelik, A. R, Livak, K. J., Rafalsk, J. A & Tingey, S. V, 1990.DNA polymorphism by arbitrary primers is useful as genetic markers. Nucl.Acids Res., 18: 6531-6535.
Biodiversity of Araneid fauna from the Trifolium alexandrinumfields at Gutwala, district Faisalabad, Pakistan
ABllllL GHAFOOR & ZAHID HUSSAIN ALVI
Department o.lZoology, GC University, I.-aisa/ahad, Pakistan
ABSTRACT
A survey of ground spiders in the Berseem, Tnlolium aJexandrinum, fieldsby using pitfall traps revealed a total of 549 spiders belongin9 to 5 families, ggenera and 21 species. The families Lycosidae and Salticidae were common asthey jointly accounted for 94.17% of the total catch. Lycosid species were moredominant as compared to others.
Key words: Biodiversity, Araneid fauna, Trifolium alexandn.num, Faisalabad.
INTRODUCTION
Biodiversity is not only an issue of curiosity, but stands firm on the politicalagenda as a resource for humanity (Heywood, 1996). Pakistan is basically anagricultural country with diverse climate and ecological background.Phytophagous insects cause serious damage to agricultural crops, vegetables,fruit trees, ornamental and wild plants. Pesticides are used to protect crops frominsecl pests, which are not only injurious to human health but also deplete bio-diversity essential for ecological stability. In order to conserve biodiversity andminimize dependence on pesticides, integrated pest management programneeds to be encouraged.
Spiders (Araneae) fall within the general area of natural control factors.They feed almost exclusively on insects and form one of the most ubiquitousgroups of predacious organisms in the animal kingdom. They are importantenemies of pest species including aphids, mites, lepidopterous larvae and theireggs. Spiders are cosmopolitan in distribution and have conquered almost allecological environments with perhaps the single exception of the air. The varioushabitats occupied are soil: houses, forests, meadows, woodlands, croplands, thepetals of flowers and even they may have adopted amphibious life.
Spiders mainly predate on insects and other arthropods. As they aregeneralist predators, they can consume all types of insects as their food. Spidersare not generally host specific and have alternate prey.
Nyffeler & Benz (1984) observed that spiders as predators had a moresignificant role in woodlands as compared to maize fields. Patel et a/. (1986)studied the ground activity, species diversity, species richness and relativeabundance of spiders caught by pitfall trap from cotton fields. They stated thatspiders playa significant role in biological control of insect pests of some crops.Spiller (1986) studied the inter-specific competition between spiders and its
42 S. A. RANA ET AL. BIOLOGIA BIOLOGIA(PAKISTAN) 2008,54 (1), 43-48PK ISSN 0006 - 3096
Krzywinski, J. & Besanki, N. K., 2003. Molecular systemics of Anopheles: FromSubgenera to sub populations ..Am. Rev. En/omol., 48: 111-139.
Lee, K. & Foster, R. C., 1991. Soil fauna and soil structure. Aus/. J. Soil Res., 29:745-776.
Nei, N. & Li, w., 1979. Mathematical model for studying genetic variation in termsof restriction endonucleases. Proc. Nail. Acad. Sci., 76: 5269-5273.
Newton, C. R & Graham, A., 1997. PCR, BIOS Scientific Pub, Ltd. U.K. pp. 18-22.
Panda, S., Sahuo, S. & Panda, S., 2000. Assessment of recovery o'i population,biomass and reproduction of the earthworm Drawida willsi following theapplication of'Malathion under field conditions. Bioi. And Fer!. of Soil .. 32:82-86.
Qureshi, J. I., Rana, S. A & Ghafoor, A., 1999. Habitat preference of earthwormspecies in Faisalabad. J.A.P.S., 9: 3-4.
Rafique, A & Rana, S. A, 2001. Species association of some earthworms in theagro-ecosystem of Faisalabad and Sargodha. Pak. J. Agri. Sci., 38: 27-28.
Rana, S. A, Rafique, A & Qureshi, J. I., 2000. Ecological distribution ofearthworm species along some water channels, water ditches, canal andriver in the agro-system of Faisalabad Division. J.A.P.S., 10: 126-130.
Rocke, T. E, Susan, R S., Amy, M. & Daniel, J. S., 2002. A serotype specificpolymerase chain reaction for identification of Pasteurella mul/ocidaserotype 1. Avian Dis., 46: 370-377,
Scheirwater, B. & Ender, A, 1993. Different thermostable DNA polymerase mayamplify different RAPD products. Nucl. Acids Res., 21: 4647-4648.
Sneath, P. H. A & Sokal, R R 1973. Numerical Taxonomy. Freeman, SanFransisco ..359pp.
Stenerson, J., 1979. Action of pesticides on earthworms. Part I. The toxicity 0/cholinesterase inhibiting insecticides to earthworm as evaluated bylaboratory test. Pes/ic. Sci., 10: 66-74.
Wells, J. D. & Sperling, F. A H., 1999. Molecular phylogeny of Chysoma albicepsand C. rufifacies ( Diptera: Calliphoridae). J. Med. Entomol., 34: 222-226.
Welsh, J. & McClieland, M., 1990. Fingerprinting genomes using PCR witharbitrary primers. Nucl. Acids Res., 18: 7213-7219.
Williams, J. G., Kubelik, A. R, Livak, K. J., Rafalsk, J. A & Tingey, S. V, 1990.DNA polymorphism by arbitrary primers is useful as genetic markers. Nucl.Acids Res., 18: 6531-6535.
Biodiversity of Araneid fauna from the Trifolium alexandrinumfields at Gutwala, district Faisalabad, Pakistan
ABllllL GHAFOOR & ZAHID HUSSAIN ALVI
Department o.lZoology, GC University, I.-aisa/ahad, Pakistan
ABSTRACT
A survey of ground spiders in the Berseem, Tnlolium aJexandrinum, fieldsby using pitfall traps revealed a total of 549 spiders belongin9 to 5 families, ggenera and 21 species. The families Lycosidae and Salticidae were common asthey jointly accounted for 94.17% of the total catch. Lycosid species were moredominant as compared to others.
Key words: Biodiversity, Araneid fauna, Trifolium alexandn.num, Faisalabad.
INTRODUCTION
Biodiversity is not only an issue of curiosity, but stands firm on the politicalagenda as a resource for humanity (Heywood, 1996). Pakistan is basically anagricultural country with diverse climate and ecological background.Phytophagous insects cause serious damage to agricultural crops, vegetables,fruit trees, ornamental and wild plants. Pesticides are used to protect crops frominsecl pests, which are not only injurious to human health but also deplete bio-diversity essential for ecological stability. In order to conserve biodiversity andminimize dependence on pesticides, integrated pest management programneeds to be encouraged.
Spiders (Araneae) fall within the general area of natural control factors.They feed almost exclusively on insects and form one of the most ubiquitousgroups of predacious organisms in the animal kingdom. They are importantenemies of pest species including aphids, mites, lepidopterous larvae and theireggs. Spiders are cosmopolitan in distribution and have conquered almost allecological environments with perhaps the single exception of the air. The varioushabitats occupied are soil: houses, forests, meadows, woodlands, croplands, thepetals of flowers and even they may have adopted amphibious life.
Spiders mainly predate on insects and other arthropods. As they aregeneralist predators, they can consume all types of insects as their food. Spidersare not generally host specific and have alternate prey.
Nyffeler & Benz (1984) observed that spiders as predators had a moresignificant role in woodlands as compared to maize fields. Patel et a/. (1986)studied the ground activity, species diversity, species richness and relativeabundance of spiders caught by pitfall trap from cotton fields. They stated thatspiders playa significant role in biological control of insect pests of some crops.Spiller (1986) studied the inter-specific competition between spiders and its
44 A. GHAFOOR & Z. H. ALVL BIOLOGIA VOL. 54 (1) BIODIVERSITY OF ARANIED FAUNA IN PAKISTAN 45
relevance to biological control. He suggested that under certain circumstances, asubject of predator species could be more effective in reduction of preypopulation than the entire natural guild.
The study of spider community, species diversity and density is a prerequisitefor any kind of attempt for studies on spiders and their role as bio-control agentsof insect pests.
This paper reports the ground activity and relative abundance of spiders, as apart of comprehensive study of spider population in different ecosystems andtheir significant role in biological control of insect pests of some crops atFaisalabad.
MATERIALS AND METHODS
The fodder crop (Trifolium a/exandrinum) fields were sampled for araneidfauna, each month for five consecutive days (Le. for 120 hours) from October2003 through April 2004 using pitfall traps comprising 14cm long and 7cm wide(at the mouth) glass jars. During operations, the traps were emptied on alternatedays and the contents were shifted to the laboratory for counting andidentification of the spiders. In the center of the field a trap line was drawndividing the field into two equal halves. On this trapping line, 20 traps were. buried. Each trap was 11 feet apart from the other. The traps were fixed in thesoil vertically in such a way that the brinks of open mouths of the traps were inlevel with the ground surface. Each trap was filled with 175ml mixture of 70%ethyl alcohol and a very little amount of kerosene oil, as a killing and preservativeagent. In each month, trapping was done fortnightly. All the contents of each trapwere filtered using mesh strainer, washed with xylene and the spiders weresorted out, brought to the laboratory and preserved in a mixture of 80% alcoholand small quantity of glycerine for identification by using the keys of Dyal (1935),Kaston (1972), Biswas (1981), Tikader (1982) and Barrion & Litsinger (1995).Spider diversity was assessed by using Hills diversity number - Ni=e H', were His Shannon's index.Evenness was calculated by using modified Hills ratio:
Es = (1/ hr1eH'.1
where H' is Shannon's index (Ludwig & Reynolds, 1988)
while 126 and 37 belonged to Pardosa and Hippasa, respectively. The speciesbelonging to genL\s Lycosa were: madani (110), kempi (43), mackenziei (90),macula/a (80). The genus Pardosa was represented by species birmanica (42),oake/eyi (50) and suma/rana (34), whereas, genus Hippasa was represented byspecies hima/ayensis (10), madraspa/ana (15) and age/enoides (12).
The family Salticidae comprised 2 g'enera, i.e., Myrmarachnae representedby 2 species, i.e., orien/a/es (3), and benga/ensis (7), while genus Rhene wasrepresented by 2 species, Le., indicus (5), and decora/us (3).
The family Thomisidae comprised 2 genera, Thomisus and Runcinia. The. genus Thomisus was represented by 3 species Le., bo/aui (13), prajec/us (9),and Thomisus spp. (5), whereas genus Runcinia was representd by only 1species, Le., benga/ensis (2)
Table 1: Spiders captured during the entire trapping sessions of October,2003 through April 2004 from Trifolium a/exandrinum fields at Gutwala,district Faisalabad.
Familv Genus SDecies SDecimensLvcosidae Lveosa L. madani 110
L kemoi 43L. makenziei 90L maculata 80
Pardosa P. binnanica 42P.oakelevi 50
-- P. sumatrana 34Hiooasa H. hima/avensis 10
H. madrasoa/ana 15H. aaelenoides 12
Salticidae Mvnmaraehnae Marien/ales 3M benaalensis 7
Rhene R. indicus 5R. decoratus 3 I
-.!homisidae Thomisus T bo/aui 13- T oroiec/us 9- Thomisus SOD. 5~. Runcinia R benaalensis 2-.Q1l-"ohosidae Zelotes Z. mandae 3-- Z. Doonaensis 9_Oxxooidae Oxyopes O. ratnae 4
Tolal' 549
(1
The species of spiders recorded throughout all sessions of trapping were:L modani, L kempi, L mackenziei, L maculate, P birmanica and P. oake/eyi.The species which were absent during OctOber, November, December, Januaryand February were: H. hima/ayensis, M bengalensis, T. projectus, R.
RESULTS
The present study was conducted from October 05, 2003 through April 25,2004 to sample out spider population from the Trifolium alexandrinum fieldslocated at Gutwala, district Faisalabad.
A total of 549 specimens of spiders belonging to 5 families, 9 genera and21 species were captured (Table 1). The family Lycosidae was represented by 3genera and 10 species; of these 320 specimens belonged to the genus Lycosa
No. of species = 21Richness index = 7.69
Diversity index = 8.61Evenness index = 0.4
:.11
44 A. GHAFOOR & Z. H. ALVL BIOLOGIA VOL. 54 (1) BIODIVERSITY OF ARANIED FAUNA IN PAKISTAN 45
relevance to biological control. He suggested that under certain circumstances, asubject of predator species could be more effective in reduction of preypopulation than the entire natural guild.
The study of spider community, species diversity and density is a prerequisitefor any kind of attempt for studies on spiders and their role as bio-control agentsof insect pests.
This paper reports the ground activity and relative abundance of spiders, as apart of comprehensive study of spider population in different ecosystems andtheir significant role in biological control of insect pests of some crops atFaisalabad.
MATERIALS AND METHODS
The fodder crop (Trifolium a/exandrinum) fields were sampled for araneidfauna, each month for five consecutive days (Le. for 120 hours) from October2003 through April 2004 using pitfall traps comprising 14cm long and 7cm wide(at the mouth) glass jars. During operations, the traps were emptied on alternatedays and the contents were shifted to the laboratory for counting andidentification of the spiders. In the center of the field a trap line was drawndividing the field into two equal halves. On this trapping line, 20 traps were. buried. Each trap was 11 feet apart from the other. The traps were fixed in thesoil vertically in such a way that the brinks of open mouths of the traps were inlevel with the ground surface. Each trap was filled with 175ml mixture of 70%ethyl alcohol and a very little amount of kerosene oil, as a killing and preservativeagent. In each month, trapping was done fortnightly. All the contents of each trapwere filtered using mesh strainer, washed with xylene and the spiders weresorted out, brought to the laboratory and preserved in a mixture of 80% alcoholand small quantity of glycerine for identification by using the keys of Dyal (1935),Kaston (1972), Biswas (1981), Tikader (1982) and Barrion & Litsinger (1995).Spider diversity was assessed by using Hills diversity number - Ni=e H', were His Shannon's index.Evenness was calculated by using modified Hills ratio:
Es = (1/ hr1eH'.1
where H' is Shannon's index (Ludwig & Reynolds, 1988)
while 126 and 37 belonged to Pardosa and Hippasa, respectively. The speciesbelonging to genL\s Lycosa were: madani (110), kempi (43), mackenziei (90),macula/a (80). The genus Pardosa was represented by species birmanica (42),oake/eyi (50) and suma/rana (34), whereas, genus Hippasa was represented byspecies hima/ayensis (10), madraspa/ana (15) and age/enoides (12).
The family Salticidae comprised 2 g'enera, i.e., Myrmarachnae representedby 2 species, i.e., orien/a/es (3), and benga/ensis (7), while genus Rhene wasrepresented by 2 species, Le., indicus (5), and decora/us (3).
The family Thomisidae comprised 2 genera, Thomisus and Runcinia. The. genus Thomisus was represented by 3 species Le., bo/aui (13), prajec/us (9),and Thomisus spp. (5), whereas genus Runcinia was representd by only 1species, Le., benga/ensis (2)
Table 1: Spiders captured during the entire trapping sessions of October,2003 through April 2004 from Trifolium a/exandrinum fields at Gutwala,district Faisalabad.
Familv Genus SDecies SDecimensLvcosidae Lveosa L. madani 110
L kemoi 43L. makenziei 90L maculata 80
Pardosa P. binnanica 42P.oakelevi 50
-- P. sumatrana 34Hiooasa H. hima/avensis 10
H. madrasoa/ana 15H. aaelenoides 12
Salticidae Mvnmaraehnae Marien/ales 3M benaalensis 7
Rhene R. indicus 5R. decoratus 3 I
-.!homisidae Thomisus T bo/aui 13- T oroiec/us 9- Thomisus SOD. 5~. Runcinia R benaalensis 2-.Q1l-"ohosidae Zelotes Z. mandae 3-- Z. Doonaensis 9_Oxxooidae Oxyopes O. ratnae 4
Tolal' 549
(1
The species of spiders recorded throughout all sessions of trapping were:L modani, L kempi, L mackenziei, L maculate, P birmanica and P. oake/eyi.The species which were absent during OctOber, November, December, Januaryand February were: H. hima/ayensis, M bengalensis, T. projectus, R.
RESULTS
The present study was conducted from October 05, 2003 through April 25,2004 to sample out spider population from the Trifolium alexandrinum fieldslocated at Gutwala, district Faisalabad.
A total of 549 specimens of spiders belonging to 5 families, 9 genera and21 species were captured (Table 1). The family Lycosidae was represented by 3genera and 10 species; of these 320 specimens belonged to the genus Lycosa
No. of species = 21Richness index = 7.69
Diversity index = 8.61Evenness index = 0.4
:.11
46 A. GHAFOOR & Z. H. ALVI. BIOLOGIA VOL. 54 (1) BIODIVERSITY OF ARANIED FAUNA IN PAKISTAN 47il',~I
I:,
bengalensis. Z mandae and 0. ratnae while those absent in November,December and January were: H. madraspatana, M. bengalensis, R. indicus. R.decoratus, T projectus, Thomisus sp., Z mandae and O. ratnae. The speciesabsent only in October and November were: H. madraspatana, M. orientales, M.bengalensis, T projectus, Thomisus sp., R. indicus, R. decoratus, Z mandaeand 0. ratnae. and absent in December were: H. himalayensis, H. agelenoides,T bloaui. T projectus T spp., R. bengalensis, Z mandae, Z poonaensis and 0.ratnae whereas absent in February were: H. madraspatana, M. bengalensis, M.orientales, T bolaui. T projectus, R. bengalensis, Z mandae, Z poonaensis and0. ratane (Table 2).
Table 2; A record of species of spiders found during October 2003 to April2004.
I-----~pecies --_.
..-
Oct.2003 Nov.2003 Doc.2003 Jan.2004 Feb.2004 Marc,2004 Apr.2004
L. madani + + + + + + + .-L. kemoi + + + + + + +L. makenziei + + + + + + + -.L. maculata . + + + + + +
fP /Jirmanica . + + + + + +P. oake/eyi . + + + + + +P. sumatranB . + + + + + +H. . + . . + + +L~;'I1~yensis
------!H . . . . + +~adrasDa/ana .
.~
+ + . . + +agelenaides. M. orientales . . . . + +'M . . . . + +
=. ;~.'~_bengi!.iensis _
~-'~:~~~I:tus'~'. . . . + +._-. . . + +T bolaui + + + ~. . +
1[TProjecti-;s- . . . . . + +f-{"SPP . . . . . + +R. . + . . . + +J?_enaalensisz. mandae . . . . . +
t=iJ_?:- (Joonae'!..!i/3-_.
. + . . . +..Q..'.B/n?e I . . . . . +.
The species absent in March was R. bolani, whereas P blrma/1/ca wasabsent in October. On comparing the diversity index, richness index andevenness index, it was evident in all the monthiy samples from October. 2003through"Aprii, 2004 (Table 2) that a lot of change in establishing community ofspiders in Trifolium alexandrinum field was observed. Diversity was high in
. February, March and April, which increased gradually to the highest in April, due. to gradual increase in species, and in October and January the diversity was thelowest. Richness was highest in March and April while lowest in the months ofOctober and December. Evenness recorded was highest in April and the lowestduring the month of October whereas during December and January the diversitywas the lowest.
The genus Zelotes of the family Gnaphosidae was represented by thespecies, Z. madnae (3) and Z poonaensis (9), while the family Oxyopidae wasrepresented by one genus Oxyopes and one species O. ratnae. The indices ofdiversity, richness and evenness were 7.69, 8.61 and 0.64, respectively. Ageneral look on the Table 2 showed that L. madani, L. mackenzie L. maculata, P.. oakeleyi and L. kempi were the dominant species, while P. birmanica and P.sUrnatrana were the moderately dominant species. All other species given in theTable 2, during the whole trapping sessions, were less frequent.
DISCUSSION
A review of the results showed that during the months of October,December and January, population of spiders continuously decreased. In themonthly samples after January, the number of spiders increased till April when. maximum number of spider species was recorded.
A total of 549 ground dwelling spiders were identified. Lycosid spiders inall the months, especially in March and April, were in abundance.
Ferguson (1984) reported the same order of dominance for familiesLycosidae and Linyphiidae. They reported that population of ground dwellingspiders peaked during June and was dominated by family Lycosidae. Snodgrass& Stadelbacher (1989) also observed that on ground, most dominant family wasLycosidae which showed dominance in total sampling during all 7 months .Whiteford et al. (1987) noted that ground dwelling spiders community was bestcharacterized as a hunting guild and consisted primarily of Lycosidae,Thomisidae and Gnaphosidae. Van (1991), in his survey recorded 112 speciesbelonging to 66 genera and 18 families and concluded that the number of pestspecies and spider diversity varied between habitats and environment factorsand also affected the community parameter including Shannon Wieners Index,Evenness Index and Simpson index.
Lycosidae was the dominant family and genus Lycosa was the dominantgenus. Soil surface habitat is a stable one (Cuiin & Rust, 1980) and can bealtered only by cultivation practices (Haskins & Shady, 1986) which results inchange of spider fauna residing there. The dominance of a genus indicates that ithas become established in habitat and may be useful in the control of insect pestof Trifolium alexandrinum (Nyffeler & Benz, 1984; Patel et al., 1986 and Spiller,1986)
I~iill
L ~!
',ii
Iii
b ~
46 A. GHAFOOR & Z. H. ALVI. BIOLOGIA VOL. 54 (1) BIODIVERSITY OF ARANIED FAUNA IN PAKISTAN 47il',~I
I:,
bengalensis. Z mandae and 0. ratnae while those absent in November,December and January were: H. madraspatana, M. bengalensis, R. indicus. R.decoratus, T projectus, Thomisus sp., Z mandae and O. ratnae. The speciesabsent only in October and November were: H. madraspatana, M. orientales, M.bengalensis, T projectus, Thomisus sp., R. indicus, R. decoratus, Z mandaeand 0. ratnae. and absent in December were: H. himalayensis, H. agelenoides,T bloaui. T projectus T spp., R. bengalensis, Z mandae, Z poonaensis and 0.ratnae whereas absent in February were: H. madraspatana, M. bengalensis, M.orientales, T bolaui. T projectus, R. bengalensis, Z mandae, Z poonaensis and0. ratane (Table 2).
Table 2; A record of species of spiders found during October 2003 to April2004.
I-----~pecies --_.
..-
Oct.2003 Nov.2003 Doc.2003 Jan.2004 Feb.2004 Marc,2004 Apr.2004
L. madani + + + + + + + .-L. kemoi + + + + + + +L. makenziei + + + + + + + -.L. maculata . + + + + + +
fP /Jirmanica . + + + + + +P. oake/eyi . + + + + + +P. sumatranB . + + + + + +H. . + . . + + +L~;'I1~yensis
------!H . . . . + +~adrasDa/ana .
.~
+ + . . + +agelenaides. M. orientales . . . . + +'M . . . . + +
=. ;~.'~_bengi!.iensis _
~-'~:~~~I:tus'~'. . . . + +._-. . . + +T bolaui + + + ~. . +
1[TProjecti-;s- . . . . . + +f-{"SPP . . . . . + +R. . + . . . + +J?_enaalensisz. mandae . . . . . +
t=iJ_?:- (Joonae'!..!i/3-_.
. + . . . +..Q..'.B/n?e I . . . . . +.
The species absent in March was R. bolani, whereas P blrma/1/ca wasabsent in October. On comparing the diversity index, richness index andevenness index, it was evident in all the monthiy samples from October. 2003through"Aprii, 2004 (Table 2) that a lot of change in establishing community ofspiders in Trifolium alexandrinum field was observed. Diversity was high in
. February, March and April, which increased gradually to the highest in April, due. to gradual increase in species, and in October and January the diversity was thelowest. Richness was highest in March and April while lowest in the months ofOctober and December. Evenness recorded was highest in April and the lowestduring the month of October whereas during December and January the diversitywas the lowest.
The genus Zelotes of the family Gnaphosidae was represented by thespecies, Z. madnae (3) and Z poonaensis (9), while the family Oxyopidae wasrepresented by one genus Oxyopes and one species O. ratnae. The indices ofdiversity, richness and evenness were 7.69, 8.61 and 0.64, respectively. Ageneral look on the Table 2 showed that L. madani, L. mackenzie L. maculata, P.. oakeleyi and L. kempi were the dominant species, while P. birmanica and P.sUrnatrana were the moderately dominant species. All other species given in theTable 2, during the whole trapping sessions, were less frequent.
DISCUSSION
A review of the results showed that during the months of October,December and January, population of spiders continuously decreased. In themonthly samples after January, the number of spiders increased till April when. maximum number of spider species was recorded.
A total of 549 ground dwelling spiders were identified. Lycosid spiders inall the months, especially in March and April, were in abundance.
Ferguson (1984) reported the same order of dominance for familiesLycosidae and Linyphiidae. They reported that population of ground dwellingspiders peaked during June and was dominated by family Lycosidae. Snodgrass& Stadelbacher (1989) also observed that on ground, most dominant family wasLycosidae which showed dominance in total sampling during all 7 months .Whiteford et al. (1987) noted that ground dwelling spiders community was bestcharacterized as a hunting guild and consisted primarily of Lycosidae,Thomisidae and Gnaphosidae. Van (1991), in his survey recorded 112 speciesbelonging to 66 genera and 18 families and concluded that the number of pestspecies and spider diversity varied between habitats and environment factorsand also affected the community parameter including Shannon Wieners Index,Evenness Index and Simpson index.
Lycosidae was the dominant family and genus Lycosa was the dominantgenus. Soil surface habitat is a stable one (Cuiin & Rust, 1980) and can bealtered only by cultivation practices (Haskins & Shady, 1986) which results inchange of spider fauna residing there. The dominance of a genus indicates that ithas become established in habitat and may be useful in the control of insect pestof Trifolium alexandrinum (Nyffeler & Benz, 1984; Patel et al., 1986 and Spiller,1986)
I~iill
L ~!
',ii
Iii
b ~
Barrion, A T. & Litsinger, J. A, 1995. Riceland spiders of Southeast Asia.International Research Institute, Philippines, pp. 716.
Biswas, B., 1981. Description of six new species of spiders of the familiesClubionidae, Gnaphosidae and Salticidae from India. Bull Zool Swv,India, 8(3): 119-127.
Culin, J. D. & Rust, R. W., 1980. Comparison of the ground surface andfoliage dwelling spider community in a soybean habitat Environ.Entomol., 9: 577-582.
Dyal, S., 1935. Fauna of Lahore, spiders of Lahore. Buff. Dept. of Zoology,Punjab Univ, Lahore, 1: 117-252.
Ferguson, H. I., 1984. Ground and foliage dwelling spiders in four soybeancropping system. Environ. Entomol., 13: 975-980.
Haskins, M. F. & Shaddy, J. H., 1986. The ecological effects of burning,mowing and ploughing on ground inhabiting spiders in an old fieldecosystem. Arachnol., 14(1): 13-33.
Heywood, V. H., 1996. The global biodiversity assessment The Globe, 30 2-4.
Kaston, B. J., 1972. How To Know The Spiders. WN. C. Brown Co.,Dubuque.
Ludwig, J. A & Reynolds, 1988. Statistical Ecology: a primer on methods andcomputing. John Wiley and Sons. pp. 337.
Nyffeler, M & Benz, G., 1984. The role of spiders as insect predators ofcereal fields near Zurich (Switzerland). at" Int Arachnid Con. Vien. pp127-131.
Patel, B. H., Pillai, G. K. & Sebastatin, P. K, 1986. The ground activity otspiders in cotton fields in Gujrat, India. Aetas Xcong. Into. Arachno/.JacalEspana, pp. 1245-1251.
Snodgrass, ..G. L. & Stadel bacher, E. A, 1989. Effect of different grass andlegume combination on spider (Araneae) and ground beetle (Coleoptera:Carabidae) population in roadside habitat in the Mississippi Delta.Southern Field Crop Insect Management Lab., ARS, USDA, Stoneville.MS38776, U. S. A, Environ. Entomo/., 18(4) 375-581.
Spiller, D. A, 1986.' Interspecific competition between spiders and itsrelevance to biological control by general predators. Environ. Entomo/.,15: 177.181.
Tikader, B. K., 1982. The fauna of India, Spiders-Araneae. Zoological Surveyof India. Calcutta, 2: 1.293._
Whiteford, F. W., Showver, B. & Edwards, G. B., 1987. Insecticide toleranceof ground and foliage dwelling spiders (Aranea: Thomisidae). Environ.Entomo/., 16(3) 779-785.
Yan, H. M., 1991. Community ecology of spiders in rice fields in south westChina Asia Soientiarum-Naturatlium University, Normalize Humamensis,14(1) 78-83.
48 A. GHA.FOOR & Z. H. A.LVL
REFERENCES
BIOLOGIA. BIOLOGIAiPAKISTANI 2008,54 (1), 49-55PK ISSN 0006 - 3096
Biodiversity and caste differences in the population of symhintieentozoic protozoans in Heterotermes iudicota
NAVEEIJA AKIHAR QURESHI, AZIZlILLAH, MUII,\MMAIl ANW,\R MALIK,MlIllAMMAIl SllARIF MliGHAL & MlIIlM1MAll ZAllIIl QlIRESl1I
IJ"IJUl'lmenl oj'Z()%gy, Ge Unh'ersily. l.ahore. Pakislan (NAQ. A. MAil". ,H.';,.\/)Depurtment (~r('hem;"'l/:\,.(lC Universify. Lahore, Pakistan (MZ{})
ABSTRACT
The lower termites harbour fiagellates for digestion of wood. The differentspecies belonging to three genera, Holomastigotes, Holomastigotoides andPseudotrichonympha, of flagellates found in Helemtermes indico/a workers andsoldiers were compared and analyzed statistically and histogram was plotted It wasfound that soldiers harbour significantly less number of flagellales as compared 10workers.
Key words: Termites, Flagellates, Holomastigotes, Holom8stigotoides,PseudotndlOnympha, Heterotennes indico/a
INTRODUCTION
Taxonomic studies of entozoic protozoa of the termite Heterotermesindico/a Wasmann were carried out by Kirby (1926), de Mello (1927) and Kudo(1966) However, none of these studies include comparison of the protozoanfauna found in soldiers and workers of H. indico/a. In fact, most of the studies onprotozoans of termites are based on protozoans of workers only (Mannesmann1972) It was, therefore, planned to study the protozoan fauna of the gut ofsoldiers as well as that of the workers of this species of termite and to comparethem with each other
MATERIALS AND METHODS
The workers and soldiers of Heterotermes indfcola were collected from theMulberry tree trunk (shehtoot) in the month of January The flagellate fauna ofsoldiers and workers of Heterotermes indico/a were studied by making temporary. slides of the gut contents of seven samples each of these castes. The numbersof each species of protozoans on each slide were counted. Mean values of theflagellate species in soldiers and workers were calculated separately Hislrogramwas plotted showing comparison of the protozoan fauna of the two castesStatistical tests, for the significance of difference in species and for thecomparison of whole fauna of the two castes ie. soldiers and workers. wereapplied
~,
l'I
I
':iii'
Barrion, A T. & Litsinger, J. A, 1995. Riceland spiders of Southeast Asia.International Research Institute, Philippines, pp. 716.
Biswas, B., 1981. Description of six new species of spiders of the familiesClubionidae, Gnaphosidae and Salticidae from India. Bull Zool Swv,India, 8(3): 119-127.
Culin, J. D. & Rust, R. W., 1980. Comparison of the ground surface andfoliage dwelling spider community in a soybean habitat Environ.Entomol., 9: 577-582.
Dyal, S., 1935. Fauna of Lahore, spiders of Lahore. Buff. Dept. of Zoology,Punjab Univ, Lahore, 1: 117-252.
Ferguson, H. I., 1984. Ground and foliage dwelling spiders in four soybeancropping system. Environ. Entomol., 13: 975-980.
Haskins, M. F. & Shaddy, J. H., 1986. The ecological effects of burning,mowing and ploughing on ground inhabiting spiders in an old fieldecosystem. Arachnol., 14(1): 13-33.
Heywood, V. H., 1996. The global biodiversity assessment The Globe, 30 2-4.
Kaston, B. J., 1972. How To Know The Spiders. WN. C. Brown Co.,Dubuque.
Ludwig, J. A & Reynolds, 1988. Statistical Ecology: a primer on methods andcomputing. John Wiley and Sons. pp. 337.
Nyffeler, M & Benz, G., 1984. The role of spiders as insect predators ofcereal fields near Zurich (Switzerland). at" Int Arachnid Con. Vien. pp127-131.
Patel, B. H., Pillai, G. K. & Sebastatin, P. K, 1986. The ground activity otspiders in cotton fields in Gujrat, India. Aetas Xcong. Into. Arachno/.JacalEspana, pp. 1245-1251.
Snodgrass, ..G. L. & Stadel bacher, E. A, 1989. Effect of different grass andlegume combination on spider (Araneae) and ground beetle (Coleoptera:Carabidae) population in roadside habitat in the Mississippi Delta.Southern Field Crop Insect Management Lab., ARS, USDA, Stoneville.MS38776, U. S. A, Environ. Entomo/., 18(4) 375-581.
Spiller, D. A, 1986.' Interspecific competition between spiders and itsrelevance to biological control by general predators. Environ. Entomo/.,15: 177.181.
Tikader, B. K., 1982. The fauna of India, Spiders-Araneae. Zoological Surveyof India. Calcutta, 2: 1.293._
Whiteford, F. W., Showver, B. & Edwards, G. B., 1987. Insecticide toleranceof ground and foliage dwelling spiders (Aranea: Thomisidae). Environ.Entomo/., 16(3) 779-785.
Yan, H. M., 1991. Community ecology of spiders in rice fields in south westChina Asia Soientiarum-Naturatlium University, Normalize Humamensis,14(1) 78-83.
48 A. GHA.FOOR & Z. H. A.LVL
REFERENCES
BIOLOGIA. BIOLOGIAiPAKISTANI 2008,54 (1), 49-55PK ISSN 0006 - 3096
Biodiversity and caste differences in the population of symhintieentozoic protozoans in Heterotermes iudicota
NAVEEIJA AKIHAR QURESHI, AZIZlILLAH, MUII,\MMAIl ANW,\R MALIK,MlIllAMMAIl SllARIF MliGHAL & MlIIlM1MAll ZAllIIl QlIRESl1I
IJ"IJUl'lmenl oj'Z()%gy, Ge Unh'ersily. l.ahore. Pakislan (NAQ. A. MAil". ,H.';,.\/)Depurtment (~r('hem;"'l/:\,.(lC Universify. Lahore, Pakistan (MZ{})
ABSTRACT
The lower termites harbour fiagellates for digestion of wood. The differentspecies belonging to three genera, Holomastigotes, Holomastigotoides andPseudotrichonympha, of flagellates found in Helemtermes indico/a workers andsoldiers were compared and analyzed statistically and histogram was plotted It wasfound that soldiers harbour significantly less number of flagellales as compared 10workers.
Key words: Termites, Flagellates, Holomastigotes, Holom8stigotoides,PseudotndlOnympha, Heterotennes indico/a
INTRODUCTION
Taxonomic studies of entozoic protozoa of the termite Heterotermesindico/a Wasmann were carried out by Kirby (1926), de Mello (1927) and Kudo(1966) However, none of these studies include comparison of the protozoanfauna found in soldiers and workers of H. indico/a. In fact, most of the studies onprotozoans of termites are based on protozoans of workers only (Mannesmann1972) It was, therefore, planned to study the protozoan fauna of the gut ofsoldiers as well as that of the workers of this species of termite and to comparethem with each other
MATERIALS AND METHODS
The workers and soldiers of Heterotermes indfcola were collected from theMulberry tree trunk (shehtoot) in the month of January The flagellate fauna ofsoldiers and workers of Heterotermes indico/a were studied by making temporary. slides of the gut contents of seven samples each of these castes. The numbersof each species of protozoans on each slide were counted. Mean values of theflagellate species in soldiers and workers were calculated separately Hislrogramwas plotted showing comparison of the protozoan fauna of the two castesStatistical tests, for the significance of difference in species and for thecomparison of whole fauna of the two castes ie. soldiers and workers. wereapplied
~,
l'I
I
':iii'
50 N. A. QURESHI ET AL BIOLOGIA VOL 54 (1) BIODIVERSITY AND CASTE DIFFERENCES !N POPULATION OF PROTOZOANS IN A TERMITE 51
L
RESULTS
The workers and soldiers.of H. indico/a were found to harbour three generaof flagellates, viz., HoJomastigotes, Holomastigotoides and Pseudotrichonymphain their gut The genus H%mastigotes was represented by five species, viz., H.campanu/a, H annandalei, H. metchnikowi, H. termites and H. biconcava. Both.workers and soldiers had all these species of Holomastigotes. The genusHolomastigotoides was also represented by five species, viz., H. kempi, Hhemigynum, H hartmanni, H metchnikowi and H. koidzumi in both soldiers andworkers. Genus Pseudotrichonympha was represented by only one species, i.e.,P. grassi in both soldiers and workers.
As mentioned above, the soldiers and workers of H. indicola had all thesespecies of flagellates in their gut However, the number of individual flagellatesdiffered significantly among the various species of the same genus and alsoamong the species of various genera within each caste, i.e., workers and soldiersand also between different castes (Tables 1 & 2). The relative number offlagellates belonging to each genus was also different in soldiers and workers of. H. indico/a. Average number of flagellates of Holomastigotes was 486.2 insoldiers and 1162.61 in workers. The average number of flagellates ofHolomastigotoides was 292.3 in soldiers and 1343.8 in workers, whereas, thenumber of flagellates of Pseudotrichonympha was 272.7 in solders and 253 inworkers. As far as the relative abundance of the protozoa of the three genera inthe workers was concerned, the genus' Holomastigotoides was the mostabundant with its population being 48.68%, whereas, the population of that of thegenus Holomastigotes was 42.12 % and the least abundant .wasPseudolrichonympha which was 9.16 % of the total flagellates. Average totalhumber of flagellates belonging to all the three genera taken together were 1051in soldiers and 2760 in workers.
The number cifflagellates of each species also showed variation in soldiersand workers. Generally, the soldiers contained fewer flagellates of each speciesas compared to the workers (Tables 1 & 2). The relative contribution of eachspecies in the total number of flagellates of the same genus and in the grandtotal of flagellates belonging to all the genera, was different in soldiers andworkers of H. indicola, e.g., in the case of Holomastigotes. campanula, thepercentage contribution of this species to the total flagellates belonging to thegenus Holomastigotes was 49.97 (581/1162,61 X 100), but in the soldiers thepercentage contribution of this species was 68.24 (331,8/486,1 X 100), but theactual average number of flagellates of H%mastigoles in workers was 1162.61,whereas, in soldiers it was only 486,2, i.e, fewer by 676,41 as compared to thatin ttie workers. The flagellates of Holomastigoles annanda/ei in workers were1328 % (1545/1162.61 X 100) of total flagellates of the genus H%mastigotes,whereas, flagellates of this species in the soldiers, were 2.01% (9.8/486.1 X 100)but the actual average number of the flagellates of this species was 154,4 inworkers and only 9.8 in soldiers, i.e., fewer by 144.6 in soldiers as compared toin workers. There was no regular. pattern of the relative abundance of eachspecies of protozoan between soldiers and workers except that flagellates of
each species in soldiers were less as compared to the flagellates of the samespecies in workers.
Table 1: Flagellate population in individual termite soldiers of Heterotermesindicola
T1 T2 T3 T4 T5 T6 T7 Ave. S.E.Genus Holomastigotes
H. annandalei 0 20 0 23 0 26 0 9.8 +4.1H. camoanula 138 736 529 115 345 230 230 331.8 +79.24H. metchnikowi 161 23 0 140 21 115 0 65,7 +24.53H, termites 45 85 0 0 0 30 40 23 +11.2H,"biconcava 98 0 115 138 0 40 0 55,8 +21Genus Holomastigotoides'"
H. hemiavnum 20 23 46 30 0 111 0 32.8 +13,33H,'Aoidzumi 92 52 40 31 70 0 15 42.71 +11,06H. metchnikowi 69 71 92 270 0 320 322 131.4 +47,67H. kemp; 20 0 0 40 0 40 130 32,85 +16.25H. hartmann; 27 0 0 65 0 146 130 52,57 +22,2Genus Pseudotrichonympha
P. grass; 161 I 223 I 322 I 350 I 210 I 250 393 I 272,7 I +29,37
Table 2: Flagellate population in individual termite workers ofHeterotermes indicola
T1 T2 T3 T4 T5 T6 T7 Avp.. S.E.
r-Genus Holomastigotes
, H. annandalei 161 184 115 253 207 92 69 154.4 +23.1H. campanula 713 550 989 644 460 391 322 5812 +209H. metchnikowi 138 115 207 161 115 115 161 144.5 +12.04H. termites 207 230 138 184 184 138 115 170.8 +14.58H. biconcava 115 92 138 92 92 115 138 111,71 +7,24Genus Holomastigotoides
H. hemigvnum 92 92 46 138 69 -- 69 85.4 +10.5H. koieizumi 598 368 943 253 414 391 253 460 +84H. metclmikowi 442 322 690 690 276 322 552 470 +61i-i-!. kemp; 276 230 161 115 276 136 115 187.2 +25.28
I H. hartmanni 184 253 115 138 115 92 92 141.2 +20.48I Genus PseudotrichonymphaI P , I 299 , 345 , 230 I 184 207 I 184 I 322 I 253 ,+23.69I . graSSJ
,'IIq:
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iii
50 N. A. QURESHI ET AL BIOLOGIA VOL 54 (1) BIODIVERSITY AND CASTE DIFFERENCES !N POPULATION OF PROTOZOANS IN A TERMITE 51
L
RESULTS
The workers and soldiers.of H. indico/a were found to harbour three generaof flagellates, viz., HoJomastigotes, Holomastigotoides and Pseudotrichonymphain their gut The genus H%mastigotes was represented by five species, viz., H.campanu/a, H annandalei, H. metchnikowi, H. termites and H. biconcava. Both.workers and soldiers had all these species of Holomastigotes. The genusHolomastigotoides was also represented by five species, viz., H. kempi, Hhemigynum, H hartmanni, H metchnikowi and H. koidzumi in both soldiers andworkers. Genus Pseudotrichonympha was represented by only one species, i.e.,P. grassi in both soldiers and workers.
As mentioned above, the soldiers and workers of H. indicola had all thesespecies of flagellates in their gut However, the number of individual flagellatesdiffered significantly among the various species of the same genus and alsoamong the species of various genera within each caste, i.e., workers and soldiersand also between different castes (Tables 1 & 2). The relative number offlagellates belonging to each genus was also different in soldiers and workers of. H. indico/a. Average number of flagellates of Holomastigotes was 486.2 insoldiers and 1162.61 in workers. The average number of flagellates ofHolomastigotoides was 292.3 in soldiers and 1343.8 in workers, whereas, thenumber of flagellates of Pseudotrichonympha was 272.7 in solders and 253 inworkers. As far as the relative abundance of the protozoa of the three genera inthe workers was concerned, the genus' Holomastigotoides was the mostabundant with its population being 48.68%, whereas, the population of that of thegenus Holomastigotes was 42.12 % and the least abundant .wasPseudolrichonympha which was 9.16 % of the total flagellates. Average totalhumber of flagellates belonging to all the three genera taken together were 1051in soldiers and 2760 in workers.
The number cifflagellates of each species also showed variation in soldiersand workers. Generally, the soldiers contained fewer flagellates of each speciesas compared to the workers (Tables 1 & 2). The relative contribution of eachspecies in the total number of flagellates of the same genus and in the grandtotal of flagellates belonging to all the genera, was different in soldiers andworkers of H. indicola, e.g., in the case of Holomastigotes. campanula, thepercentage contribution of this species to the total flagellates belonging to thegenus Holomastigotes was 49.97 (581/1162,61 X 100), but in the soldiers thepercentage contribution of this species was 68.24 (331,8/486,1 X 100), but theactual average number of flagellates of H%mastigoles in workers was 1162.61,whereas, in soldiers it was only 486,2, i.e, fewer by 676,41 as compared to thatin ttie workers. The flagellates of Holomastigoles annanda/ei in workers were1328 % (1545/1162.61 X 100) of total flagellates of the genus H%mastigotes,whereas, flagellates of this species in the soldiers, were 2.01% (9.8/486.1 X 100)but the actual average number of the flagellates of this species was 154,4 inworkers and only 9.8 in soldiers, i.e., fewer by 144.6 in soldiers as compared toin workers. There was no regular. pattern of the relative abundance of eachspecies of protozoan between soldiers and workers except that flagellates of
each species in soldiers were less as compared to the flagellates of the samespecies in workers.
Table 1: Flagellate population in individual termite soldiers of Heterotermesindicola
T1 T2 T3 T4 T5 T6 T7 Ave. S.E.Genus Holomastigotes
H. annandalei 0 20 0 23 0 26 0 9.8 +4.1H. camoanula 138 736 529 115 345 230 230 331.8 +79.24H. metchnikowi 161 23 0 140 21 115 0 65,7 +24.53H, termites 45 85 0 0 0 30 40 23 +11.2H,"biconcava 98 0 115 138 0 40 0 55,8 +21Genus Holomastigotoides'"
H. hemiavnum 20 23 46 30 0 111 0 32.8 +13,33H,'Aoidzumi 92 52 40 31 70 0 15 42.71 +11,06H. metchnikowi 69 71 92 270 0 320 322 131.4 +47,67H. kemp; 20 0 0 40 0 40 130 32,85 +16.25H. hartmann; 27 0 0 65 0 146 130 52,57 +22,2Genus Pseudotrichonympha
P. grass; 161 I 223 I 322 I 350 I 210 I 250 393 I 272,7 I +29,37
Table 2: Flagellate population in individual termite workers ofHeterotermes indicola
T1 T2 T3 T4 T5 T6 T7 Avp.. S.E.
r-Genus Holomastigotes
, H. annandalei 161 184 115 253 207 92 69 154.4 +23.1H. campanula 713 550 989 644 460 391 322 5812 +209H. metchnikowi 138 115 207 161 115 115 161 144.5 +12.04H. termites 207 230 138 184 184 138 115 170.8 +14.58H. biconcava 115 92 138 92 92 115 138 111,71 +7,24Genus Holomastigotoides
H. hemigvnum 92 92 46 138 69 -- 69 85.4 +10.5H. koieizumi 598 368 943 253 414 391 253 460 +84H. metclmikowi 442 322 690 690 276 322 552 470 +61i-i-!. kemp; 276 230 161 115 276 136 115 187.2 +25.28
I H. hartmanni 184 253 115 138 115 92 92 141.2 +20.48I Genus PseudotrichonymphaI P , I 299 , 345 , 230 I 184 207 I 184 I 322 I 253 ,+23.69I . graSSJ
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iii
Flagellates Species In Soldiers In Workers
Genus Ho/omastlgotes
H. annandalei 98 +4.1 154.4 +23.1H. campanula 331.8 +79.24 581.2 +209H. metchnikowi 657+24.53 1445+12.04H. termites 23 +11.2 170.8 +14.58H. biconcava 55.8 +21 111.71+7.24Grand total 486.1 1162.61. Genus Holomastigotoides
H. hemigynum 32.8 +13.33 85.4 +10.5H. koidzumi 4271+11.06 460 +84H. metchnikowi 131.4+4767 .' 470 +61H. kempi 3285 +16.25 1872+2528H. hartmanni . 52.57+22.2 141.2+20.48Grand total 292.33 1343.8Genus Pseudotrichonympha
P.strassi I 272.7+29.39 I 253+2369Grand total 1272.7 1253
Some species of flagellates were significantly fewer in soldiers ascompared to those of the same species of flagellates in workers, but in someother cases the number of flagellates of a species was only slightly and insignificantly less in soldiers as compared to workers (Table 3, Fig. 1). Amongflagellates of genus Ho/omastigotes, the species H. campanula was the mostabundant species. It ranked as number one .as far as the average number offlagellates occurring in both soldiers and workers was concerned (Table 3).
In the genus Holomastigotoides, the species H. metchnikowi and H.koidzumi were represented in larger numbers than the other species of genusHo/omastigotoides in workers. In soldiers. these two species were alsorepresented in greater numbers than the other species of the genus. However,the number of H. metchnikowi and H. koidzumi was significantly andconsiderably less in soldiers as compared to workers. The other three species ofthe same genus also occurred in more numbers in workers than in soldiers(Table 3)
The genus Pseudotrichonympha was represented, in both workers andsoldiers, by only one species viz. P. grassi. In workers it was 10.5% of the total. pqpulation of flagellates, whereas in soldiers it was 36.17% of the tot,,1 populationof flagella'tes in the gut. However, the average number of flagellates of thisspecies in workers was 253 whereas, in soldiers it was 272.7, i.e., it was onlyslightly and insignificantly less in workers as compared to that in soldiers (Table3, Fig. 1).
Table 3: Comparison of average flagellate population in soldiers andworkers of Heterotermes indicola
53
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VOL. 54 (1) BIODIVERSITY AND CASTE DIFFERENCES IN POPULATION OF PROTOZOANS IN A TERMITE
tJ
900800
., J
DISCUSSION
Fig. 1: Species-wise comparison of average flagellate population insoldiers and workers of Heterotermes indicola
The termites feed on wooden matter, which is assimilated by termites afterbeing digested by the flagellates, living in the digestive tract, particularly in therectum of the termites. The enzyme cellulase required for digestion of wood hasbeen found to be present in digestive tract of the termites (Metcalf, 1992 andGuyton, 1988). The termites eat wood and wood material. The particles of woodon reaching the hind gut are engulfed by these flagellates present in the hind gut.The flagellates secrete cellulase enzyme which digests the wood particles andmakes them ready for further digestion by the termites. How the wood particlesare taken into their bodies by flagellates, is yet another riddle. In this respectthere is no direct evidence reported so far. However, it has been suggested byKudo (1966) that flagellates feed by throwing out pseudopodia from any part oftheir body which engulf the particles. These particles are digested within the bodyof flagellates. A part of the digested food is consumed by the flagellate itself,while the rest of it is expelled out by exocytosis being the mode 'If excretion inprotozoans (De Robertis & De Robertis, 1980). The digested food is released byflagellates in the rectum of the gut. There are little chances of the same being. assimilated there, as the rectum is lined internally with chitin (Richards & Davies,1984). So, the rectal nutritive fluid (semi-digested) is perhaps moved back intothe intestine where its digestion is completed and assimilation is carried out.
BIOLOGIAN. A. QURESHI ET AL52
Flagellates Species In Soldiers In Workers
Genus Ho/omastlgotes
H. annandalei 98 +4.1 154.4 +23.1H. campanula 331.8 +79.24 581.2 +209H. metchnikowi 657+24.53 1445+12.04H. termites 23 +11.2 170.8 +14.58H. biconcava 55.8 +21 111.71+7.24Grand total 486.1 1162.61. Genus Holomastigotoides
H. hemigynum 32.8 +13.33 85.4 +10.5H. koidzumi 4271+11.06 460 +84H. metchnikowi 131.4+4767 .' 470 +61H. kempi 3285 +16.25 1872+2528H. hartmanni . 52.57+22.2 141.2+20.48Grand total 292.33 1343.8Genus Pseudotrichonympha
P.strassi I 272.7+29.39 I 253+2369Grand total 1272.7 1253
Some species of flagellates were significantly fewer in soldiers ascompared to those of the same species of flagellates in workers, but in someother cases the number of flagellates of a species was only slightly and insignificantly less in soldiers as compared to workers (Table 3, Fig. 1). Amongflagellates of genus Ho/omastigotes, the species H. campanula was the mostabundant species. It ranked as number one .as far as the average number offlagellates occurring in both soldiers and workers was concerned (Table 3).
In the genus Holomastigotoides, the species H. metchnikowi and H.koidzumi were represented in larger numbers than the other species of genusHo/omastigotoides in workers. In soldiers. these two species were alsorepresented in greater numbers than the other species of the genus. However,the number of H. metchnikowi and H. koidzumi was significantly andconsiderably less in soldiers as compared to workers. The other three species ofthe same genus also occurred in more numbers in workers than in soldiers(Table 3)
The genus Pseudotrichonympha was represented, in both workers andsoldiers, by only one species viz. P. grassi. In workers it was 10.5% of the total. pqpulation of flagellates, whereas in soldiers it was 36.17% of the tot,,1 populationof flagella'tes in the gut. However, the average number of flagellates of thisspecies in workers was 253 whereas, in soldiers it was 272.7, i.e., it was onlyslightly and insignificantly less in workers as compared to that in soldiers (Table3, Fig. 1).
Table 3: Comparison of average flagellate population in soldiers andworkers of Heterotermes indicola
53
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" ::> c: .~ ",~ c: c: '" '" '" c: c:
~l1J .c: CD <.> .>1> " .c: .~
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l1J <.> -Q .c: "'" "'" .c: C>:r: :r: :r: :r: :r: :r: :r: :r: :r: :r: Q
VOL. 54 (1) BIODIVERSITY AND CASTE DIFFERENCES IN POPULATION OF PROTOZOANS IN A TERMITE
tJ
900800
., J
DISCUSSION
Fig. 1: Species-wise comparison of average flagellate population insoldiers and workers of Heterotermes indicola
The termites feed on wooden matter, which is assimilated by termites afterbeing digested by the flagellates, living in the digestive tract, particularly in therectum of the termites. The enzyme cellulase required for digestion of wood hasbeen found to be present in digestive tract of the termites (Metcalf, 1992 andGuyton, 1988). The termites eat wood and wood material. The particles of woodon reaching the hind gut are engulfed by these flagellates present in the hind gut.The flagellates secrete cellulase enzyme which digests the wood particles andmakes them ready for further digestion by the termites. How the wood particlesare taken into their bodies by flagellates, is yet another riddle. In this respectthere is no direct evidence reported so far. However, it has been suggested byKudo (1966) that flagellates feed by throwing out pseudopodia from any part oftheir body which engulf the particles. These particles are digested within the bodyof flagellates. A part of the digested food is consumed by the flagellate itself,while the rest of it is expelled out by exocytosis being the mode 'If excretion inprotozoans (De Robertis & De Robertis, 1980). The digested food is released byflagellates in the rectum of the gut. There are little chances of the same being. assimilated there, as the rectum is lined internally with chitin (Richards & Davies,1984). So, the rectal nutritive fluid (semi-digested) is perhaps moved back intothe intestine where its digestion is completed and assimilation is carried out.
BIOLOGIAN. A. QURESHI ET AL52
In order to test the above hypothesis, the hind gut at its junction with theintestine was cut and separated from the stomach also. When it was opened, itdid have the flagellates, though lesser in numbers. This can- be further supportedby histological studies.
According to different authors, defaunated termites (workers) do not survivef{)r more than two days without flagellates and die of starvation. This suggeststhat termites were totally dependent for the digestion of their food on theseflagellates. Termites and flagellates thus have developed a mutualistic type ofassociation with each other (Odum, 1971). Termites are social insects with acaste system comprising queen, king, workers and soldiers in the colony. Thequeen and king are reproductive members, while soldiers defend the colony fromintruders and guard the workers in their work. They have strong jaws and are fedby the workers. Among the various castes, only the worker termites are capableof digesting food. Oily exudates are secreted through the cuticle, which are freelycirculated from caste to caste and individual to individual throughout the colony(Metcalf, 1992)
The smaller number of flagellates in the gut of soldiers than in the workers. being reported in this investigation may be explained to be the result of feeding ofthe soldiers on the digested food from the bodies of the workers. The fopdexudating from the bodies of workers probably also contains the protozoans
It was noticed that the most abundant genus ()f the flagellates in workerswas Ho/omastigotoides whereas the most abundant genus of flagellates insoldiers was Holomastigotes. The most abundant species, in both workers andsoldiers, however, belonged to genus Holomastigotes. The reason for theabundance of the particular genus and the species of flagellates over othergenera and species remains obscure as far as termites are concerned. It mayhave been the result of different rates of reproduction among the flagellates, forinstance, the genus Holomastigotoides may be the most rapidly reproducinggenus of all the genera occurring in the termite gut. Of the species,Holomastiogotes campanula may be the most rapidly reproducing species of allthe species of the three genera present in Heterotermes indico/a.
The genus Pseudotrichonympha and its species P. grassi althoughpresent in lesser number as compared to some species of other genera, is tl)elargest of all the species present. It is almost 2 to 3 times as big a3 any of theother species of flagellates present in H. indicola.
The bodies of P. grassi were found to be replete with wood particles,. which indicates that this species of the flagellates feeds on the wood. The bodiesof the other flagellates were found to be largely empty of the wood particles. Thismeans that most of the supply of digested food for the termite is obtained fromPseudotrichonympha grassi. In these circumstances, the role of the otherflagellates species becomes less important and in the view of the authors thisspecies of flagellates, i.e., P. grassi may be fulfilling the entire nutritional needs ofthe termite. If so, then what is the role of other species of flagellates? The answeris not clear. In 'act a number of facts regarding the biodiversity of flagellates intermites remain unanswered. One of these questions is why there should bemore than one species of flagellates? Why cannot just one species, in this caseP. grassi, do the job? A great deal of more research work is required to answerthese questions.
REFERENCES
The existence of flagellates in soldiers seems to be the result of .theparticular mode of feeding of the soldiers, i.e., only licking the fluid exuding fromthe rectum of the workers as this fluid contains the protozoan (flagellates)suspended in it. The flagellates in the bodies of soldiers, however, do not help inthe food digestion because the soldiers themselves do not feed on the wood.They depend entirely on the digested food from the bodies of the workers. It was. observed by the present authors that when the soldiers were kept on the filterpaper in a petridish without workers in it, the soldiers did not survive beyond the2' day, although the food source, i.e., filter paper was available and theflagellates were also present in the bodies of the soldiers. This clearly showedthat soldiers did not even attempt to eat filter paper even when they werestarving to death. The flagellates in the digestive tracts of soldiers did not seemto survive for very long as was shown by the entire absence of the flagellates insome soldiers. These were perhaps the soldiers for whom a considerable timehad elapsed since they fed on rectal fluid of the workers. This would imply thatthe more freshly fed a soldier is, the higher the numbers of the flagellates it wouldcontain.
IIjI
;IiqlI'55VOL54,(1), BIODNERSITY AND CASTE DIFFERENCES IN POPUL~T.ION OF PROTOZOANS IN A TERMITE
;..:••• !il' ' • '"
de Mello, I. F., 1927. "Revision des trichonympides du Leucotermes indicolaWasm" Arquivos da Escola medico." Ser Cirugica de nova Goa., 1: 1-28.
De Robertis, E D, P, & De Robertis, E. M. F., 1980, Cell and Molecular Biology,7'h ed., Lee & Fabiger, London.
,Guyton, A. C., 1988, Textbook of Medical Physiology, 7'h ed. W.B.SaundersCompany, London.
Kirby, M. 1926. The intestinal flagellates of the termite Cryptotermes hermsiKirby. Univ. Calif Pub/. Zool, , 29: 103-120
Kudo, R. R. 1966. Protozoology, 5'h ed. Springfield, U,S.A.Mannesmann, R. 1972, Relationship between different wood species as a termite
food source and the reproduction rates of termite symbionts. Z Ang.Entomo/. 72: 116-128
Metcalf, G. L., 1992. Destructive and Useful Insects. Their habits and contro/. 5'"ed., McGraw Hill Book Co. Inc, New York, U. S. A.Odum, E. P. 1971. Fundamentals of Ecology, W, B, Saunders Company,
Philadelphila, U, S A.Richards, O. W. & Davies, R. G" 1984. Imm's General Text Book of Entomology,
Vols I & II, 10'h ed., Chapmann & Hall, London, U. K.
BIOLOGIAN. A. QURESHI ET AL.54
.~
In order to test the above hypothesis, the hind gut at its junction with theintestine was cut and separated from the stomach also. When it was opened, itdid have the flagellates, though lesser in numbers. This can- be further supportedby histological studies.
According to different authors, defaunated termites (workers) do not survivef{)r more than two days without flagellates and die of starvation. This suggeststhat termites were totally dependent for the digestion of their food on theseflagellates. Termites and flagellates thus have developed a mutualistic type ofassociation with each other (Odum, 1971). Termites are social insects with acaste system comprising queen, king, workers and soldiers in the colony. Thequeen and king are reproductive members, while soldiers defend the colony fromintruders and guard the workers in their work. They have strong jaws and are fedby the workers. Among the various castes, only the worker termites are capableof digesting food. Oily exudates are secreted through the cuticle, which are freelycirculated from caste to caste and individual to individual throughout the colony(Metcalf, 1992)
The smaller number of flagellates in the gut of soldiers than in the workers. being reported in this investigation may be explained to be the result of feeding ofthe soldiers on the digested food from the bodies of the workers. The fopdexudating from the bodies of workers probably also contains the protozoans
It was noticed that the most abundant genus ()f the flagellates in workerswas Ho/omastigotoides whereas the most abundant genus of flagellates insoldiers was Holomastigotes. The most abundant species, in both workers andsoldiers, however, belonged to genus Holomastigotes. The reason for theabundance of the particular genus and the species of flagellates over othergenera and species remains obscure as far as termites are concerned. It mayhave been the result of different rates of reproduction among the flagellates, forinstance, the genus Holomastigotoides may be the most rapidly reproducinggenus of all the genera occurring in the termite gut. Of the species,Holomastiogotes campanula may be the most rapidly reproducing species of allthe species of the three genera present in Heterotermes indico/a.
The genus Pseudotrichonympha and its species P. grassi althoughpresent in lesser number as compared to some species of other genera, is tl)elargest of all the species present. It is almost 2 to 3 times as big a3 any of theother species of flagellates present in H. indicola.
The bodies of P. grassi were found to be replete with wood particles,. which indicates that this species of the flagellates feeds on the wood. The bodiesof the other flagellates were found to be largely empty of the wood particles. Thismeans that most of the supply of digested food for the termite is obtained fromPseudotrichonympha grassi. In these circumstances, the role of the otherflagellates species becomes less important and in the view of the authors thisspecies of flagellates, i.e., P. grassi may be fulfilling the entire nutritional needs ofthe termite. If so, then what is the role of other species of flagellates? The answeris not clear. In 'act a number of facts regarding the biodiversity of flagellates intermites remain unanswered. One of these questions is why there should bemore than one species of flagellates? Why cannot just one species, in this caseP. grassi, do the job? A great deal of more research work is required to answerthese questions.
REFERENCES
The existence of flagellates in soldiers seems to be the result of .theparticular mode of feeding of the soldiers, i.e., only licking the fluid exuding fromthe rectum of the workers as this fluid contains the protozoan (flagellates)suspended in it. The flagellates in the bodies of soldiers, however, do not help inthe food digestion because the soldiers themselves do not feed on the wood.They depend entirely on the digested food from the bodies of the workers. It was. observed by the present authors that when the soldiers were kept on the filterpaper in a petridish without workers in it, the soldiers did not survive beyond the2' day, although the food source, i.e., filter paper was available and theflagellates were also present in the bodies of the soldiers. This clearly showedthat soldiers did not even attempt to eat filter paper even when they werestarving to death. The flagellates in the digestive tracts of soldiers did not seemto survive for very long as was shown by the entire absence of the flagellates insome soldiers. These were perhaps the soldiers for whom a considerable timehad elapsed since they fed on rectal fluid of the workers. This would imply thatthe more freshly fed a soldier is, the higher the numbers of the flagellates it wouldcontain.
IIjI
;IiqlI'55VOL54,(1), BIODNERSITY AND CASTE DIFFERENCES IN POPUL~T.ION OF PROTOZOANS IN A TERMITE
;..:••• !il' ' • '"
de Mello, I. F., 1927. "Revision des trichonympides du Leucotermes indicolaWasm" Arquivos da Escola medico." Ser Cirugica de nova Goa., 1: 1-28.
De Robertis, E D, P, & De Robertis, E. M. F., 1980, Cell and Molecular Biology,7'h ed., Lee & Fabiger, London.
,Guyton, A. C., 1988, Textbook of Medical Physiology, 7'h ed. W.B.SaundersCompany, London.
Kirby, M. 1926. The intestinal flagellates of the termite Cryptotermes hermsiKirby. Univ. Calif Pub/. Zool, , 29: 103-120
Kudo, R. R. 1966. Protozoology, 5'h ed. Springfield, U,S.A.Mannesmann, R. 1972, Relationship between different wood species as a termite
food source and the reproduction rates of termite symbionts. Z Ang.Entomo/. 72: 116-128
Metcalf, G. L., 1992. Destructive and Useful Insects. Their habits and contro/. 5'"ed., McGraw Hill Book Co. Inc, New York, U. S. A.Odum, E. P. 1971. Fundamentals of Ecology, W, B, Saunders Company,
Philadelphila, U, S A.Richards, O. W. & Davies, R. G" 1984. Imm's General Text Book of Entomology,
Vols I & II, 10'h ed., Chapmann & Hall, London, U. K.
BIOLOGIAN. A. QURESHI ET AL.54
.~
56 BIOLOGIA(PAKISTAN) 2008, 54 (1), 57-64PK ISSN 0006 - 3096
Gcnus Telluil'alp/ls Donnadieu (Acarina: Tenuipalpidae) fromGuj ranwala, Pakistan, with the description of a new species
NASREEN KAUSER, SHAMSHAD AKBAR & IMRANA NAZ
Departincnf qjZou/ogy, GC University. Lahore, Pakistan
ABSTRACT
Genus TenuipaJpus Donnadieu is an important genus of familyTenuipalpidae. To record species of this genus, some localities of GujranwalaDivision were surveyed which resulted in the collection of Tenuipalpusjandialensis n, sp., Tenuipalpus ortus Chaudhri and Tenuipalpus pJacitusChaudhri. As new setal nomenclature has been adopted, therefore, alongwiththe new species, both already known species have been fe-described andillustrated in detail.
Key words.: Tenuipalpus, New species, Gujranwala
INTRODUCTION
Mites of the genus Tenuipalpus of the family Tenuipalpidae (Acarina) are. serious pests throughout the world and do a lot of damage to the plants of alltypes, e,g., Tenuipalpus haveae causes serious damage to the rubber trees inBrazil. Tenuipafpus punicae Sayed and Tenuipafpus granati Sayed cause injuryto pomegranate in Eastern Mediterranean region and Pakistan (Pritchard &Baker, 1958 and Chaudhri et al., 1974), whereas Tenuipalpus reticu/us Siddiquiand Chaudhri, Tenuipafpus p/acitus Chaudhri, Tenuipafpus dimensus Chaudhriand Tenuipa/pus mustus Chaudhri are pests of pomegranate, guava, apple andcitrus, respectively, in Pakistan (Chaudhri et al., 1974).
Keeping in view the importance of this genus, a lot of work has been donethroughout the world, of which the contributions of Pritchard & Baker (1958),Mitrofanove (1973), Baker et ai, (1975), Meyer (1979) and Reinaldo et at. (2006)are worth mentioning; whereas Chaudhri et ai, (1974), Akbar & Chaudhri (1981),Chaudhri & Akbar (1985) and Hassan et al. (2003) recorded mites specis of thegenus Tenuipalpus from Pakistan. .
MATERIALS AND METHODS,Different localities of Gujranwala Division were, selected at random and
. surveyed for the collection of mites of the genus Tenuipalpus of the familyTenuipalpidae. The samples of flowers, leaves, twigs and grasses from variouslocalities were collected and processed by sieve collection method to collect themites of the family Tenuipalpidae. Specimens thus collected were preserved in70% alcohol and a few drops of glycerine. Permanent slides were prepared inHoyer's medium. For identification of species, Chaudhri et al. (1974) and
I,Ii
I,I"I
'II
I'
I'
I'!,
58 N. KAUSER ET AL BIOLOGIA VOL 54 (1) GENUS TENUIPALPUS DONNADIEU FROM GUJRANWALA 59
Chaudhri & Akbar (1985) were followed and for setal nomenclature, Lindquist-Evans system (Lindquist, 1985) has been adopted.
RESULTS AND DISCUSSION
As a result of the survey, one new species, i.e., Tenuipalpus jandialensis and twoalready known species, i.e., Tenuipalpus ortus Chaudhri and Tenuipalpusplacitus Chaudhri have been recorded. As Lindquist-Evans system (Lindquist,1985) has been followed, so the already known species have been re-describedaccording to this new system.
Key to the species of the genus Tenuipalpus Donnadieu fromGujranwala Division
1. Posterior mediovenlral setae 2 pairs .-------------------- T jandialensis n. sp.Posterior medioventral setae 1 pair ----------------------------------------- 2
2. Dorso-central setae, long, barbed,anterior medioventral setae 2 pairs------------------------ T. o.rtus Chaudhri
Dorso-central. setae small, simple,anterior medioventral setae 1 pair ------------------------ T. placilus Chaudhri
Tenuipalpus jandialensis n. sp.(Fig. 1 A-D)
GnathosomaRostrum pointed at the tip; extends to the base of femur 1. RosJral shield
medially notched, striated, with 1 prominent lobe medially and 1 minute lobelaterally on each side (Fig. 1 C). Palpus 2 segmented, terminal segment with 1eupathidium and 1 simple seta, 'II segment wrinkled, without seta (Fig. 1 D).
DorsumBody 225 11min length (without rostrum), 170 11mwide. Prodorsum with few
reticulations near the .sejugal furrow, reticulations change into broken striationstowards anterior and lateral sides. One pair of eyes on each side. Prodorsalsetae .three pairs, v2, se1, sc2 measuring r;; pm each. Setae sc2 slightlylanceolate, serrate. humeral setae c3, simple; measuring 9 ~m in length.Opisthosoma with broken striations, changing into reticulations between central.setae d1. and 111.longitudinal striations towards the lateral sides. Opisthosomalcentral setae three .pairs, all s'imple, setae c1 measuring 7~m, d1 and e1 each 611m. Opistho.so.mal lateral setae six pairs, d3 and e3 5 11m, '2 and '3 slightlylanceo.late, 10 11meach, h111~m in length, setae h2 fiagellate, (Fig. 1A).
VenterVenter simple. Anterio.r medioventral prodo.rsal setae le1a 36 ~m, le2a. 38
11m,and Ic3a 18~m in length; posterior medioventral metapodosomal setae Ic4a1. and Ic4a2, both measuring 36 11m,simple, do not reach the base of ventral shieldsetae. Ventral shield setae ag measuring 34 ~m fall short o.f distance to. the baseo.f genital shield setae. Genital shield with sparsely serrate setae, g1 and 92,measuring 14 11meach, anal shield with a few striations, setae two pairs, ps1 andps2, measuring 8 ~m each.
o
8
c
Figure 1: Tenuiplapus jandialensis n. sp., A-Dorsal aspect, B-Ventralaspect, C-Rostral shield, D-Palpus
LegsLegs four pairs, legs segments slightly wrinkled. Setae on legs segments;
coxae 1-1-1-1, trochanters 1-1-2-1, femora 3-3-2-1, genua 2-2-0-0, tibiae 4-4-3-3,setae on tarsi not clear.
MaleNot came in collection.
Type
Holotype female, collected at Jandiala Bagh Wala, on 18-05-2006 frommulberry (Morus alba) (Nasreen) and deposited in the Museum, Department ofZoology, GC University, Lahore, Pakistan.
!
58 N. KAUSER ET AL BIOLOGIA VOL 54 (1) GENUS TENUIPALPUS DONNADIEU FROM GUJRANWALA 59
Chaudhri & Akbar (1985) were followed and for setal nomenclature, Lindquist-Evans system (Lindquist, 1985) has been adopted.
RESULTS AND DISCUSSION
As a result of the survey, one new species, i.e., Tenuipalpus jandialensis and twoalready known species, i.e., Tenuipalpus ortus Chaudhri and Tenuipalpusplacitus Chaudhri have been recorded. As Lindquist-Evans system (Lindquist,1985) has been followed, so the already known species have been re-describedaccording to this new system.
Key to the species of the genus Tenuipalpus Donnadieu fromGujranwala Division
1. Posterior mediovenlral setae 2 pairs .-------------------- T jandialensis n. sp.Posterior medioventral setae 1 pair ----------------------------------------- 2
2. Dorso-central setae, long, barbed,anterior medioventral setae 2 pairs------------------------ T. o.rtus Chaudhri
Dorso-central. setae small, simple,anterior medioventral setae 1 pair ------------------------ T. placilus Chaudhri
Tenuipalpus jandialensis n. sp.(Fig. 1 A-D)
GnathosomaRostrum pointed at the tip; extends to the base of femur 1. RosJral shield
medially notched, striated, with 1 prominent lobe medially and 1 minute lobelaterally on each side (Fig. 1 C). Palpus 2 segmented, terminal segment with 1eupathidium and 1 simple seta, 'II segment wrinkled, without seta (Fig. 1 D).
DorsumBody 225 11min length (without rostrum), 170 11mwide. Prodorsum with few
reticulations near the .sejugal furrow, reticulations change into broken striationstowards anterior and lateral sides. One pair of eyes on each side. Prodorsalsetae .three pairs, v2, se1, sc2 measuring r;; pm each. Setae sc2 slightlylanceolate, serrate. humeral setae c3, simple; measuring 9 ~m in length.Opisthosoma with broken striations, changing into reticulations between central.setae d1. and 111.longitudinal striations towards the lateral sides. Opisthosomalcentral setae three .pairs, all s'imple, setae c1 measuring 7~m, d1 and e1 each 611m. Opistho.so.mal lateral setae six pairs, d3 and e3 5 11m, '2 and '3 slightlylanceo.late, 10 11meach, h111~m in length, setae h2 fiagellate, (Fig. 1A).
VenterVenter simple. Anterio.r medioventral prodo.rsal setae le1a 36 ~m, le2a. 38
11m,and Ic3a 18~m in length; posterior medioventral metapodosomal setae Ic4a1. and Ic4a2, both measuring 36 11m,simple, do not reach the base of ventral shieldsetae. Ventral shield setae ag measuring 34 ~m fall short o.f distance to. the baseo.f genital shield setae. Genital shield with sparsely serrate setae, g1 and 92,measuring 14 11meach, anal shield with a few striations, setae two pairs, ps1 andps2, measuring 8 ~m each.
o
8
c
Figure 1: Tenuiplapus jandialensis n. sp., A-Dorsal aspect, B-Ventralaspect, C-Rostral shield, D-Palpus
LegsLegs four pairs, legs segments slightly wrinkled. Setae on legs segments;
coxae 1-1-1-1, trochanters 1-1-2-1, femora 3-3-2-1, genua 2-2-0-0, tibiae 4-4-3-3,setae on tarsi not clear.
MaleNot came in collection.
Type
Holotype female, collected at Jandiala Bagh Wala, on 18-05-2006 frommulberry (Morus alba) (Nasreen) and deposited in the Museum, Department ofZoology, GC University, Lahore, Pakistan.
!
60 N. KAUSER ET AL. BIOLOGIA VOL 54 (1). GENUS TENUIPALPUS DONNADIEU FROM GUJRANWALA 61
EtymologyThe name of this species has been proposed after the name of the place of itscollection.Remarks. This :neli#'species of Tenuipalpus is closely related to T. reticulus Siddiqui &Chaudhri; 'however, can be differentiated from it on the basis of the followingcharacters:
i). The median area in between central setae d1 and e1 with reticulations in thisnew species whereas entire area medially, from sejugal furrow to caudal end,is reticulated in T. reticulus .
. iii. Genital and anal shields not reticulated in this new species, whereasreticulated in T. ret/culus.
iii). Leg chaetotaxy of femora, genua and tibiae differs in both the species.
This new species can also be separated from T. granati Sayed due to thefollowing characters:
i). Dorsum with few reticulation mediolaterally and in between setae d1 and e1 inthis new' species, whereas it is entirely reticulated in T. granati.
iii. Genital setae 2 pairs in this new species but 1 pair in T. granati.
TenuipaJpus onus Chaudhri(Fig. 2 A-D)
TelJuipalpus ortus Chaudhri 1972c:21, Chaudhri et al., 1974:80, Chaudhri &Akbar 1985 98.
Gnathosoma
Rostrum conical reaching about the middle of femur I. Rostral shielddeeply notched medially, with 1 median and 1 prominent lateral lobe on eachside (Fig. 2C). Palpus 3 segmented, wrinkled, terminal segment with 1eupathidium and 1 seta, II segment with 1 long barbed seta (Fig. 2 D).
Dorsum
Body 208 IJm long (without rostrum), 141 IJm wide, Prodorsum withlongitudinal irregular broken striations mediolaterally and laterally, median areawithout striations. Prodorsal setae 3 pairs, long, setiform, serrate, seta v2measuring 131Jm, sc1 and sc2 both 19 IJm. Eyes 1 pairs on each side. Humeral
. setae, c3 1 pair, serrate, 11 IJm in length. Opisthosoma with irregular brokenlongitudinal striations laterally and transversally directed medially. Dorso-centralopisthosomal setae 3 pairs, setiform. serrate, c1 measuring 16 IJm, d1, 14 IJmand e1, 10 IJm. Two pores posterior to central setae e1 present. Dorso-lateralopisthosomal setae 6 pairs, seliform, serrate. d3 measuring 14 IJm, e3 91Jm, /211IJm, f3and h1 11IJm each, h2 flagellate, serrate (Fig 2 A).
o
B
cFig. 2: Tenuiplapus onus Chaudhri, A-Dorsal aspect, B-Ventral aspect, C-
Rostral shield, 0- Palpus
VenterVenter with transverse striations at the base of coxae I and'il and lateral
to anterior medioventral, prodorsal setae and medially posterior to medioventralmetapodosomal setae 1c2a and 1c3a and between coxae III and IV. Setae 1c2;,measuring 52 IJm, 1c3a 7IJm and 1c3b 45 IJm present Posterior medioventralmetapodosomal setae 1c4a 1 pair, 46 IJm in length, reaches the base of setae1c3a. Ventral shield setae ag 1 pair, 18 IJm, crosses the bases of genital shieldsetae. Genital shield setae 2 pairs, g1 and g2, measuring 8IJm and 121Jm,Justreach the base of anal shield setae. Anal shield setae 2 pairs, ps1 and pS2, bothsimple, measuring 5 IJm and 6 IJm, respectively (Fig. 26).
II
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60 N. KAUSER ET AL. BIOLOGIA VOL 54 (1). GENUS TENUIPALPUS DONNADIEU FROM GUJRANWALA 61
EtymologyThe name of this species has been proposed after the name of the place of itscollection.Remarks. This :neli#'species of Tenuipalpus is closely related to T. reticulus Siddiqui &Chaudhri; 'however, can be differentiated from it on the basis of the followingcharacters:
i). The median area in between central setae d1 and e1 with reticulations in thisnew species whereas entire area medially, from sejugal furrow to caudal end,is reticulated in T. reticulus .
. iii. Genital and anal shields not reticulated in this new species, whereasreticulated in T. ret/culus.
iii). Leg chaetotaxy of femora, genua and tibiae differs in both the species.
This new species can also be separated from T. granati Sayed due to thefollowing characters:
i). Dorsum with few reticulation mediolaterally and in between setae d1 and e1 inthis new' species, whereas it is entirely reticulated in T. granati.
iii. Genital setae 2 pairs in this new species but 1 pair in T. granati.
TenuipaJpus onus Chaudhri(Fig. 2 A-D)
TelJuipalpus ortus Chaudhri 1972c:21, Chaudhri et al., 1974:80, Chaudhri &Akbar 1985 98.
Gnathosoma
Rostrum conical reaching about the middle of femur I. Rostral shielddeeply notched medially, with 1 median and 1 prominent lateral lobe on eachside (Fig. 2C). Palpus 3 segmented, wrinkled, terminal segment with 1eupathidium and 1 seta, II segment with 1 long barbed seta (Fig. 2 D).
Dorsum
Body 208 IJm long (without rostrum), 141 IJm wide, Prodorsum withlongitudinal irregular broken striations mediolaterally and laterally, median areawithout striations. Prodorsal setae 3 pairs, long, setiform, serrate, seta v2measuring 131Jm, sc1 and sc2 both 19 IJm. Eyes 1 pairs on each side. Humeral
. setae, c3 1 pair, serrate, 11 IJm in length. Opisthosoma with irregular brokenlongitudinal striations laterally and transversally directed medially. Dorso-centralopisthosomal setae 3 pairs, setiform. serrate, c1 measuring 16 IJm, d1, 14 IJmand e1, 10 IJm. Two pores posterior to central setae e1 present. Dorso-lateralopisthosomal setae 6 pairs, seliform, serrate. d3 measuring 14 IJm, e3 91Jm, /211IJm, f3and h1 11IJm each, h2 flagellate, serrate (Fig 2 A).
o
B
cFig. 2: Tenuiplapus onus Chaudhri, A-Dorsal aspect, B-Ventral aspect, C-
Rostral shield, 0- Palpus
VenterVenter with transverse striations at the base of coxae I and'il and lateral
to anterior medioventral, prodorsal setae and medially posterior to medioventralmetapodosomal setae 1c2a and 1c3a and between coxae III and IV. Setae 1c2;,measuring 52 IJm, 1c3a 7IJm and 1c3b 45 IJm present Posterior medioventralmetapodosomal setae 1c4a 1 pair, 46 IJm in length, reaches the base of setae1c3a. Ventral shield setae ag 1 pair, 18 IJm, crosses the bases of genital shieldsetae. Genital shield setae 2 pairs, g1 and g2, measuring 8IJm and 121Jm,Justreach the base of anal shield setae. Anal shield setae 2 pairs, ps1 and pS2, bothsimple, measuring 5 IJm and 6 IJm, respectively (Fig. 26).
II
~'
ii'
62 N. KAUSER ET AL. BloLoGIA VOL 54(1) GENUS TENUIPALPUS DoNNADIEU FROM GUJRANWALA 63
LegsLegs 4 pairs, segments wrinkled. Setae on legs segment: coxae 2-2-1-1,trochanters 1-1-2-1, femora 4-4-2-1, genua 3-3-1-0, tibiae 5-5-3-3, setae on tarsinot clear; dorsai setae on femora I and II each, very long (Fig. 2A).
MALENot came in collection.
Known DistributionAlipur, Bahawalpur, Balakot, Chichawattni, Mansehra, Mian Channu,
Multan, Naushera, Rajana Forest, Sukheki and Wahwa.
Known Host PlantsAcacia nilotica, Aerua javanica, Aerua sp., Cordia obliqua, Gisekia
pharnecoides, Morus alba, Salvadora oleoides, Tegets tenuifolia, Cardia rothi,Arundodonax sp., Tecoma grandis.
New Collection DataThe present authors have collected 2 females of this species on 11-01-
2006 from "Gainda" (Tegets tenuifolia) and 1 from Sadabahar (Vinca rosea) atNandipur and deposited in the Museum, Department of Zoology, GC University,Lahore, Pakistan.
TenuipaJpus pJacitus Chaudhri(Fig. 3 A-D)
Tenuipalpus placitus Chaudhri 1971b:209, Chaudhri et al., 1974:95, Chaudhri &Akbar 1985: 105 .
. GnathosomaRostrum narrow at the anterior end, broad at the base reaching the distal
end of the femur 1. Rostral shield deeply notched medially, striated with 1. median and 1 lateral lobes on each side (Fig. 3 C). Palpus 3 segmented, terminalsegment with 1 eupathidium only, II segment with a long barbed seta (Fig. 3 D).
Dorsum
Body 269 >1m long (without rostrum), 151 >1m wide. Proaorsum withbroken, irregular striations, striations slightly semicircular near sejugal furrow.Eyes 1 pair on each side. Prodorsal setae 3 pairs, setae v2 and sc1 minute, sc2setiform, simple, 11>1m.Humeral setae c3, 1 pair, simple, 6 >1m.Opisthosomawith irregular broken striations. Opisthosomal central setae 3 pairs c1, dl and el,simple, 5 >1m,3 >1mand 5 >1mlong, respectively. Dorsolateral opisthosomal setae6 pairs: d33 >1m,e36 >1m,f2 and f3 8 >1meach, h1 9 >1mlong, h2 nagellate, (Fig.3A)
VenterVenterwith transverse striation, posterior to bases of coxae I and II, and
posterior to setae Ic3a. Anterior medioventral prodorsal setae Ic1a 41~m, Ic2a 39>1m, Ic3a small, 5 >1m, posterior medioventral prodorsal setae Ic4a 1 44 >1mcrossing the base of ventral shield setae, ventral shield satae ag 1 pair, 26 >1m,crosses the base of genital shield setae. Genital shield setae 2 pairs gl and g2,14 ~m and 12 ~m, respectively. Anal shield setae 2 pairs, simple, ps116 >1m,ps211~m (Fig. 3 B).
LegsLegs 4 pairs, segment wrinkled. Setae on leg's segments: coxae 2-2-1-1,
trochanters 1-1-2-1, femora 4-4-2-1, genua 2-2-0-0, tibiae 5-5"3-3, setae on tarsinot clear.
MaleNot came in collection.
8
Fig. 3: TenuipJapus pJacitus Chaudhri, A-Dorsal aspect, B-Ventral aspect,C-Rostral shield, D-Palpus
III
62 N. KAUSER ET AL. BloLoGIA VOL 54(1) GENUS TENUIPALPUS DoNNADIEU FROM GUJRANWALA 63
LegsLegs 4 pairs, segments wrinkled. Setae on legs segment: coxae 2-2-1-1,trochanters 1-1-2-1, femora 4-4-2-1, genua 3-3-1-0, tibiae 5-5-3-3, setae on tarsinot clear; dorsai setae on femora I and II each, very long (Fig. 2A).
MALENot came in collection.
Known DistributionAlipur, Bahawalpur, Balakot, Chichawattni, Mansehra, Mian Channu,
Multan, Naushera, Rajana Forest, Sukheki and Wahwa.
Known Host PlantsAcacia nilotica, Aerua javanica, Aerua sp., Cordia obliqua, Gisekia
pharnecoides, Morus alba, Salvadora oleoides, Tegets tenuifolia, Cardia rothi,Arundodonax sp., Tecoma grandis.
New Collection DataThe present authors have collected 2 females of this species on 11-01-
2006 from "Gainda" (Tegets tenuifolia) and 1 from Sadabahar (Vinca rosea) atNandipur and deposited in the Museum, Department of Zoology, GC University,Lahore, Pakistan.
TenuipaJpus pJacitus Chaudhri(Fig. 3 A-D)
Tenuipalpus placitus Chaudhri 1971b:209, Chaudhri et al., 1974:95, Chaudhri &Akbar 1985: 105 .
. GnathosomaRostrum narrow at the anterior end, broad at the base reaching the distal
end of the femur 1. Rostral shield deeply notched medially, striated with 1. median and 1 lateral lobes on each side (Fig. 3 C). Palpus 3 segmented, terminalsegment with 1 eupathidium only, II segment with a long barbed seta (Fig. 3 D).
Dorsum
Body 269 >1m long (without rostrum), 151 >1m wide. Proaorsum withbroken, irregular striations, striations slightly semicircular near sejugal furrow.Eyes 1 pair on each side. Prodorsal setae 3 pairs, setae v2 and sc1 minute, sc2setiform, simple, 11>1m.Humeral setae c3, 1 pair, simple, 6 >1m.Opisthosomawith irregular broken striations. Opisthosomal central setae 3 pairs c1, dl and el,simple, 5 >1m,3 >1mand 5 >1mlong, respectively. Dorsolateral opisthosomal setae6 pairs: d33 >1m,e36 >1m,f2 and f3 8 >1meach, h1 9 >1mlong, h2 nagellate, (Fig.3A)
VenterVenterwith transverse striation, posterior to bases of coxae I and II, and
posterior to setae Ic3a. Anterior medioventral prodorsal setae Ic1a 41~m, Ic2a 39>1m, Ic3a small, 5 >1m, posterior medioventral prodorsal setae Ic4a 1 44 >1mcrossing the base of ventral shield setae, ventral shield satae ag 1 pair, 26 >1m,crosses the base of genital shield setae. Genital shield setae 2 pairs gl and g2,14 ~m and 12 ~m, respectively. Anal shield setae 2 pairs, simple, ps116 >1m,ps211~m (Fig. 3 B).
LegsLegs 4 pairs, segment wrinkled. Setae on leg's segments: coxae 2-2-1-1,
trochanters 1-1-2-1, femora 4-4-2-1, genua 2-2-0-0, tibiae 5-5"3-3, setae on tarsinot clear.
MaleNot came in collection.
8
Fig. 3: TenuipJapus pJacitus Chaudhri, A-Dorsal aspect, B-Ventral aspect,C-Rostral shield, D-Palpus
III
64 N. KAUSER ET AL. BIOLOGIABIOLOGIA(PAKISTAN) 2008.54 (1), 65-72PK ISSN 0006 - 3096
Known Distribution
Alipur, 'Dadar, Hassan AbdaJ, Nia Lahore (Jhang), Ochali, Okara, PakkaAnna, Rahim Var Khan, Salooni Jhal (Lyallpur), Sukkur.
Known Host Plants
Cassia purpurea. Chenopodium ambrosioides, Punica granatum, Pyruscummunis.
New Collection Data
1 female collected from Alipur Chatha on 21-09-2006 from Anar (Punicagranatum) (Nasreen) and deposited in the Museum, Department of Zoology, GCUniversity, Lahore, Pakistan.
REFERENCES
Akbar, S., & Chaudhri, W. M., 1981. New species of the genus Tenuipalpus(Acarina: Tenuipalpidae) from Pakistan. Pak. Entomol., 3(1-2): 1-20.
Baker, E. W, Tuttle, D. M. & Abbatiello, M. J., 1975. The false spider mites ofNorthwestern and North Central Mexico (Acarina:Tenuipalpidae).Smithsonian Contrib. Zool., 194: 1-23.
Chaudhri, W M. & Akbar, S., 1985. Studies on the biosystematics and control ofmites of field crops, vegetables and fruit plants in Pakistan. Monograph No.3, Univ. of AgrL Faisalabad, 314pp.
Chaudhri, W. M., Akbar, S. & Rasool, A, 1974. Taxonomic studies of the mitesbelonging to the families Tenuipalpidae, Tetranychidae, Tuckerellidae,Caligonellidae, Stigmaeidae and Phytoseiidae. Monograph NO.1. UnL Agri.Faisalabad. 250pp.
Hassan, M. U.~Akbar, S.& Bashir, F., 2003. New species of genus Tenuipalpus(Acarina:' Tenuipalpidae) from the upper Punjab, Pakistan. Pak. J. Zool.,35(3): 205-210.
Lindquist, E. E., 1985. External Anatomy, In: Spider Mites, Their Biology, NaturalEnemies and Control. (eds Helle, W & Sabelis, M. W). 1A:3-26.
Meyer, M. K. P., 1979. The Tenuipalpidae (Acari) of Africa with keys to the worldfauna. Ento. Memoir Dept. AgrL Tech. Servo Republic S. Africa, 50: 11-15.
Mitrofanove, V. I., 1973. Revision of the system of phytophagous mites of thesubfamily Tenuipalpini (Trombidiformes: Tenuipalpidae). Zool. Zh., 52(9)1315-1319. .
Pritchard, A E. & Baker, E. W., 1958. The .false spider mites (Acarina:Tenuipalpidae). Univ. Calif Publ. Ent., 14(3): 175-274.
Reinaldo, J., Feres, R. J. F. & Hernandez, F. A, 2006. Three new species ofTenuipalpus Donnadieu (Acari: Tenuipalpidae) from the state of Sao Paulo,
"""Bra.zil. Zootaxa, 1125: 57-68. .
Selenoportax vexillarius from Dhokpathan formation of theMiddle Siwaliks, Pakistan
MUHAMMAD AKBAR KHAN, ZAHEER AHMED& MUHHAMADAKHTAR
Department ~rZoology.GC University, Faisa/abacl, Pakistan (MAK)Department o(Zoology, GC University. Lahore, Pakistan (ZA)
Department o/Zo%gy, Punjab UniverSily, Lahore, Pakislan (MA)
ABSTRACT
The upper and lower teeth of the species Selenopottax vexillariuscollected from the Miocene of Hasnot and Dhokpathan formation (MiddleSiwaliks) are described to determine the individual varialions in lhe dentalmorphology. The specimens studied have been compared with the relevantspecimens in the American Museum of Natural History (AMNH). The uppermolars are moderately hypsodont, quadrate with strong diverged styles, medianribs and entostyle. The entostyle is not much extending transversely. The crown'is narrow at the base and broad at the apex. The enamel is rugose, theroughness of the enamel is also variable in the dental material of Selenopottaxvexillarius. In most of the specimens, upper molars are not reduced and there is-a trend towards the heightening of lhe crown from Nagr; to Dhokpathanformation.
Key words: Dhokpathan. Hosnol, Selenopottax vexillanus, Boselaphines,Siwaliks, EntoJstyle
INTRODUCTION
Boselaphini (Mammalia, Bovidae) are represented by two living species,i.e.. Nilgauy (Boselaphus tragocamelus) and Tetracere (Tetracerusquadricornis). Pilgrim (1937, 1939) reported six species of Boselaphines in theLower Siwaliks and fourteen species in the Middle Siwaliks and consequentlystressed the high density of Boselaphines in the Siwaliks group. Boselaphinesare abundantly found in the Late Miocene of the Siwaliks. They consist of small. and large sized animals (Thomas, 1984). The large sized Siwaliks Boselaphinesinclude the genera Selenoportax and Pachyportax, as Ivell as allied forms suchas Austropottax lat/frons from the Middle Miocene of Europe (Gentry, 1999).
The material under study was collected from the Hasnot and theDhokpathan villages which comprised teeth of a large sized boselaphineSelenoportax, only present in the continental deposits of the Siwaliks. The aim ofthis work is to study the intraspecific variations and paleobiogeography ofSelenoportax in the Siwaliks sediments of Pakistan.
The Siwaliks Group in Pakistan can be clearly divided, according to thelithological characters, into the usual three subgroups: Lower, Middle and Upper
J
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.1
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'I
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64 N. KAUSER ET AL. BIOLOGIABIOLOGIA(PAKISTAN) 2008.54 (1), 65-72PK ISSN 0006 - 3096
Known Distribution
Alipur, 'Dadar, Hassan AbdaJ, Nia Lahore (Jhang), Ochali, Okara, PakkaAnna, Rahim Var Khan, Salooni Jhal (Lyallpur), Sukkur.
Known Host Plants
Cassia purpurea. Chenopodium ambrosioides, Punica granatum, Pyruscummunis.
New Collection Data
1 female collected from Alipur Chatha on 21-09-2006 from Anar (Punicagranatum) (Nasreen) and deposited in the Museum, Department of Zoology, GCUniversity, Lahore, Pakistan.
REFERENCES
Akbar, S., & Chaudhri, W. M., 1981. New species of the genus Tenuipalpus(Acarina: Tenuipalpidae) from Pakistan. Pak. Entomol., 3(1-2): 1-20.
Baker, E. W, Tuttle, D. M. & Abbatiello, M. J., 1975. The false spider mites ofNorthwestern and North Central Mexico (Acarina:Tenuipalpidae).Smithsonian Contrib. Zool., 194: 1-23.
Chaudhri, W M. & Akbar, S., 1985. Studies on the biosystematics and control ofmites of field crops, vegetables and fruit plants in Pakistan. Monograph No.3, Univ. of AgrL Faisalabad, 314pp.
Chaudhri, W. M., Akbar, S. & Rasool, A, 1974. Taxonomic studies of the mitesbelonging to the families Tenuipalpidae, Tetranychidae, Tuckerellidae,Caligonellidae, Stigmaeidae and Phytoseiidae. Monograph NO.1. UnL Agri.Faisalabad. 250pp.
Hassan, M. U.~Akbar, S.& Bashir, F., 2003. New species of genus Tenuipalpus(Acarina:' Tenuipalpidae) from the upper Punjab, Pakistan. Pak. J. Zool.,35(3): 205-210.
Lindquist, E. E., 1985. External Anatomy, In: Spider Mites, Their Biology, NaturalEnemies and Control. (eds Helle, W & Sabelis, M. W). 1A:3-26.
Meyer, M. K. P., 1979. The Tenuipalpidae (Acari) of Africa with keys to the worldfauna. Ento. Memoir Dept. AgrL Tech. Servo Republic S. Africa, 50: 11-15.
Mitrofanove, V. I., 1973. Revision of the system of phytophagous mites of thesubfamily Tenuipalpini (Trombidiformes: Tenuipalpidae). Zool. Zh., 52(9)1315-1319. .
Pritchard, A E. & Baker, E. W., 1958. The .false spider mites (Acarina:Tenuipalpidae). Univ. Calif Publ. Ent., 14(3): 175-274.
Reinaldo, J., Feres, R. J. F. & Hernandez, F. A, 2006. Three new species ofTenuipalpus Donnadieu (Acari: Tenuipalpidae) from the state of Sao Paulo,
"""Bra.zil. Zootaxa, 1125: 57-68. .
Selenoportax vexillarius from Dhokpathan formation of theMiddle Siwaliks, Pakistan
MUHAMMAD AKBAR KHAN, ZAHEER AHMED& MUHHAMADAKHTAR
Department ~rZoology.GC University, Faisa/abacl, Pakistan (MAK)Department o(Zoology, GC University. Lahore, Pakistan (ZA)
Department o/Zo%gy, Punjab UniverSily, Lahore, Pakislan (MA)
ABSTRACT
The upper and lower teeth of the species Selenopottax vexillariuscollected from the Miocene of Hasnot and Dhokpathan formation (MiddleSiwaliks) are described to determine the individual varialions in lhe dentalmorphology. The specimens studied have been compared with the relevantspecimens in the American Museum of Natural History (AMNH). The uppermolars are moderately hypsodont, quadrate with strong diverged styles, medianribs and entostyle. The entostyle is not much extending transversely. The crown'is narrow at the base and broad at the apex. The enamel is rugose, theroughness of the enamel is also variable in the dental material of Selenopottaxvexillarius. In most of the specimens, upper molars are not reduced and there is-a trend towards the heightening of lhe crown from Nagr; to Dhokpathanformation.
Key words: Dhokpathan. Hosnol, Selenopottax vexillanus, Boselaphines,Siwaliks, EntoJstyle
INTRODUCTION
Boselaphini (Mammalia, Bovidae) are represented by two living species,i.e.. Nilgauy (Boselaphus tragocamelus) and Tetracere (Tetracerusquadricornis). Pilgrim (1937, 1939) reported six species of Boselaphines in theLower Siwaliks and fourteen species in the Middle Siwaliks and consequentlystressed the high density of Boselaphines in the Siwaliks group. Boselaphinesare abundantly found in the Late Miocene of the Siwaliks. They consist of small. and large sized animals (Thomas, 1984). The large sized Siwaliks Boselaphinesinclude the genera Selenoportax and Pachyportax, as Ivell as allied forms suchas Austropottax lat/frons from the Middle Miocene of Europe (Gentry, 1999).
The material under study was collected from the Hasnot and theDhokpathan villages which comprised teeth of a large sized boselaphineSelenoportax, only present in the continental deposits of the Siwaliks. The aim ofthis work is to study the intraspecific variations and paleobiogeography ofSelenoportax in the Siwaliks sediments of Pakistan.
The Siwaliks Group in Pakistan can be clearly divided, according to thelithological characters, into the usual three subgroups: Lower, Middle and Upper
J
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II !l }
'I
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66 M. A. KHAN ET AL BIOLOGIA VOL. 54 (1) SELENOPORTAX VEXILLARIUS FROM DHOKPATHAN, PAKISTAN 67
Type Species: Selenoportax vexillarius Pilgrim, 1937,
Distribution: Middle and Upper Siwalik.
SELENOPORTAX VEX/LLARIUS Pilgrim, 1937(Plate 1, Figs. 1-5)
I
I:
: I
SYSTEMATIC PALAEONTOLOGY
SOVIDAE Gray, 1821BOVINAE Gray, 1821SOSELAPHINI Knottnerus-Meyer, 1907
Genus SELENOPORTAX Pilgrim, 1937
FamilySubfamilyTribe
Stratigraphic Range: Middle and Upper Siwaliks (Akhtar, 1992; Pilgrim, 1939;Khan, 2008)Diagnosis: Cheek teeth large and strongly hypsodont, enamel rugose, uppermolars quadrate with strong and divergent styles near the neck of crown, ribs
Material: PUPC 86/241-left second upper molar, Dhokpathan; PUPC 04/28-left;second upper ",olar, Hasnot; PUPC 96/44-left first lower molar; Hasnot; PUPC,86/20-right first lower molar, Dhokpathan; PUPC 83/644-right second lowermolar, Dhokpathan.
Generic Diagnosis: Moderate to large sized Siwalik Sovid; with hypsodont toextremely hypsodont teeth, upper .molars quadrate with strong divergent styles,median ribs well developed, entostyle strongly developed and ectostylidmoderately developed, enamel very rugose, crown narrow at the base and broadat the apex (Pilgrim, 1937),
Included Species: Selenoportax vexillarius Pilgrim, 1937; SelenoportaxIydekkeri Pilgrim, 1937; Selenoportax dhokpathanensis Akhtar, 1992,
AbbreviationsPUPC Punjab University Palaeontological CollectionAMNH American Museum of Natural HistoryMa Million years agoH HasnotDP DhokpathanL LeftR RightM,' First lower and upper MolarM,' Second lower and upper mola'r,
Type Specimen: A skull lacking maxilla and dentition and most of thebasicranium (AMNH 19748).
-
Siwaliks. The Lower Siwaliks (Kamlial, Chinji) consists of a sequence ofsandstone-mudstone couplets with a marked dominance of the mudstones overthe sandstones. The development of paleosol horizons is also fairly frequent The. Middle Siwaliks (Nagri, Dhokpathan) are dominantly arenaceous, consisting ofmultistoried coarse to medium-grained, blue-gray, massive sandstones (30 to >60m) with subordinate representation of clays, mudstones and siltstones. TheMiddle Siwaliks sediments are known to produce scattered fossils. The Upp.erSiwalik (Tatrot, Pinjor, Boulder Conglomerate) subgroup is classified into threelithostratigraphic formations. The three units comprise the sequence of thesandstone-mudstone couplets, the Parmandal sandstone and the BoulderConglomerate formation, the upper most lithostratigraphic unit (Quade & Cerling,1995)
The Dhokpathan formation is composed of gray sandstone and red-brownmudstone with a few thin conglomerate interbeds. Sandstone and superposedred mudstone often form fining-upward couplets where the lower contact iserosional'and lined with ripped-up clasts of the underlying mottlej and red-brown claystone. At a few places, thin crevasse-splay sheets, around 30 cmthick, clast-supported conglomerates occur. These conglomerate beds oftencontain .unidentifiable bone and tooth fragments. Sandstone beds section9radually:. gets thicker as well as multistoried. These substantially thicker,vertic.al}y~\~ctea and laterally extensive individual gray sandstone units form afining-upward'"sequence with thinner dull red to brown siltstones on top.Varicolored" mottled, highly bioturbated paleosol horizons form distinct andlaterally, extensive units within the siltstone or at the transition of the sandstoneto siltstone facies.
In the lower part, there are pinkish to light brown, hard, mottled, pseudo-nodular calcrete beds that are developed usually at the top of sandstone units,which are heavily bioturbated near the calcrete beds.
The middle part of the formation has alternating buff and light graysandstone units, very much reminiscent of the Litra formation of the SulaimanRange (Behrensmeyer & Tauxe, 1982), Conglomerate inter-beds containingextra-formational pebbles of sandstone, quartzite, limestone and igneous rocksusually define the erosional base of the fining-upward sequences, but towardsthe up-section they form fairly thick laterally extensive independent bodies. Theupper contact of the middle portion of the formation is gradational, usually placedwhen the conglomerates frequently occur as massive to crudely bedded units, 1-3 m thick, These sand bodies also commonly have conglomerate lenses andscattered pebbles along the cross bed. The upper part of the formation consistsof thick massive to stratified conglomerate and pebbly gray sandstone withsubordinate medium-to fine-grained friable gray sandstone and grayish-brownsiltstone. Finer facies appear to be more common in the middle portion in theBhandar area where they also contain some vertebrate fossils (Bhattacharya &Misra, 1963; Bhattacharya, 1970; Chaudhri & Gill, 1983; Bagati & KCJmar, 1994,Bi~~was,1994; Raiverman & Suresh, 1997 and Raiverman, 2002).
The two villages of the Dhokpathan formation are well known for having theTertiary vertebrate" fauna. The Hasnot village has younger fauna and alsocomparatively ricil'ttertiary faunal biodiversity than the Dhokpathan village,
66 M. A. KHAN ET AL BIOLOGIA VOL. 54 (1) SELENOPORTAX VEXILLARIUS FROM DHOKPATHAN, PAKISTAN 67
Type Species: Selenoportax vexillarius Pilgrim, 1937,
Distribution: Middle and Upper Siwalik.
SELENOPORTAX VEX/LLARIUS Pilgrim, 1937(Plate 1, Figs. 1-5)
I
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SYSTEMATIC PALAEONTOLOGY
SOVIDAE Gray, 1821BOVINAE Gray, 1821SOSELAPHINI Knottnerus-Meyer, 1907
Genus SELENOPORTAX Pilgrim, 1937
FamilySubfamilyTribe
Stratigraphic Range: Middle and Upper Siwaliks (Akhtar, 1992; Pilgrim, 1939;Khan, 2008)Diagnosis: Cheek teeth large and strongly hypsodont, enamel rugose, uppermolars quadrate with strong and divergent styles near the neck of crown, ribs
Material: PUPC 86/241-left second upper molar, Dhokpathan; PUPC 04/28-left;second upper ",olar, Hasnot; PUPC 96/44-left first lower molar; Hasnot; PUPC,86/20-right first lower molar, Dhokpathan; PUPC 83/644-right second lowermolar, Dhokpathan.
Generic Diagnosis: Moderate to large sized Siwalik Sovid; with hypsodont toextremely hypsodont teeth, upper .molars quadrate with strong divergent styles,median ribs well developed, entostyle strongly developed and ectostylidmoderately developed, enamel very rugose, crown narrow at the base and broadat the apex (Pilgrim, 1937),
Included Species: Selenoportax vexillarius Pilgrim, 1937; SelenoportaxIydekkeri Pilgrim, 1937; Selenoportax dhokpathanensis Akhtar, 1992,
AbbreviationsPUPC Punjab University Palaeontological CollectionAMNH American Museum of Natural HistoryMa Million years agoH HasnotDP DhokpathanL LeftR RightM,' First lower and upper MolarM,' Second lower and upper mola'r,
Type Specimen: A skull lacking maxilla and dentition and most of thebasicranium (AMNH 19748).
-
Siwaliks. The Lower Siwaliks (Kamlial, Chinji) consists of a sequence ofsandstone-mudstone couplets with a marked dominance of the mudstones overthe sandstones. The development of paleosol horizons is also fairly frequent The. Middle Siwaliks (Nagri, Dhokpathan) are dominantly arenaceous, consisting ofmultistoried coarse to medium-grained, blue-gray, massive sandstones (30 to >60m) with subordinate representation of clays, mudstones and siltstones. TheMiddle Siwaliks sediments are known to produce scattered fossils. The Upp.erSiwalik (Tatrot, Pinjor, Boulder Conglomerate) subgroup is classified into threelithostratigraphic formations. The three units comprise the sequence of thesandstone-mudstone couplets, the Parmandal sandstone and the BoulderConglomerate formation, the upper most lithostratigraphic unit (Quade & Cerling,1995)
The Dhokpathan formation is composed of gray sandstone and red-brownmudstone with a few thin conglomerate interbeds. Sandstone and superposedred mudstone often form fining-upward couplets where the lower contact iserosional'and lined with ripped-up clasts of the underlying mottlej and red-brown claystone. At a few places, thin crevasse-splay sheets, around 30 cmthick, clast-supported conglomerates occur. These conglomerate beds oftencontain .unidentifiable bone and tooth fragments. Sandstone beds section9radually:. gets thicker as well as multistoried. These substantially thicker,vertic.al}y~\~ctea and laterally extensive individual gray sandstone units form afining-upward'"sequence with thinner dull red to brown siltstones on top.Varicolored" mottled, highly bioturbated paleosol horizons form distinct andlaterally, extensive units within the siltstone or at the transition of the sandstoneto siltstone facies.
In the lower part, there are pinkish to light brown, hard, mottled, pseudo-nodular calcrete beds that are developed usually at the top of sandstone units,which are heavily bioturbated near the calcrete beds.
The middle part of the formation has alternating buff and light graysandstone units, very much reminiscent of the Litra formation of the SulaimanRange (Behrensmeyer & Tauxe, 1982), Conglomerate inter-beds containingextra-formational pebbles of sandstone, quartzite, limestone and igneous rocksusually define the erosional base of the fining-upward sequences, but towardsthe up-section they form fairly thick laterally extensive independent bodies. Theupper contact of the middle portion of the formation is gradational, usually placedwhen the conglomerates frequently occur as massive to crudely bedded units, 1-3 m thick, These sand bodies also commonly have conglomerate lenses andscattered pebbles along the cross bed. The upper part of the formation consistsof thick massive to stratified conglomerate and pebbly gray sandstone withsubordinate medium-to fine-grained friable gray sandstone and grayish-brownsiltstone. Finer facies appear to be more common in the middle portion in theBhandar area where they also contain some vertebrate fossils (Bhattacharya &Misra, 1963; Bhattacharya, 1970; Chaudhri & Gill, 1983; Bagati & KCJmar, 1994,Bi~~was,1994; Raiverman & Suresh, 1997 and Raiverman, 2002).
The two villages of the Dhokpathan formation are well known for having theTertiary vertebrate" fauna. The Hasnot village has younger fauna and alsocomparatively ricil'ttertiary faunal biodiversity than the Dhokpathan village,
68 M. A. KHAN ET At. BIOlOGIA VOL. 54 (1) SELENOPORTAX VEXILLARIUS FROM DHOKPATHAN. PAKISTAN 69
"'.!
2b
~r.
2a
•lb '"...• ~1a
The enamel is thick, shiny and rugose in pupe 86/20. The molars have almostsame length anteroposteriorly.
pupe 83/644 (plate1 ,Fig. Sa, b) is in early wear and well preserved exceptthe apex of the metaconid and entoconid which are missing. The tooth isextremely hypsodont and narrow crowned. The median basal pillar is present inthe transverse valley between the protoconid and hypoconid, and damaged atthe apex. The goat fold is moderately developed and more prominent .at the baseof the molar. The metastylid is strongly developed while the others aremoderately developed. The comparative measurements of the molars areprovided in Table 1.
Fossettes withoutgoat folds poorly
Description: pupe 86/241 (plate1,Figs. la, b) is well preserved indicating the.complete development of the major cusps and the roots are also present at thebase of the ~rown. The central cavities are very narrow due to early state of wearand filled with hard sandstone. The median basal pillar is embedded in thesandstone. The parastyle is well developed but damaged at the apex of thecrown. Vertical cracks are present in the protocone and metacone.
pupe 04/28 (platel,Figs. 2a, b) is not in a good state of preservation. Out ofthe four major cusps, paracone and metacone are damaged on the buccal side.Therefore, all the styles and median ribs are missing. The median basal pillar isvery strong and isolated. The transverse goat of paracone is missing. Theenamel is very rugose and the rugosity is very prominent around the protoconeand hypocone. The central cavities are crescentic in their general appearanceand deeper.
pupe 96/44 (plate1 ,Figs. 3a, b) and pupe 86/20 (Figs. 4a, b) are the firstlower molars of Se/enoportax vexillarius. Both the molars are poorly preserved,the tooth is half worn out. The median basal pillar is very prominent in pupe86/20 but in pupe 96/44 it is just present at the base of the crown.
quite large, entostyle/ectostylid strongly developed.indentations and simple in outline, transverse anteriordeveloped at front of lower molars.
iII:
.-~'"
til4b
.".. "0'-
. ,- -'. - -~: ..
3b
5b
Selenoportax vexillariusJ
Fig. 1. LM' (PUPC 86/241). (a) occlusal view (b) buccal view.Fig. 2. LM' (PUPC 04/28), (a) occlusal view (b) lingual view.Fig. 3. LM, (PUPC 96/44). (a) buccal view (b) lingual view.Fig. 4. RM, (PUPC 86/20), (a) occlusal view (b) lingual view.Fig. 5. RM, (PUPC 83/644), (a) occlusal view (b) buccal view.
Scale bar 10 mm.
3a
Table.1: Measurements of the cheek teeth of SeJenoportax vexiJIarius inmm (millimeters).
.,
pu pe 86/20 is broken anteroposteriorly and goat fold is not preserved init. However, pupe 96/44 has well developed goat fold. styles and median nbs.r'
Number Nature Position Length Width WIL ratio(L) (W)
PUPC 86/241 (OP)' left molar M2 26.0 22.0 0.84PUPC 04/28 (H)' left molar M' 260 178 068PUPC 96/44 (H)' left molar M, 20 13.5 0.67PUPC 86/20 (DP)' right molar M, 20 14.0 0.70PUPC 83/644 (OP)' right molar M, 28 14.7 0.52PUPC 01/23 left molar M' 23.5 19.5 0.82PUPC 85/40 left molar M, 19.7 12.5 0.63PUPC 04/12 left molar M, 20:0 12.5 0.62AMNH 29917 left molar M, 18 13 0.72AMNH 19844 right molar M' 25.7 240 0.93AMNH 19844 left molar M, 25.9 165 0.63AMNH 19514 left molar M, 22.0 15.5 070AMNH 29917 left molar M, 21.0 15.0 0.71AMNH 19514 left molar M3 33.0 21.5 0.65PUPC 87/19 left molar M' 29 21 0.72* The specimens studied and compared with those of referred material fron:JPilgrim 1937, • Plate 1.1939. The locality code (from where the specimen was collected) is given in parentheses
68 M. A. KHAN ET At. BIOlOGIA VOL. 54 (1) SELENOPORTAX VEXILLARIUS FROM DHOKPATHAN. PAKISTAN 69
"'.!
2b
~r.
2a
•lb '"...• ~1a
The enamel is thick, shiny and rugose in pupe 86/20. The molars have almostsame length anteroposteriorly.
pupe 83/644 (plate1 ,Fig. Sa, b) is in early wear and well preserved exceptthe apex of the metaconid and entoconid which are missing. The tooth isextremely hypsodont and narrow crowned. The median basal pillar is present inthe transverse valley between the protoconid and hypoconid, and damaged atthe apex. The goat fold is moderately developed and more prominent .at the baseof the molar. The metastylid is strongly developed while the others aremoderately developed. The comparative measurements of the molars areprovided in Table 1.
Fossettes withoutgoat folds poorly
Description: pupe 86/241 (plate1,Figs. la, b) is well preserved indicating the.complete development of the major cusps and the roots are also present at thebase of the ~rown. The central cavities are very narrow due to early state of wearand filled with hard sandstone. The median basal pillar is embedded in thesandstone. The parastyle is well developed but damaged at the apex of thecrown. Vertical cracks are present in the protocone and metacone.
pupe 04/28 (platel,Figs. 2a, b) is not in a good state of preservation. Out ofthe four major cusps, paracone and metacone are damaged on the buccal side.Therefore, all the styles and median ribs are missing. The median basal pillar isvery strong and isolated. The transverse goat of paracone is missing. Theenamel is very rugose and the rugosity is very prominent around the protoconeand hypocone. The central cavities are crescentic in their general appearanceand deeper.
pupe 96/44 (plate1 ,Figs. 3a, b) and pupe 86/20 (Figs. 4a, b) are the firstlower molars of Se/enoportax vexillarius. Both the molars are poorly preserved,the tooth is half worn out. The median basal pillar is very prominent in pupe86/20 but in pupe 96/44 it is just present at the base of the crown.
quite large, entostyle/ectostylid strongly developed.indentations and simple in outline, transverse anteriordeveloped at front of lower molars.
iII:
.-~'"
til4b
.".. "0'-
. ,- -'. - -~: ..
3b
5b
Selenoportax vexillariusJ
Fig. 1. LM' (PUPC 86/241). (a) occlusal view (b) buccal view.Fig. 2. LM' (PUPC 04/28), (a) occlusal view (b) lingual view.Fig. 3. LM, (PUPC 96/44). (a) buccal view (b) lingual view.Fig. 4. RM, (PUPC 86/20), (a) occlusal view (b) lingual view.Fig. 5. RM, (PUPC 83/644), (a) occlusal view (b) buccal view.
Scale bar 10 mm.
3a
Table.1: Measurements of the cheek teeth of SeJenoportax vexiJIarius inmm (millimeters).
.,
pu pe 86/20 is broken anteroposteriorly and goat fold is not preserved init. However, pupe 96/44 has well developed goat fold. styles and median nbs.r'
Number Nature Position Length Width WIL ratio(L) (W)
PUPC 86/241 (OP)' left molar M2 26.0 22.0 0.84PUPC 04/28 (H)' left molar M' 260 178 068PUPC 96/44 (H)' left molar M, 20 13.5 0.67PUPC 86/20 (DP)' right molar M, 20 14.0 0.70PUPC 83/644 (OP)' right molar M, 28 14.7 0.52PUPC 01/23 left molar M' 23.5 19.5 0.82PUPC 85/40 left molar M, 19.7 12.5 0.63PUPC 04/12 left molar M, 20:0 12.5 0.62AMNH 29917 left molar M, 18 13 0.72AMNH 19844 right molar M' 25.7 240 0.93AMNH 19844 left molar M, 25.9 165 0.63AMNH 19514 left molar M, 22.0 15.5 070AMNH 29917 left molar M, 21.0 15.0 0.71AMNH 19514 left molar M3 33.0 21.5 0.65PUPC 87/19 left molar M' 29 21 0.72* The specimens studied and compared with those of referred material fron:JPilgrim 1937, • Plate 1.1939. The locality code (from where the specimen was collected) is given in parentheses
70 M. A. KHAN ET AL BIOLOGIA VOL 54 (1) SELENOPORTAX VEXILLARIUS FROM DHOKPATHAN, PAKISTAN 71
DISCUSSION
The specimens under study have moderate goat folds and advancedmedian basal, pillars that confirm their inclusion in the genus Selenoportax. Thegenus has three species, Selenoportax vexillarius, Selenoportax dhokgaalensis,Selenoportax Iydekkeri, found in the Middle Siwaliks of Pakistan. TheSelenoportax Iydekkeri is larger one and has strong divergent stYles (Pilgrim,1939). The described dental material is moderate in size and has no divergentstyles, which confirms that the material belongs to the species Selenoportaxvexillarius. The specimens were metrically compared to the type specimens atAmerican Museum of Natural History and found resemblance in theirmeasurements (Table 1). The specimens, with divergent styles, median ribs welldeveloped, median basal pillar strongly developed in upper molars andmoderately developed in lower molars, enamel largely rugose and the crownnarrow at the base and broad at the apex, show all the characteristics exhibitedin the species Selenoportax vexillarius (Pilgrim, 1937). A comparison betweenthe American and Pakistani specimens attributed to Selenoportax vexillarius,shows that the indices of the molars do not differ and the molars are very similarin dimensions and morphology (Table 1). From the above comparison, we. attribute the PUPC 86/241, PUPC 04/28, PUPC 96/44, PUPC 86/20 and PUPC83/644 to the species Selenoportax vexillarius.
Boselaphines are known from Neogene rocks and Selenoportax is onlyknown from the Neogene rocks of the Siwaliks whereas Pachyportax is alsoknown from the late Miocene of Abu Dahbi and Saudi Arabia (Gentry, 1999).Small sized Boselaphines are rare in the late Miocene strata, however, somefossils are found in the late Miocene rocks. Such small species probably werepresent in the late Miocene, but hitherto unrepresented for reasons of taphonomyor COllecting bias (Gentry & Heinzmann, 1996).
The large Boselaphine Selenoportax are found abundantly in the MiddleSiwaliks of the subcontinent (Pilgrim, 1937, 1939; Akhtar, 1996 and Khan et aI.,2006, 2007), but one specimen has been recovered. in the sediments of theUpper Siwaliks (Akhtar, 1992). This is based on lithostratigraphic position of thespecimen. Some uncertain occurrences are mentioned out of the Siwaliks, e.g.,Solounias (1981) referred a Selenoportax horn core from Pikermi; according tohim the material is not diagnostic enough for a more specific determination.Nevertheless, the Selenoportax fauna suggests a relative stability of theecosystem in the Middle Siwaliks for the Boselaphines, especially for the largesized Boselaphines, Selenoportax and Pachyportax. At the end of the Middle. Siwaiiks period, Boselaphines disappeared from the Siwaliks and are todayrepresented by a single living species Nilgauy (Boselaphus tragocamelus) in theIndian Peninsula.
ACKNOWLEDGEMENTS
We are thankful to Mr. Altaf, Mr. Sajjad from the village Dhokpathan andMr. Anaar Khan from the village Hasnot for their assistance in field-work and thehospitality during our stay. We are also grateful to Mr. Maskeen Ali for his
efficient help during field-work. We offer special thanks to Mr. Nadeem Fazal forhis help in the photography.
REFERENCES
Akhtar, M., 1992. Taxonomy and Distribution of the Siwalik Bovids. PhD.disserlation (unpublished), University of the Punjab, Lahore, Pakistan.
Akhtar, M., 1996. A new species of the genus Selenoportax (Mammalia,Artiodactyla, Bovidae) from DhokPathan, district Chakwal, Punjab, Pakistan.Proc. Pak Congo Zool., 16: 91-96.
Bagati, T. N. & Kumar, R, 1994. Clay mineralogy of Middle Siwalik sequence inMohand area, Dehra Dun: implication for climate and source area. In: Kumar,R, Ghosh, S. K., Phadtare, N.R (Eds.), Siwalik Foreland Basin of Himalaya.Himalayan Geology, 15: 219-228.
Behrensmeyer, A K. & Tauxe, L, 1982. Isochronous fluvial systems in Miocenedeposits of northern Pakistan. Sedimentology, 29: 331-352.
Bhattacharya, N., 1970. Clay mineralogy and trace element geochemistry ofSubathu, Dharmsala and Siwalik sediments in Himalayan foothills ofnorthwest India. J Geol. Soc. India, 11: 309-332.
Bhattacharya, N., & Misra, S. S., 1963. Petrology and sedimentation of MiddleSiwalik clays at Dhokhand, Saharanpur district U.P. India. Beit. Zur Miner.Und Petgr, 9 139-147.
Biswas, S. K., 1994. Status of exploration for hydrocarbons in Siwalik Basin ofIndia and future trends. In: Symposium on Siwalik Basin, 1991. GeologicalSociety of India, Dehra Dun. pp. 283-300
Chaudhri, R S & Gill, G. T. S., 1983. Clay mineralogy of the Siwalik Group ofSimla Hills, northwestern Himalaya. J Geol Soc. India, 24: 159-165
Gentry, A W., 1999. Fossil Pecorans from the Baynunah Formation, Emirate ofAbu Dhabi, United Arab Emirates. Chap. 22 in :Abu Dhabi pecorans, YaleUniversity Press, New Haven, pp. 290-316.
Gentry, A W., & Heinzmann, E. P. J., 1996. Miocene ruminants of the Centraland Eastern Tethys and Paratethys, In: The evolution of Western EurasianNeogene Mammal Faunas (eds Bernor, R L, Fahlbusch, V. & Mittmann, HW) Columbia University Press, New York, pp 378-391.
Khan, M. A, 2008. Fossil bovids from the late Miocene of Padri, Jhelum,Pakistan. Pak. J Zool. 40(1): 25-29.
Khan, M. A, Khan, A M, Farooq, U. & Akhtar, M., 2006. Selenoportax vexillarius(Mammalia, Boselaphini, Bovidae) from the Siwaliks. Pak. J Zool 38(4):321-326.
Khan, M. A, Ghaffar, A, Farooq, U., & Akhtar, M, 2007. New fossil remains ofSelenoportax vexillarius from the late Miocene of Hasnot Pak. J Zool. 39(5):333-338.Pilgrim, G. E, 1937. Siwalik antelopes and oxen in the American Museum of
Natural History. Bull. Amer. Mus. Nat His!., 72: 729-874.Pilgrrm, G. E, 1939. The fossil Bovidae of India. Pal. Ind, NS., 26(1): 1-356.
II"
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I
II
I:
I
I
70 M. A. KHAN ET AL BIOLOGIA VOL 54 (1) SELENOPORTAX VEXILLARIUS FROM DHOKPATHAN, PAKISTAN 71
DISCUSSION
The specimens under study have moderate goat folds and advancedmedian basal, pillars that confirm their inclusion in the genus Selenoportax. Thegenus has three species, Selenoportax vexillarius, Selenoportax dhokgaalensis,Selenoportax Iydekkeri, found in the Middle Siwaliks of Pakistan. TheSelenoportax Iydekkeri is larger one and has strong divergent stYles (Pilgrim,1939). The described dental material is moderate in size and has no divergentstyles, which confirms that the material belongs to the species Selenoportaxvexillarius. The specimens were metrically compared to the type specimens atAmerican Museum of Natural History and found resemblance in theirmeasurements (Table 1). The specimens, with divergent styles, median ribs welldeveloped, median basal pillar strongly developed in upper molars andmoderately developed in lower molars, enamel largely rugose and the crownnarrow at the base and broad at the apex, show all the characteristics exhibitedin the species Selenoportax vexillarius (Pilgrim, 1937). A comparison betweenthe American and Pakistani specimens attributed to Selenoportax vexillarius,shows that the indices of the molars do not differ and the molars are very similarin dimensions and morphology (Table 1). From the above comparison, we. attribute the PUPC 86/241, PUPC 04/28, PUPC 96/44, PUPC 86/20 and PUPC83/644 to the species Selenoportax vexillarius.
Boselaphines are known from Neogene rocks and Selenoportax is onlyknown from the Neogene rocks of the Siwaliks whereas Pachyportax is alsoknown from the late Miocene of Abu Dahbi and Saudi Arabia (Gentry, 1999).Small sized Boselaphines are rare in the late Miocene strata, however, somefossils are found in the late Miocene rocks. Such small species probably werepresent in the late Miocene, but hitherto unrepresented for reasons of taphonomyor COllecting bias (Gentry & Heinzmann, 1996).
The large Boselaphine Selenoportax are found abundantly in the MiddleSiwaliks of the subcontinent (Pilgrim, 1937, 1939; Akhtar, 1996 and Khan et aI.,2006, 2007), but one specimen has been recovered. in the sediments of theUpper Siwaliks (Akhtar, 1992). This is based on lithostratigraphic position of thespecimen. Some uncertain occurrences are mentioned out of the Siwaliks, e.g.,Solounias (1981) referred a Selenoportax horn core from Pikermi; according tohim the material is not diagnostic enough for a more specific determination.Nevertheless, the Selenoportax fauna suggests a relative stability of theecosystem in the Middle Siwaliks for the Boselaphines, especially for the largesized Boselaphines, Selenoportax and Pachyportax. At the end of the Middle. Siwaiiks period, Boselaphines disappeared from the Siwaliks and are todayrepresented by a single living species Nilgauy (Boselaphus tragocamelus) in theIndian Peninsula.
ACKNOWLEDGEMENTS
We are thankful to Mr. Altaf, Mr. Sajjad from the village Dhokpathan andMr. Anaar Khan from the village Hasnot for their assistance in field-work and thehospitality during our stay. We are also grateful to Mr. Maskeen Ali for his
efficient help during field-work. We offer special thanks to Mr. Nadeem Fazal forhis help in the photography.
REFERENCES
Akhtar, M., 1992. Taxonomy and Distribution of the Siwalik Bovids. PhD.disserlation (unpublished), University of the Punjab, Lahore, Pakistan.
Akhtar, M., 1996. A new species of the genus Selenoportax (Mammalia,Artiodactyla, Bovidae) from DhokPathan, district Chakwal, Punjab, Pakistan.Proc. Pak Congo Zool., 16: 91-96.
Bagati, T. N. & Kumar, R, 1994. Clay mineralogy of Middle Siwalik sequence inMohand area, Dehra Dun: implication for climate and source area. In: Kumar,R, Ghosh, S. K., Phadtare, N.R (Eds.), Siwalik Foreland Basin of Himalaya.Himalayan Geology, 15: 219-228.
Behrensmeyer, A K. & Tauxe, L, 1982. Isochronous fluvial systems in Miocenedeposits of northern Pakistan. Sedimentology, 29: 331-352.
Bhattacharya, N., 1970. Clay mineralogy and trace element geochemistry ofSubathu, Dharmsala and Siwalik sediments in Himalayan foothills ofnorthwest India. J Geol. Soc. India, 11: 309-332.
Bhattacharya, N., & Misra, S. S., 1963. Petrology and sedimentation of MiddleSiwalik clays at Dhokhand, Saharanpur district U.P. India. Beit. Zur Miner.Und Petgr, 9 139-147.
Biswas, S. K., 1994. Status of exploration for hydrocarbons in Siwalik Basin ofIndia and future trends. In: Symposium on Siwalik Basin, 1991. GeologicalSociety of India, Dehra Dun. pp. 283-300
Chaudhri, R S & Gill, G. T. S., 1983. Clay mineralogy of the Siwalik Group ofSimla Hills, northwestern Himalaya. J Geol Soc. India, 24: 159-165
Gentry, A W., 1999. Fossil Pecorans from the Baynunah Formation, Emirate ofAbu Dhabi, United Arab Emirates. Chap. 22 in :Abu Dhabi pecorans, YaleUniversity Press, New Haven, pp. 290-316.
Gentry, A W., & Heinzmann, E. P. J., 1996. Miocene ruminants of the Centraland Eastern Tethys and Paratethys, In: The evolution of Western EurasianNeogene Mammal Faunas (eds Bernor, R L, Fahlbusch, V. & Mittmann, HW) Columbia University Press, New York, pp 378-391.
Khan, M. A, 2008. Fossil bovids from the late Miocene of Padri, Jhelum,Pakistan. Pak. J Zool. 40(1): 25-29.
Khan, M. A, Khan, A M, Farooq, U. & Akhtar, M., 2006. Selenoportax vexillarius(Mammalia, Boselaphini, Bovidae) from the Siwaliks. Pak. J Zool 38(4):321-326.
Khan, M. A, Ghaffar, A, Farooq, U., & Akhtar, M, 2007. New fossil remains ofSelenoportax vexillarius from the late Miocene of Hasnot Pak. J Zool. 39(5):333-338.Pilgrim, G. E, 1937. Siwalik antelopes and oxen in the American Museum of
Natural History. Bull. Amer. Mus. Nat His!., 72: 729-874.Pilgrrm, G. E, 1939. The fossil Bovidae of India. Pal. Ind, NS., 26(1): 1-356.
II"
II
I
I
II
I:
I
I
72 M. A. KHAN ET AL. BIOLOGIA . BIOLOGIA(PAKISTAN) 2008,54 (1). 73.76PK ISSN 0006 - 3096
III, II ,
I
1>
Quade, J. & Cerling, T. E., 1995. Expansion of C, grasses in the Late Miocene ofNorthern Pakistan: evidence .from stable isotopes in: paleosoles.Palaeogeography, Palaeoclimatology, Palaeoecology, 115: 91-116 .
. Raiverman, V., 2002. Foreland Sedimentation in Himalayan Tectonic Regime, In:A Relook At The Orogenic Process. Bishen Singh Mahendra, Pal SinghPublishers, Dehra Dun, India, 378pp.
Raiverman, V. & Suresh, N., 1997. Clay mineral distribution in the Cenozoicsequence of the western Himalayan Foothills. J. Indian Assoc. Sediment.,16: 63-75.
Solounias, N., 1981. The turolian fauna from the Island of Samos, Greece; withspecial emphasis on the hyaenids and the bovids. Cont. Vert. Evo!., 6: 1-248.
Thomas, H., 1984. Les bovides ante-hipparions des Siwaliks inferieurs (plateaudu Potwar), Pakistan. Memoires de la Societe Geologique de France, 145: 1-68.
(..
•
An assessment of sulphate ions (8042.) in the fog samples of rural'and urban areas of Gujranwala
AMIR SHAHZAD, MUHHAMAD WASEEM MUMTAZ, HAMID MUKHTAR,MUHAMMAD ADIL YAQUB & GHULAM MUSTAFA
Dep0rll1lenJ (~lChemjslry, GC University, Lahore, Pakistan (AS, A1WM,MAY. GM)
IIH/illlle o/lnc/lIslriu/ Biotechnology, GC University, Lohore. Pakistan (HM)
ABSTRACT
The amount of sulphate ions (SO.'") present in the fog samples, collected bydifferent techniques, has been calculated. Gravimetric method was applied bywhich the sulphate ions in acidified sample solution were made to react. withBarium chloride solution to form Barium sulphate. From this Barium sulphate, theamount of sulphate ions was determined. The analytical data showed that theconcentration of sulphate ions varied from 12.34 to 152.50 ppm in the collectedsamples. The main objective was to determine the atmospheric pollution load'caused by the sulphate ions. Concentration of SO/- ions was higher in urbanareas than in rural ones.
Key words: Fog, Sulphate ions, Determination, Atmospheric pollution
INTRODUCTION
The atmosphere can be regarded as a blanket of gases surrounding theearth which sustains life on earth and save it from the hostile environment ofouter space. It absorbs most of the cosmic rays from outer space and a majorportion of electromagnetic radiation from the sun. The atmosphere plays a key. role in maintaining the heat balance of the earth. It is a source of oxygen, carbondioxide and nitrogen, which nitrogen fixing bacteria utilize to yield chemicallybound nitrogen essential for life. Furthermore, it is a vital carrier of water fromoceans to land as part of the hydrologic cycle (Dix, 1997). Unfortunately, withprogress in science and technology, man .has been dumping waste materials intothe atmosphere, which are posing a problem for survival of mankind itself on theearth (Anil, 2001)
Fog is a cloud comprising water droplets or less commonly ice crystalsformed near the ground, resulting in reduction in visibility. It is the result ofcondensation and subsequent formation of water droplets or ice crystals in theatmosphere. Formation of fog involves 3 main factors: optimum temperature forcondensation, sufficient humidity and condensation nuclei. Fogs of all typesOCCUI' when temperature and dew point of air become identical, provided thatsufficient condensation nuclei are available (Chiras & Deniel, 1994).Condensation nuclei are suspended particles, either solid or liquid. Their
IIIi
72 M. A. KHAN ET AL. BIOLOGIA . BIOLOGIA(PAKISTAN) 2008,54 (1). 73.76PK ISSN 0006 - 3096
III, II ,
I
1>
Quade, J. & Cerling, T. E., 1995. Expansion of C, grasses in the Late Miocene ofNorthern Pakistan: evidence .from stable isotopes in: paleosoles.Palaeogeography, Palaeoclimatology, Palaeoecology, 115: 91-116 .
. Raiverman, V., 2002. Foreland Sedimentation in Himalayan Tectonic Regime, In:A Relook At The Orogenic Process. Bishen Singh Mahendra, Pal SinghPublishers, Dehra Dun, India, 378pp.
Raiverman, V. & Suresh, N., 1997. Clay mineral distribution in the Cenozoicsequence of the western Himalayan Foothills. J. Indian Assoc. Sediment.,16: 63-75.
Solounias, N., 1981. The turolian fauna from the Island of Samos, Greece; withspecial emphasis on the hyaenids and the bovids. Cont. Vert. Evo!., 6: 1-248.
Thomas, H., 1984. Les bovides ante-hipparions des Siwaliks inferieurs (plateaudu Potwar), Pakistan. Memoires de la Societe Geologique de France, 145: 1-68.
(..
•
An assessment of sulphate ions (8042.) in the fog samples of rural'and urban areas of Gujranwala
AMIR SHAHZAD, MUHHAMAD WASEEM MUMTAZ, HAMID MUKHTAR,MUHAMMAD ADIL YAQUB & GHULAM MUSTAFA
Dep0rll1lenJ (~lChemjslry, GC University, Lahore, Pakistan (AS, A1WM,MAY. GM)
IIH/illlle o/lnc/lIslriu/ Biotechnology, GC University, Lohore. Pakistan (HM)
ABSTRACT
The amount of sulphate ions (SO.'") present in the fog samples, collected bydifferent techniques, has been calculated. Gravimetric method was applied bywhich the sulphate ions in acidified sample solution were made to react. withBarium chloride solution to form Barium sulphate. From this Barium sulphate, theamount of sulphate ions was determined. The analytical data showed that theconcentration of sulphate ions varied from 12.34 to 152.50 ppm in the collectedsamples. The main objective was to determine the atmospheric pollution load'caused by the sulphate ions. Concentration of SO/- ions was higher in urbanareas than in rural ones.
Key words: Fog, Sulphate ions, Determination, Atmospheric pollution
INTRODUCTION
The atmosphere can be regarded as a blanket of gases surrounding theearth which sustains life on earth and save it from the hostile environment ofouter space. It absorbs most of the cosmic rays from outer space and a majorportion of electromagnetic radiation from the sun. The atmosphere plays a key. role in maintaining the heat balance of the earth. It is a source of oxygen, carbondioxide and nitrogen, which nitrogen fixing bacteria utilize to yield chemicallybound nitrogen essential for life. Furthermore, it is a vital carrier of water fromoceans to land as part of the hydrologic cycle (Dix, 1997). Unfortunately, withprogress in science and technology, man .has been dumping waste materials intothe atmosphere, which are posing a problem for survival of mankind itself on theearth (Anil, 2001)
Fog is a cloud comprising water droplets or less commonly ice crystalsformed near the ground, resulting in reduction in visibility. It is the result ofcondensation and subsequent formation of water droplets or ice crystals in theatmosphere. Formation of fog involves 3 main factors: optimum temperature forcondensation, sufficient humidity and condensation nuclei. Fogs of all typesOCCUI' when temperature and dew point of air become identical, provided thatsufficient condensation nuclei are available (Chiras & Deniel, 1994).Condensation nuclei are suspended particles, either solid or liquid. Their
IIIi
MATERIALS AND METHODS
The concentration of inorganic sulphate ion (50/') was determinedgravimetrically from fog samples collected from the different areas of districtGujranwala, including Kandan Sian, Bubban Sundwa, Liaqat Bagh, GulshanIqbal,'Wapda Town and Peoples Colony.
. Sample Collection
Samples were collected by the method as adopted by Department ofAtmospheric Science, Colorado University, Colorado, U.S.A (AtmosphereChemistryl Air Quality Program, 2000) and Stachelin (1997) In this method,clean and dried aluminum foils and polythene sheets were spread on the groundin the form of an inverted canopy and fog water was collected. It was stored inthoroughly washed reagent bottles at DoC.
\"\ ., \
75
RESULTS AND DISCUSSION
ASSESSMENT OF SULPHATE IONS (sot) IN THE FOG SAMPLESVOL 54 (1)
.....
SO; SO; 50/ . ;...~ .•tt. .Sr, S;lmpling sites . "S!i1riClartlNo. (Rural or (ppm) (ppm) (ppm) deviaii~ii
Urban) Maximum Minimum Meanvalue value value
1 Kandan Sian 53.50 12.34 28.22 . ' 13,09,.~(Rural)c" ,...,.:. .~,; >'.
2 Bubban Sundwa 53.50 20.57 38.25 10,93(Rural)
3 Liaqat Bagh 11110 69.64 90.53 1356, "(Urban)
4 Gulshan Iqbal 54.50 32.92 42.95 2.65(Urban)
5 Wapda Town 98.76 70.00 84.07 847(Urban).
6 Poeples Colony 152.25 11522 13463 11.63(Urban)
.,.~
Table 1: Concentration of sulphate ion (SO;') in fog samples at various" :sites in district Gujranwala,
The analytical data showed that the concentration of sulphate ions varied. from 12.34 to 152.25 ppm in the collected samples. This variation inconcentration is from rural to urban areas. Therefore, it is quite obvious that theconcentration of sulphate ions, which .is considered nucleus for fog formation,was' higher in urban areas than in rural areas (Table 1).
The higher concentration of sulphate ions in the urban areas is due toanthropogenic activities leading towards the concentration of the Sulphur dioxidealong with other aerosols in the atmosphere. It was also noted that the maximumvalues were attributed to the wind currents because the chemistry of atmosphereaerosol is also deeply embedded in dynamic phenomena of wind.
BIOLOGIAA. SHAHZAD ET AL.74
Determination of (SO;') in fog samples
The sample of water was filtered through filter paper (Whatman No. 42) toremove the dust particles. After filtration, 100 ml of the filtered sample was takeninto 3 250 ml conical flak and boiled by heating and 1.5 ml of HCI solution wasadded to the conical flask to make the medium acidic. Then 50 ml of boilingBaCl, solution was added to hot acidified sample solution containing sulph,ateions. Barium (Ba
2+) and sulphate ions (50/') reacted to form Barium sulphate
(BaS04). Precipitates, thus formed, were collected by filteration and washed withdistilled water until the washing was free of chloride ions (0) as indicated bytesting with AgN031 HN03 solution. The precipitates thus obtained were dried inoven equipped with thermostat and the amount of sulphate ions (50/') wascalculated using the following formula:
50,2' (mgll) = BaSO, (mg) x 4116
Sample (ml)The amount of BaSO, was calculated from difference in weight of filter paperbefore and after filtration.
diameters range from a few microns to few tenths of a micron. There are muchsmaller nuclei in the atmosphere, called Aitkin nuclei.
Nuclei that h.ave diameters of several microns and are composed ofhygroscopic or moisture attracting substances (e.g. sea salt) are called giantcondensation nuclei (Harrison & Grilker, 1998). The nature of the condensationnuclei originates from the chemistry of atmosphere. The atmosphere aerosols. originate both from physical as well as chemical processes. Dispersion aerosols,such as dust formed from the disintegration of larger particles, are usually abovemicron in size. From condensation, aerosols such as nitrate (NO;), nitrite (NO,")and sulphate ions (50/) play vital role in the formation of fog by providingcondensation nuclei (Anil, 2001),
MATERIALS AND METHODS
The concentration of inorganic sulphate ion (50/') was determinedgravimetrically from fog samples collected from the different areas of districtGujranwala, including Kandan Sian, Bubban Sundwa, Liaqat Bagh, GulshanIqbal,'Wapda Town and Peoples Colony.
. Sample Collection
Samples were collected by the method as adopted by Department ofAtmospheric Science, Colorado University, Colorado, U.S.A (AtmosphereChemistryl Air Quality Program, 2000) and Stachelin (1997) In this method,clean and dried aluminum foils and polythene sheets were spread on the groundin the form of an inverted canopy and fog water was collected. It was stored inthoroughly washed reagent bottles at DoC.
\"\ ., \
75
RESULTS AND DISCUSSION
ASSESSMENT OF SULPHATE IONS (sot) IN THE FOG SAMPLESVOL 54 (1)
.....
SO; SO; 50/ . ;...~ .•tt. .Sr, S;lmpling sites . "S!i1riClartlNo. (Rural or (ppm) (ppm) (ppm) deviaii~ii
Urban) Maximum Minimum Meanvalue value value
1 Kandan Sian 53.50 12.34 28.22 . ' 13,09,.~(Rural)c" ,...,.:. .~,; >'.
2 Bubban Sundwa 53.50 20.57 38.25 10,93(Rural)
3 Liaqat Bagh 11110 69.64 90.53 1356, "(Urban)
4 Gulshan Iqbal 54.50 32.92 42.95 2.65(Urban)
5 Wapda Town 98.76 70.00 84.07 847(Urban).
6 Poeples Colony 152.25 11522 13463 11.63(Urban)
.,.~
Table 1: Concentration of sulphate ion (SO;') in fog samples at various" :sites in district Gujranwala,
The analytical data showed that the concentration of sulphate ions varied. from 12.34 to 152.25 ppm in the collected samples. This variation inconcentration is from rural to urban areas. Therefore, it is quite obvious that theconcentration of sulphate ions, which .is considered nucleus for fog formation,was' higher in urban areas than in rural areas (Table 1).
The higher concentration of sulphate ions in the urban areas is due toanthropogenic activities leading towards the concentration of the Sulphur dioxidealong with other aerosols in the atmosphere. It was also noted that the maximumvalues were attributed to the wind currents because the chemistry of atmosphereaerosol is also deeply embedded in dynamic phenomena of wind.
BIOLOGIAA. SHAHZAD ET AL.74
Determination of (SO;') in fog samples
The sample of water was filtered through filter paper (Whatman No. 42) toremove the dust particles. After filtration, 100 ml of the filtered sample was takeninto 3 250 ml conical flak and boiled by heating and 1.5 ml of HCI solution wasadded to the conical flask to make the medium acidic. Then 50 ml of boilingBaCl, solution was added to hot acidified sample solution containing sulph,ateions. Barium (Ba
2+) and sulphate ions (50/') reacted to form Barium sulphate
(BaS04). Precipitates, thus formed, were collected by filteration and washed withdistilled water until the washing was free of chloride ions (0) as indicated bytesting with AgN031 HN03 solution. The precipitates thus obtained were dried inoven equipped with thermostat and the amount of sulphate ions (50/') wascalculated using the following formula:
50,2' (mgll) = BaSO, (mg) x 4116
Sample (ml)The amount of BaSO, was calculated from difference in weight of filter paperbefore and after filtration.
diameters range from a few microns to few tenths of a micron. There are muchsmaller nuclei in the atmosphere, called Aitkin nuclei.
Nuclei that h.ave diameters of several microns and are composed ofhygroscopic or moisture attracting substances (e.g. sea salt) are called giantcondensation nuclei (Harrison & Grilker, 1998). The nature of the condensationnuclei originates from the chemistry of atmosphere. The atmosphere aerosols. originate both from physical as well as chemical processes. Dispersion aerosols,such as dust formed from the disintegration of larger particles, are usually abovemicron in size. From condensation, aerosols such as nitrate (NO;), nitrite (NO,")and sulphate ions (50/) play vital role in the formation of fog by providingcondensation nuclei (Anil, 2001),
REFERENCES
.~-~'")..~;.•.
ABSTRACT
A thorough survey of the Punjab, Pakistan was done to explore the mitefauna of the family Cunaxidae. For collection at the spot, sieve collection methodwas adopted. The mites received on the paper were sorted out with the help of a
MATERIALS AND METHODS
INTRODUCTION
Key words: New species, Pseudobonzia ashfaqi. Cunaxidae, Punjab, Pakistan
Adult female of a new cunaxid mite, Pseudobonzia ashfaqi, was collectedduring the taxonomic exploration of the mite fauna of the family Cunaxidae fromPunjab, Pakistan. This species was compared with three already describedspecies of this genus from Pakistan. Illustrations and description of this newspecies, alongwith its distribution and distinguishing remarks, are also ,given.
Department of Agri. Entomology, University of Agriculture. Faisalabad,Pakistan (MHB, MA)
Department a/Zoology, GC University. Lahore. Pakistan (SA)
BIOLOGIA(PAKISTAN) 2008,54 (1), 77-82PK ISSN 0006 - 3096
A new species of genus Pseudobonzia Smiley (Prostigmata: Acari)from Punjab, Pakistan
MUHAMMAD HAMID BASHtR, MUHAMMAD AFZAL & SHAMSHAD AKBAR
Members of the family Cunaxidae are important predators of other harmfulmites and small soft bodied insects (Smiley, 1992). Many members of this familyare known to predate upon different mites, e.g., Cunaxoides oliveri predatesupon grape vine mite Cleptitremerus vitus (Schruft, 1971), Coleoscirus. simplex
. feeds on rootknot nematodes (Meloidogyne spp) (Walter & Kaplan, 1991). They. have diverse types of habitats and are reported from different parts of the world(Arbabi et al., 2002; Tagore & Putatunda, 2003 and de-Oliveria & Daemon,
. 2003)
The genus Pseudobonzia was erected by Smiley in 1975 with Cunaxareticulata Heryford as its type species. Later on Chaudhri (1977), Chaudhri et at.(1979), den Heyer (1977, 1980), Luxton (1982), Liang (1983, 1984);Sepasgosarian (1984) and Smiley (1992) made significant contribution to thefauna of this family world over.
From Pakistan, 4 species had been described previously. The authors havenow described and illustrated one new species of this genus, thus making a total. of 5 species of genus Pseudobonzia from Pakistan.
BIOLOGIAA. SHAHZAD ET AL.76
Anil, K: D, 2001.Environmental Chemistry, Wiley Eastern Ltd, 2"' ed, pp 3, 4, 6,12.
Atmoshpere Chemistryl Air Quality Program, 2000. Atmoshpere ScienceDepartment, Colorado State University, Fort Collins, Colorado, U. S. A.
Chiras, & Dahiel, D., 1994. Environmental Science, Wordswort~ PublishingCompany, 5th ed. pp 223-224.
oix. H. M., 1997. Environmental Pollution, 1" ed. John Wiley & Sons, New York,USA
Harrison. R. M. & Grilker, R. V., 1998. Atmoshpere Particles, John Wiley,Chichester. U. K.
Stachelin, J., 1997. Collection of winter precipitation at Mont Rigi, An overview.Water, Air and Soil Pollution, 68: 1-14.
The highest concentration of sulphate ions was recorded in the samplescollected from People Colony, Gujranwala which can be attributed to a number offactors: Firstly, presence.of factories in the peripheral areas of People Colony,where there is high consumption of furnace oil and other fuels, which results inthe ,.re'lease of Sulphur dioxide along with other aerosols in the atmosphere.secori'dly', Peoples Colony is situated about 1 KM from GT Road and is adjacentto by-pass where there is a large flow of heavy traffic coming from Rawalpindiand Sialkol. This contributes heavily towards the addition of sulphate ions.Thirdly, the main source of public transportation from the city to the PeopleColony is "Motorcycle Rikshaw". The main railway-line passes near the entranceof Peoples Colony, due to which, most of the time, the traffic remains blocked.which results in considerable increase of CO2, CO and S02 in the atmospheredue to emission from these rikshaws. Lastly, the dense fog in this area can alsobe attributed to the presence of a canal link with Upper Chenab on the easternside of ~y-pass which is a source of humidity.
,I
REFERENCES
.~-~'")..~;.•.
ABSTRACT
A thorough survey of the Punjab, Pakistan was done to explore the mitefauna of the family Cunaxidae. For collection at the spot, sieve collection methodwas adopted. The mites received on the paper were sorted out with the help of a
MATERIALS AND METHODS
INTRODUCTION
Key words: New species, Pseudobonzia ashfaqi. Cunaxidae, Punjab, Pakistan
Adult female of a new cunaxid mite, Pseudobonzia ashfaqi, was collectedduring the taxonomic exploration of the mite fauna of the family Cunaxidae fromPunjab, Pakistan. This species was compared with three already describedspecies of this genus from Pakistan. Illustrations and description of this newspecies, alongwith its distribution and distinguishing remarks, are also ,given.
Department of Agri. Entomology, University of Agriculture. Faisalabad,Pakistan (MHB, MA)
Department a/Zoology, GC University. Lahore. Pakistan (SA)
BIOLOGIA(PAKISTAN) 2008,54 (1), 77-82PK ISSN 0006 - 3096
A new species of genus Pseudobonzia Smiley (Prostigmata: Acari)from Punjab, Pakistan
MUHAMMAD HAMID BASHtR, MUHAMMAD AFZAL & SHAMSHAD AKBAR
Members of the family Cunaxidae are important predators of other harmfulmites and small soft bodied insects (Smiley, 1992). Many members of this familyare known to predate upon different mites, e.g., Cunaxoides oliveri predatesupon grape vine mite Cleptitremerus vitus (Schruft, 1971), Coleoscirus. simplex
. feeds on rootknot nematodes (Meloidogyne spp) (Walter & Kaplan, 1991). They. have diverse types of habitats and are reported from different parts of the world(Arbabi et al., 2002; Tagore & Putatunda, 2003 and de-Oliveria & Daemon,
. 2003)
The genus Pseudobonzia was erected by Smiley in 1975 with Cunaxareticulata Heryford as its type species. Later on Chaudhri (1977), Chaudhri et at.(1979), den Heyer (1977, 1980), Luxton (1982), Liang (1983, 1984);Sepasgosarian (1984) and Smiley (1992) made significant contribution to thefauna of this family world over.
From Pakistan, 4 species had been described previously. The authors havenow described and illustrated one new species of this genus, thus making a total. of 5 species of genus Pseudobonzia from Pakistan.
BIOLOGIAA. SHAHZAD ET AL.76
Anil, K: D, 2001.Environmental Chemistry, Wiley Eastern Ltd, 2"' ed, pp 3, 4, 6,12.
Atmoshpere Chemistryl Air Quality Program, 2000. Atmoshpere ScienceDepartment, Colorado State University, Fort Collins, Colorado, U. S. A.
Chiras, & Dahiel, D., 1994. Environmental Science, Wordswort~ PublishingCompany, 5th ed. pp 223-224.
oix. H. M., 1997. Environmental Pollution, 1" ed. John Wiley & Sons, New York,USA
Harrison. R. M. & Grilker, R. V., 1998. Atmoshpere Particles, John Wiley,Chichester. U. K.
Stachelin, J., 1997. Collection of winter precipitation at Mont Rigi, An overview.Water, Air and Soil Pollution, 68: 1-14.
The highest concentration of sulphate ions was recorded in the samplescollected from People Colony, Gujranwala which can be attributed to a number offactors: Firstly, presence.of factories in the peripheral areas of People Colony,where there is high consumption of furnace oil and other fuels, which results inthe ,.re'lease of Sulphur dioxide along with other aerosols in the atmosphere.secori'dly', Peoples Colony is situated about 1 KM from GT Road and is adjacentto by-pass where there is a large flow of heavy traffic coming from Rawalpindiand Sialkol. This contributes heavily towards the addition of sulphate ions.Thirdly, the main source of public transportation from the city to the PeopleColony is "Motorcycle Rikshaw". The main railway-line passes near the entranceof Peoples Colony, due to which, most of the time, the traffic remains blocked.which results in considerable increase of CO2, CO and S02 in the atmospheredue to emission from these rikshaws. Lastly, the dense fog in this area can alsobe attributed to the presence of a canal link with Upper Chenab on the easternside of ~y-pass which is a source of humidity.
,I
hand lens and the mites of the family Cunaxidae were sorted in small vials'containing 70% alcohol with few drops of glycerin. The materials which could notbe processed in the field were brought to laboratory in small cellophane bags andprocessed through the Berlese's funnel for at least 24 hours. Cunaxid mites weresorted from the bulk collection and preserved in separate vials for further studies.Permanent mounts were prepared by using the Hoyer's medium. The mountedspecimens were examined under a high power phase contrast microscope.Sketches of different body parts were prepared by using the ocular grid. Thespecies was identified with the help of available literature and keys of Chaudhri(1977), Chaudhri et al. (1979) and Smiley (1992).
Following abbreviations have been used in this manuscript:
Asl attenuate solenidion
Bsl Blunt ended solenidion
Sts Simple tactile setae
T trichobothrium
Legs
Legs I-IV measuring (from trochanter base to the tip of tarsus) 185 J.lm, 175~m, 200 ~m and 205 ~, respectively. All legs pitted with papillae, without bilobedflanges .. Chaetotaxy of legs I-IV as follows: Coxae 3-3-3-3; trochanters 1-1-2-1;basifemora 4-5-4-0; telofemora 5-5-4-3; genua 9 (4 asl + 5 sts)-6-6-7, tibiae 7-6-6-5, tarsi 21 (bsl + 1 bsl + 14 sts)-20(1bsl + 19 sts)-15-14 (Fig. 1-F).
79NEW SPECIES OF GENUS PSEUDOBONZIA FROM PAKISTAN
Venter
VOL 54 (1)
Venter with dotted striations. Coxae I-II and III-IV contiguous, III-IV basebroader than coxae I-II base. Hysterosoma with 6 pairs, simple setae betweencoxae II and distal part of the body in addition to setae of genital and anal region.Genital shield with two valves having random lobe-like dots. each valve with 4simple genital setae (g,-g;v) in a row and 2 genital suckers. Anal setae (a,-all) 2pairs, para-anal setae (pa) 1 pair. One pair minute pores near anal shield (Fig. 1-B).
BIOLOGIAM. H. BASHIR ET AL.78
RESULTS AND DISCUSSION Male
Pseudobonzia ashfaqi, new species, (Fig. 1 A-F)
Not came in collection.
Type
Female
Gnathosoma
Holotype female, collected from Okara (Punjab) from leaf debris on 30-05-2004(Hamid) and deposited in Acarology Research Laboratory, Department of Agri.Entomology, University of Agriculture, Faisalabad, Pakistan.
Remarks
This species is named in honor of Prof. Dr. M. Ashfaq, for his outstandingcontributions in the field of Acarology in Pakistan.
This new species keys out closely .to Pseudobonzia numida Chaudhri on thebasis of different body characters but can be separated from it due to followingcharacters:
Gnathosoma 160 ~m long and 70~m wide. Hypostome sub-rectangular andcone shaped distally, with 4 pairs hypognathal setae (hg,-h94) (Fig. 1-E).
Palp 5 segmented, measuring 125~m. Chaetotaxy of palp is as follows:trochanter none; basifemur with 1 simple seta; telofemur with 1 simple seta:tibiotarsus terminating in a claw, with 6 simple setae and 1 small lateral medianthorn-like seta (Fig. 1-C). Chelicerae 125 ~m long, terminating in a claw, with 1dorso-medial simple seta, dorsal and ventral sides with lobes (Fig. 1-0).
Dorsum
Etymology ~
Body 340 ~m long (without gnathosoma) and 200 ~m wide. Propodosomawith a weakly sclerotized sub-rectangular shield bearing randomly scatteredpapillae. Propodosomal shield with sensillae PS, and PS2 measuring 115 ~m and100 ~m, respectively and propodosomal setae P, and P2 each measuring 20 ~m
Hysterosoma with papillate striae. Hysterosoma with setae 0,-0, measuring12 ~m, 12 ~m, 12 ~m, 20 ~m and 25 ~m, respectively; setae L,. L4 and L" each12 ~m long., simple. Hysterosomal integument with one pair of pores anterior tosetae 04 (Fig. 1-A)
1. Ventral hysterosoma with 7 pairs, simple setae between coxae II and distalpart of the body in addition to setae in anal and genital r1'gion in Pseudobonzianumida as against 6 pairs in this new species.
2 Chaetotaxy of legs I-IV in Pseudobonzia numida is: Coxae 3-3-3-2; basifemora5-6-5-2; genua 8-8-6-6; tibiae 6-6-5-5 and tarsi 20-20-19-19 as against: 3-3-3-3;4-5-4-0; 9-6-6-7; 7-6-6.5 and 21-20-15-14 on same segments in this newspecies.
-.•...1:1
hand lens and the mites of the family Cunaxidae were sorted in small vials'containing 70% alcohol with few drops of glycerin. The materials which could notbe processed in the field were brought to laboratory in small cellophane bags andprocessed through the Berlese's funnel for at least 24 hours. Cunaxid mites weresorted from the bulk collection and preserved in separate vials for further studies.Permanent mounts were prepared by using the Hoyer's medium. The mountedspecimens were examined under a high power phase contrast microscope.Sketches of different body parts were prepared by using the ocular grid. Thespecies was identified with the help of available literature and keys of Chaudhri(1977), Chaudhri et al. (1979) and Smiley (1992).
Following abbreviations have been used in this manuscript:
Asl attenuate solenidion
Bsl Blunt ended solenidion
Sts Simple tactile setae
T trichobothrium
Legs
Legs I-IV measuring (from trochanter base to the tip of tarsus) 185 J.lm, 175~m, 200 ~m and 205 ~, respectively. All legs pitted with papillae, without bilobedflanges .. Chaetotaxy of legs I-IV as follows: Coxae 3-3-3-3; trochanters 1-1-2-1;basifemora 4-5-4-0; telofemora 5-5-4-3; genua 9 (4 asl + 5 sts)-6-6-7, tibiae 7-6-6-5, tarsi 21 (bsl + 1 bsl + 14 sts)-20(1bsl + 19 sts)-15-14 (Fig. 1-F).
79NEW SPECIES OF GENUS PSEUDOBONZIA FROM PAKISTAN
Venter
VOL 54 (1)
Venter with dotted striations. Coxae I-II and III-IV contiguous, III-IV basebroader than coxae I-II base. Hysterosoma with 6 pairs, simple setae betweencoxae II and distal part of the body in addition to setae of genital and anal region.Genital shield with two valves having random lobe-like dots. each valve with 4simple genital setae (g,-g;v) in a row and 2 genital suckers. Anal setae (a,-all) 2pairs, para-anal setae (pa) 1 pair. One pair minute pores near anal shield (Fig. 1-B).
BIOLOGIAM. H. BASHIR ET AL.78
RESULTS AND DISCUSSION Male
Pseudobonzia ashfaqi, new species, (Fig. 1 A-F)
Not came in collection.
Type
Female
Gnathosoma
Holotype female, collected from Okara (Punjab) from leaf debris on 30-05-2004(Hamid) and deposited in Acarology Research Laboratory, Department of Agri.Entomology, University of Agriculture, Faisalabad, Pakistan.
Remarks
This species is named in honor of Prof. Dr. M. Ashfaq, for his outstandingcontributions in the field of Acarology in Pakistan.
This new species keys out closely .to Pseudobonzia numida Chaudhri on thebasis of different body characters but can be separated from it due to followingcharacters:
Gnathosoma 160 ~m long and 70~m wide. Hypostome sub-rectangular andcone shaped distally, with 4 pairs hypognathal setae (hg,-h94) (Fig. 1-E).
Palp 5 segmented, measuring 125~m. Chaetotaxy of palp is as follows:trochanter none; basifemur with 1 simple seta; telofemur with 1 simple seta:tibiotarsus terminating in a claw, with 6 simple setae and 1 small lateral medianthorn-like seta (Fig. 1-C). Chelicerae 125 ~m long, terminating in a claw, with 1dorso-medial simple seta, dorsal and ventral sides with lobes (Fig. 1-0).
Dorsum
Etymology ~
Body 340 ~m long (without gnathosoma) and 200 ~m wide. Propodosomawith a weakly sclerotized sub-rectangular shield bearing randomly scatteredpapillae. Propodosomal shield with sensillae PS, and PS2 measuring 115 ~m and100 ~m, respectively and propodosomal setae P, and P2 each measuring 20 ~m
Hysterosoma with papillate striae. Hysterosoma with setae 0,-0, measuring12 ~m, 12 ~m, 12 ~m, 20 ~m and 25 ~m, respectively; setae L,. L4 and L" each12 ~m long., simple. Hysterosomal integument with one pair of pores anterior tosetae 04 (Fig. 1-A)
1. Ventral hysterosoma with 7 pairs, simple setae between coxae II and distalpart of the body in addition to setae in anal and genital r1'gion in Pseudobonzianumida as against 6 pairs in this new species.
2 Chaetotaxy of legs I-IV in Pseudobonzia numida is: Coxae 3-3-3-2; basifemora5-6-5-2; genua 8-8-6-6; tibiae 6-6-5-5 and tarsi 20-20-19-19 as against: 3-3-3-3;4-5-4-0; 9-6-6-7; 7-6-6.5 and 21-20-15-14 on same segments in this newspecies.
-.•...1:1
•
I,
81NEW SPECIES OF GENUS PSEUDOBONZIA FROM PAKISTANVOL. 54 (1)
REFERENCES
1. Palp tibiotarsus with 5 setae and a small spine-like seta in P.newzealandicus while palp tibiotarsus with 6 setae and a small tuberclein this new species
2, Ventral hysterosoma with 5 pairs of simple set~e adjacent to lateralplates and distal part of body in addition to seiae of anal and genitalregion in P. newzea/andicus as against 6 pairs in this new species.
3. Setal counts on legs I-IV differ in both the species.
Arbabi, M., Golmohammadzadeh, N, K. & Askari, M., 2002. Plant mite fauna ofSistan-Baluchestan and Hormozgan provinces. J. Entomol Soci. Iran, 22(1):87-105
Chaudhri, W. M., 1977. Description of the mite of the family Cunaxidae (Acarina)from Pakistan, Pak, J. Agric. Sci., 14: 41-52 .
. Chaudhri, W. M., Akbar, S. & Rasool, A., 1979. Studies on the predatory leafinhabiting mites of Pakistan ..Monograph" NO.2: Univ. of Agri., Faisalabad.234pp
de-Oliveria, A. J. & Daemon, E., 2003. Qualitative and quantitative assessmentof mites (Acari) in domiciliary dust in rural dwellings in the "Zona da Mata"Region, Minas Gerias, Brazil. Revista-Brazifeira-de-Zooi., 20(4): 675.679 .
den Heyer, J., 1977. A new genus Neosciru/a (Cunaxidae: Prostigmata: Acari)from the Ethiopian region. Jour. Enlomo/, Soc. S, Afr., 40(1): 73-86.
den Heyer, J., 1980. Sculopalpus, a new cunaxid genus from the EthiopianRegion (Prostigmata: Acari). Acar%gia, 21(2): 187-193.
Liang, G, W., 1983. Notes on four species of mites (Acarina: Cunaxidae) inChina Agric. Bur, Guangdong Prov., 5(2): 104-107.
Liang, G.W., 1984, A new species and a new record of the genus Pseudobonziafrom China. Acla Zoolaxonomica Sinica, 9: 49-51,
'Luxton, M., 1982. Some new species of mites from New Zealand peat soils. N Z.J Zool. 9: 325-332,
Schruft, G., 1971. Haleupa/us oliveri new species, a "Thorn-palped" mite ongrape vines (Vitis spp.) Acari: Cunaxidae. Disch. Entomol. z., 18(4-5): 379-382.
. Sepasgosarian, H., 1984. The world genera and species of the family Cunaxidae(Actinedida: Acarida). Zeif, Ang. Zool. 71: 135-150.
Smiley, R L., 1975, A generic revision of the mites of the family Cunaxidae(Acarina). Ann. Entomol. Soc. Am, 68(2): 227-244.
Smiley, R L., 1992. The Predatory Mite Family Cunaxidae (Acari) Of The WorldWith A New Classification. Indira Publishing House, Michigan, U. S. A.356pp
. This new species comes closer to P, newzea/andicus Smiley, however, differsfrom it on the basis of following characters:
BIOLOGIAM. H. BASHIR ET AL
"
l.l~ f{, [;'1j': ;)1. .....•.....
!.'f.
JiB!f);" J'ff': ..<. •
III IV
Fig. 1: Pseudobonzia ashfaqi, n. sp.
A- Dorsal side; B-Ventral side; C-Palp; D-Chelicera; E-Hypostome;F-Iegs '-IV
\ i .!:t.~ ~ ~'l(.' D &
This new species can also be separated from Pseudobonzia reliculata(Heryford) on the basis of following characters:
1. Palp. tibiotarsus without thorn-like seta in Pseudobonzia reticulata but with athorn-like seta in this new species.
2. Propodosomal shield and coxal plates are reticulated in Pseudobonziareticulata while pitted in this new species.
80
3. Leg segments I-IV basifemora; telofemora; genua; tibiae and tarsi with 3-3-2-1;6-5-4-3; 9-7-6-6; 7-6-6-6 and 27-21-18-19, setae, respectively, in Pseudobonziareticulala as against 4-5-4-0; 5-5-4-3; 9-6-6-7; 7-6-6-5 and 21-20-15-14 setae,respectively, in this new species,
~\.
•
I,
81NEW SPECIES OF GENUS PSEUDOBONZIA FROM PAKISTANVOL. 54 (1)
REFERENCES
1. Palp tibiotarsus with 5 setae and a small spine-like seta in P.newzealandicus while palp tibiotarsus with 6 setae and a small tuberclein this new species
2, Ventral hysterosoma with 5 pairs of simple set~e adjacent to lateralplates and distal part of body in addition to seiae of anal and genitalregion in P. newzea/andicus as against 6 pairs in this new species.
3. Setal counts on legs I-IV differ in both the species.
Arbabi, M., Golmohammadzadeh, N, K. & Askari, M., 2002. Plant mite fauna ofSistan-Baluchestan and Hormozgan provinces. J. Entomol Soci. Iran, 22(1):87-105
Chaudhri, W. M., 1977. Description of the mite of the family Cunaxidae (Acarina)from Pakistan, Pak, J. Agric. Sci., 14: 41-52 .
. Chaudhri, W. M., Akbar, S. & Rasool, A., 1979. Studies on the predatory leafinhabiting mites of Pakistan ..Monograph" NO.2: Univ. of Agri., Faisalabad.234pp
de-Oliveria, A. J. & Daemon, E., 2003. Qualitative and quantitative assessmentof mites (Acari) in domiciliary dust in rural dwellings in the "Zona da Mata"Region, Minas Gerias, Brazil. Revista-Brazifeira-de-Zooi., 20(4): 675.679 .
den Heyer, J., 1977. A new genus Neosciru/a (Cunaxidae: Prostigmata: Acari)from the Ethiopian region. Jour. Enlomo/, Soc. S, Afr., 40(1): 73-86.
den Heyer, J., 1980. Sculopalpus, a new cunaxid genus from the EthiopianRegion (Prostigmata: Acari). Acar%gia, 21(2): 187-193.
Liang, G, W., 1983. Notes on four species of mites (Acarina: Cunaxidae) inChina Agric. Bur, Guangdong Prov., 5(2): 104-107.
Liang, G.W., 1984, A new species and a new record of the genus Pseudobonziafrom China. Acla Zoolaxonomica Sinica, 9: 49-51,
'Luxton, M., 1982. Some new species of mites from New Zealand peat soils. N Z.J Zool. 9: 325-332,
Schruft, G., 1971. Haleupa/us oliveri new species, a "Thorn-palped" mite ongrape vines (Vitis spp.) Acari: Cunaxidae. Disch. Entomol. z., 18(4-5): 379-382.
. Sepasgosarian, H., 1984. The world genera and species of the family Cunaxidae(Actinedida: Acarida). Zeif, Ang. Zool. 71: 135-150.
Smiley, R L., 1975, A generic revision of the mites of the family Cunaxidae(Acarina). Ann. Entomol. Soc. Am, 68(2): 227-244.
Smiley, R L., 1992. The Predatory Mite Family Cunaxidae (Acari) Of The WorldWith A New Classification. Indira Publishing House, Michigan, U. S. A.356pp
. This new species comes closer to P, newzea/andicus Smiley, however, differsfrom it on the basis of following characters:
BIOLOGIAM. H. BASHIR ET AL
"
l.l~ f{, [;'1j': ;)1. .....•.....
!.'f.
JiB!f);" J'ff': ..<. •
III IV
Fig. 1: Pseudobonzia ashfaqi, n. sp.
A- Dorsal side; B-Ventral side; C-Palp; D-Chelicera; E-Hypostome;F-Iegs '-IV
\ i .!:t.~ ~ ~'l(.' D &
This new species can also be separated from Pseudobonzia reliculata(Heryford) on the basis of following characters:
1. Palp. tibiotarsus without thorn-like seta in Pseudobonzia reticulata but with athorn-like seta in this new species.
2. Propodosomal shield and coxal plates are reticulated in Pseudobonziareticulata while pitted in this new species.
80
3. Leg segments I-IV basifemora; telofemora; genua; tibiae and tarsi with 3-3-2-1;6-5-4-3; 9-7-6-6; 7-6-6-6 and 27-21-18-19, setae, respectively, in Pseudobonziareticulala as against 4-5-4-0; 5-5-4-3; 9-6-6-7; 7-6-6-5 and 21-20-15-14 setae,respectively, in this new species,
~\.
Tagore, A & Putatunda, B. N., 2003. Mites associated with some ornamentalplants at Hisar, Haryana. Pest Manag. Econ. Zool, 11(1): 37-44.
Walter, D. E. & Kaplan, D. T., 1991. Observations on Coleoscirus simplex(Acarina: Prostigmata), a predatory mite that colonizes greenhouse culturesof rootknot nematode (Meloidogyne spp.), and a review of feeding behaviorin the Cunaxidae. Exp. Appl Acarol., 12(1-2): 47-59.
82 M. H. BASHIR ET AL BIOLOGIA BIOLOGIA(PAKISTAN) 2008,54 (1),83-90PK ISSN 0006 - 3096
Seasonal variations in Proteocep1lalus filicollis (Rudolphi) eggsin a natural population of Gasterosteus aculeatus L.
ZAFAR IQBAL & R. WOOTTEN
Zoology Department, University ({the Pw?iab, Lahore, Pakistan (ZI)Universilya/Stirling, Scotland, U K. (RW)
ABSTRACT
A study on the seasonal variations of Proteocephalus filicollis eggs hasshown that eggs were larger in winter and spring and smaller in summer. Therewas a tendency for smaller worms to contain larger eggs. Eggs with a smallerdiameter were obselVed in larger worms. The size of the hexacanth showed moreconsistency in its diameter and was positively correlated to the diameter of theegg. The winter eggs were found viable and infective to the copepod intermediatehost. The structure of the egg is described and compared with previous studies onthe eggs of this parasite.
Key words: Proteocephalas fiticollis, Cestode, E99S, Hexacanth, Diameter,Gasterosteus aculeatus
INTRODUCTION
The gravid worm ProteocephaJus filicoilis sheds its eggs in water. There arethree to five membranes, which surround the egg. The egg is bilaterallysymmetrical and contains a hexacanth embryo with three pairs of embryonichooks. The egg is ingested by a copepod, which hatches within the intestine ofthe cope pod and develops into infective stage larva for the next host of its lifecycle.
The studies on Proteocephalus species, by Hunter (1928, 1929) on Pambiopfitis and P pingius, by Wagner (1954) on P. tumidocoilius, by Freeman(1964) on P parailacticus, by Fischer (1968) on P. fluviJitis, by Wootten (1974) onP percae, by Priemer (1987) on P. exiguous, by Scholz (1Q91, 1993) on Pneglectus and P. torulosus, have provided detailed information on the shape,structure and membranes surrounding the eggs of these species. The studies byMeggitt (1914) and Freze (1965) gave information on the morphology of Pfificoilis eggs, which is surrounded by 2 to 3 membranes, and vary in diameterfrom 50-75 ~m, 58 ~m and 13 ~m.
Iqbal & Vliootten (2001) described the structure of the P fificoilis egg indetail. According to them the .(liameters of P filicoilis eggs in expanded. staterange from 37.5 ~m to 118.7 ~m (mean diameter 55.31 ~m). The hexacanthembryo is bilaterally symmetrical and measures 17.5 ~m x 27.5 ~m (mean 21.34~m). It is reported that eggs of P. fit/collis are surrounded by at least four mainembryonic envelopes, Le., the capsule; outer envelope; inner envelope (granular.Iayers) and oncospheral membrane. The oncospheral membrane immediately
Tagore, A & Putatunda, B. N., 2003. Mites associated with some ornamentalplants at Hisar, Haryana. Pest Manag. Econ. Zool, 11(1): 37-44.
Walter, D. E. & Kaplan, D. T., 1991. Observations on Coleoscirus simplex(Acarina: Prostigmata), a predatory mite that colonizes greenhouse culturesof rootknot nematode (Meloidogyne spp.), and a review of feeding behaviorin the Cunaxidae. Exp. Appl Acarol., 12(1-2): 47-59.
82 M. H. BASHIR ET AL BIOLOGIA BIOLOGIA(PAKISTAN) 2008,54 (1),83-90PK ISSN 0006 - 3096
Seasonal variations in Proteocep1lalus filicollis (Rudolphi) eggsin a natural population of Gasterosteus aculeatus L.
ZAFAR IQBAL & R. WOOTTEN
Zoology Department, University ({the Pw?iab, Lahore, Pakistan (ZI)Universilya/Stirling, Scotland, U K. (RW)
ABSTRACT
A study on the seasonal variations of Proteocephalus filicollis eggs hasshown that eggs were larger in winter and spring and smaller in summer. Therewas a tendency for smaller worms to contain larger eggs. Eggs with a smallerdiameter were obselVed in larger worms. The size of the hexacanth showed moreconsistency in its diameter and was positively correlated to the diameter of theegg. The winter eggs were found viable and infective to the copepod intermediatehost. The structure of the egg is described and compared with previous studies onthe eggs of this parasite.
Key words: Proteocephalas fiticollis, Cestode, E99S, Hexacanth, Diameter,Gasterosteus aculeatus
INTRODUCTION
The gravid worm ProteocephaJus filicoilis sheds its eggs in water. There arethree to five membranes, which surround the egg. The egg is bilaterallysymmetrical and contains a hexacanth embryo with three pairs of embryonichooks. The egg is ingested by a copepod, which hatches within the intestine ofthe cope pod and develops into infective stage larva for the next host of its lifecycle.
The studies on Proteocephalus species, by Hunter (1928, 1929) on Pambiopfitis and P pingius, by Wagner (1954) on P. tumidocoilius, by Freeman(1964) on P parailacticus, by Fischer (1968) on P. fluviJitis, by Wootten (1974) onP percae, by Priemer (1987) on P. exiguous, by Scholz (1Q91, 1993) on Pneglectus and P. torulosus, have provided detailed information on the shape,structure and membranes surrounding the eggs of these species. The studies byMeggitt (1914) and Freze (1965) gave information on the morphology of Pfificoilis eggs, which is surrounded by 2 to 3 membranes, and vary in diameterfrom 50-75 ~m, 58 ~m and 13 ~m.
Iqbal & Vliootten (2001) described the structure of the P fificoilis egg indetail. According to them the .(liameters of P filicoilis eggs in expanded. staterange from 37.5 ~m to 118.7 ~m (mean diameter 55.31 ~m). The hexacanthembryo is bilaterally symmetrical and measures 17.5 ~m x 27.5 ~m (mean 21.34~m). It is reported that eggs of P. fit/collis are surrounded by at least four mainembryonic envelopes, Le., the capsule; outer envelope; inner envelope (granular.Iayers) and oncospheral membrane. The oncospheral membrane immediately
84 Z. IQBAL & R. WOOTIEN BIOLOGIA VOL. 54 (1) VARIATIONS IN OCCURRENCE OF P. FILfCOLUS EGGS IN G ACULEATUS 85
Infectivity of winter eggs
1.''<,Mean diameter of P. fiJicoJlis hexacanth:
ranged from 25-401Jm during September 1994. February, April and first twoweeks of May 1995. However, the mean diameter of the eggs was reduced andnarrowed to 16 to 21 IJm from second week of May to second week of July 1995.
The length of the gravid worms from which eggs were obtained rangedfrom 5mm to 25mm. the majority of gravid worms were between 5mm and 17mmin length. There was a significant inverse relationship between egg diameter andlength of the worm (r" = 0.363; r = 0.602; P'; 0.001). It is clear that the diameter ofeggs was greater in smaller worms as compared to larger worms (Fig. 2).
A weak positive significant relationship was observed between diameter ofthe egg and the infra-population size of the worm in the intestine of the fish (r' =0.259; r = 0.508; P'; 0.001). This relationship may be due to the presence of eggsamples from two infra-population size ranges (1 to 11 and 25 to 27) as in Fig.3.
The mean diameter of P. filieol/is hexacanth observed is shown in Fig 1; Themaximum mean diameter of the hexacanth (15.4 IJm) was observed. inSeptember 1994 and the smallest eggs (7.5 IJm diameter) in the second week ofJuly 1995. Analysis of variance demonstrated a significant variation in' thediameter of hexacanth over sampling period (F= 121.97; P,;0.001; Faas(1)149=3.90). Mean diameter of the hexacanth showed a considerable range (13-15 .11m)from September 1994 to May 1995 and a narrow range (11.5-12.5 IJm) Jromsecond week of June 1995 to second week of July 1995. The diameter ofhexacanth was significantly positively correlated to egg diameter (r' = 0.646; r =0.80; P = 0.001; F = 270.53; F = 005(1)149=3.90)(Fig. 4).
The eggs obtained in February 1995 were stored at 4'oC for 24 hours Theseeggs were fed to five Acanthocyelops robustus. These infected <;opepods weremaintained at 15-16°C. Out of these five A. robustus specimens, two were. infected, each bearing two procecoids. Observations under microscope showedthat by day 13'h these larvae in the copepods measured 72.5,141.3, 1205 and96.8 IJm Unfortunately no fully developed larva was obtained and the copepoddid not survive beyond 14 days post-infection.
encircles the embryo (Iqbal & Wootten, 2002). The P. filieol/is eggs are shed inwater mostly in summer; however, some eggs are also shed in autumn, winterand spring too (Iqbal, 1998). Hence this study was undertaken to investigate thebiology of the eggs in relation to season, length of the gravid worm. and infra-. population size of the worm and compare it with the previous studies.
The fish, the three-spined stickleback, Gasterosteus aculeatus. wassampled from Airthray Loch, Scotland, U.K. Iqbal & Wootten (2004, 2005) havedescribed fish sampling methods. The gravid worms were extracted from theintestine of the fish. These worms were kept in separate petri dishes containingwarm water (20-25°C). The worms released eggs in water. A few drops of waterfrom petri dish containing eggs were taken on clean glass slide and covered withclean cover slip. Morphometric observations of the eggs were taken undermicroscope fitted with an 'eye piece graticule. Readings were taken in IJm.
After shedding eggs, the gravid worms were released in tapl distilled waterfor one hour or till the movement of the worm stopped. The worms were fixed in5% buffered formalin. The fixed specimens were stained with Mayer'sparacarmine and differentiated in 50% acid alcohol, dehydrated, cleared in cedarwood oil and mounted in Canada balsam. The mounted worms were measuredin IJm under microscope and length was converted into millimeter.
The eggs obtained in winter were stored at 4°C for 24 hours and were thenfed to previously starved copepod, Acanthoeyclops rubustus, which were rearedin the laboratory according to Iqbal (1998), to check the infectivity of these eggsThese infected copepods were kept at 15-16°C to observe the growth ofprocercoids in them. The rearing, maintenance and observations of the infectedcopepod were made following Iqbal & Wootten (2001)
RESULTS
The dimensions of the eggs presented here are based on the measurementof 150 individual eggs obtained from sixteen gravid worms from sixteen differentfish. The gravid worms were obtained in different months of the year (September,1994; February, April, May, June and July, 1995)
MATERIALS AND METHODS
Seasonal variation in diameter of P. filicoJlis eggs:,
The mean diameter of P. filieol/is eggs, recorded from September 1994 toJuly 1995, is shown in Fig. 1. A total of 150 eggs were measured from sixteen. different gravid worms, each sample included 8-10 eggs. The maximum meandiameter (40IJm) was observed in September 1994 and minimum mean diameter(15IJm) in the last week of June 1995. Analysis of variance showed a significantvariation in the diameter of eggs throughout the sampling period (F=135.52, P';0.001). The eggs showed two distinct diameter ranges. The mean egg diameter
84 Z. IQBAL & R. WOOTIEN BIOLOGIA VOL. 54 (1) VARIATIONS IN OCCURRENCE OF P. FILfCOLUS EGGS IN G ACULEATUS 85
Infectivity of winter eggs
1.''<,Mean diameter of P. fiJicoJlis hexacanth:
ranged from 25-401Jm during September 1994. February, April and first twoweeks of May 1995. However, the mean diameter of the eggs was reduced andnarrowed to 16 to 21 IJm from second week of May to second week of July 1995.
The length of the gravid worms from which eggs were obtained rangedfrom 5mm to 25mm. the majority of gravid worms were between 5mm and 17mmin length. There was a significant inverse relationship between egg diameter andlength of the worm (r" = 0.363; r = 0.602; P'; 0.001). It is clear that the diameter ofeggs was greater in smaller worms as compared to larger worms (Fig. 2).
A weak positive significant relationship was observed between diameter ofthe egg and the infra-population size of the worm in the intestine of the fish (r' =0.259; r = 0.508; P'; 0.001). This relationship may be due to the presence of eggsamples from two infra-population size ranges (1 to 11 and 25 to 27) as in Fig.3.
The mean diameter of P. filieol/is hexacanth observed is shown in Fig 1; Themaximum mean diameter of the hexacanth (15.4 IJm) was observed. inSeptember 1994 and the smallest eggs (7.5 IJm diameter) in the second week ofJuly 1995. Analysis of variance demonstrated a significant variation in' thediameter of hexacanth over sampling period (F= 121.97; P,;0.001; Faas(1)149=3.90). Mean diameter of the hexacanth showed a considerable range (13-15 .11m)from September 1994 to May 1995 and a narrow range (11.5-12.5 IJm) Jromsecond week of June 1995 to second week of July 1995. The diameter ofhexacanth was significantly positively correlated to egg diameter (r' = 0.646; r =0.80; P = 0.001; F = 270.53; F = 005(1)149=3.90)(Fig. 4).
The eggs obtained in February 1995 were stored at 4'oC for 24 hours Theseeggs were fed to five Acanthocyelops robustus. These infected <;opepods weremaintained at 15-16°C. Out of these five A. robustus specimens, two were. infected, each bearing two procecoids. Observations under microscope showedthat by day 13'h these larvae in the copepods measured 72.5,141.3, 1205 and96.8 IJm Unfortunately no fully developed larva was obtained and the copepoddid not survive beyond 14 days post-infection.
encircles the embryo (Iqbal & Wootten, 2002). The P. filieol/is eggs are shed inwater mostly in summer; however, some eggs are also shed in autumn, winterand spring too (Iqbal, 1998). Hence this study was undertaken to investigate thebiology of the eggs in relation to season, length of the gravid worm. and infra-. population size of the worm and compare it with the previous studies.
The fish, the three-spined stickleback, Gasterosteus aculeatus. wassampled from Airthray Loch, Scotland, U.K. Iqbal & Wootten (2004, 2005) havedescribed fish sampling methods. The gravid worms were extracted from theintestine of the fish. These worms were kept in separate petri dishes containingwarm water (20-25°C). The worms released eggs in water. A few drops of waterfrom petri dish containing eggs were taken on clean glass slide and covered withclean cover slip. Morphometric observations of the eggs were taken undermicroscope fitted with an 'eye piece graticule. Readings were taken in IJm.
After shedding eggs, the gravid worms were released in tapl distilled waterfor one hour or till the movement of the worm stopped. The worms were fixed in5% buffered formalin. The fixed specimens were stained with Mayer'sparacarmine and differentiated in 50% acid alcohol, dehydrated, cleared in cedarwood oil and mounted in Canada balsam. The mounted worms were measuredin IJm under microscope and length was converted into millimeter.
The eggs obtained in winter were stored at 4°C for 24 hours and were thenfed to previously starved copepod, Acanthoeyclops rubustus, which were rearedin the laboratory according to Iqbal (1998), to check the infectivity of these eggsThese infected copepods were kept at 15-16°C to observe the growth ofprocercoids in them. The rearing, maintenance and observations of the infectedcopepod were made following Iqbal & Wootten (2001)
RESULTS
The dimensions of the eggs presented here are based on the measurementof 150 individual eggs obtained from sixteen gravid worms from sixteen differentfish. The gravid worms were obtained in different months of the year (September,1994; February, April, May, June and July, 1995)
MATERIALS AND METHODS
Seasonal variation in diameter of P. filicoJlis eggs:,
The mean diameter of P. filieol/is eggs, recorded from September 1994 toJuly 1995, is shown in Fig. 1. A total of 150 eggs were measured from sixteen. different gravid worms, each sample included 8-10 eggs. The maximum meandiameter (40IJm) was observed in September 1994 and minimum mean diameter(15IJm) in the last week of June 1995. Analysis of variance showed a significantvariation in the diameter of eggs throughout the sampling period (F=135.52, P';0.001). The eggs showed two distinct diameter ranges. The mean egg diameter
VARIATIONS IN OCCURRENCE OF P. FILlCOLLIS EGGS IN G. ACULEATUS
~------------_._---------
II! 86
40
,--j
z. IQBAL & R. WOOTIEN B/OlOGIA
-&'J-Dla ot egg-O-Oia of hexaoan1h
VOL 54 (1)
3.9
I •• ••
87
y = 3.532. 2.90E-02X
R-Squared = 0.366
! y = 3.01257 + 1.72E-02XR-Squared :; 0.259
diameter ofsampled in
•
•I•
i •
Length of worm (mm)
Fig.2 Relationship between length of PrOleocephalus jilico/[is (mrn)-;;;id d'ameter of egg ( "m), These eggs Were sampled in September 1994,Fehruary, April. May, June and July 1995.
Infrapopulation Si7...e
... .• •...• I I -----.....---------,--:
L"-~--;-------- . :I
•• • •I. I.... ..I.:' :.'"r ••••••
i •. : ••••, .,
--1-----------. --.-.- ...-....-----.---,
Fig. 3 Relationship between infrapopularion sjze andj5~rJie-ocepJr{llus fi/ieollis eggs (11m), These eggs wereScptemher 1994, February, April, May, June and July 1995.
-1
2.5
-i'----------.-.-r-- ..-.-----.----.---T-__'_ . . . ..)
o 10 20 30
4.0~Eid'"'-',u 3.5~0,u;:;E'"i5
30
34Ed'"0»"
2.9 .1~0
lJ"
24 JEi'"is
.1;1
j~1
rAjT
•
Sampling week
i{j---~~~-
-T ! I I I ITl:-TiTl ! I
•L
Fig. I Mean diametcl" of Proteocephrdflsjilicollis eeg [Inc! hexi'lc;;nth indifterent months. The letters S, F. A, M. J, lL srand. [nr Seplemhe,",Fcbru::lry, J\pril, May, !L1~e.July ar;.c [he number on the. right of tbe. k:l..:rindicates YCilf and sampling week. Bill'S repres~nt Sti.lIld"t:,J devlnlioll.",
S94 F95 A1 A2 M1 M2 M3 J2 J2 J3 J3 J4 J4 J4 J4 JU
30
20
10
0'
~~"g~u
'".:;~,1:1'~"@~:3R".~
VARIATIONS IN OCCURRENCE OF P. FILlCOLLIS EGGS IN G. ACULEATUS
~------------_._---------
II! 86
40
,--j
z. IQBAL & R. WOOTIEN B/OlOGIA
-&'J-Dla ot egg-O-Oia of hexaoan1h
VOL 54 (1)
3.9
I •• ••
87
y = 3.532. 2.90E-02X
R-Squared = 0.366
! y = 3.01257 + 1.72E-02XR-Squared :; 0.259
diameter ofsampled in
•
•I•
i •
Length of worm (mm)
Fig.2 Relationship between length of PrOleocephalus jilico/[is (mrn)-;;;id d'ameter of egg ( "m), These eggs Were sampled in September 1994,Fehruary, April. May, June and July 1995.
Infrapopulation Si7...e
... .• •...• I I -----.....---------,--:
L"-~--;-------- . :I
•• • •I. I.... ..I.:' :.'"r ••••••
i •. : ••••, .,
--1-----------. --.-.- ...-....-----.---,
Fig. 3 Relationship between infrapopularion sjze andj5~rJie-ocepJr{llus fi/ieollis eggs (11m), These eggs wereScptemher 1994, February, April, May, June and July 1995.
-1
2.5
-i'----------.-.-r-- ..-.-----.----.---T-__'_ . . . ..)
o 10 20 30
4.0~Eid'"'-',u 3.5~0,u;:;E'"i5
30
34Ed'"0»"
2.9 .1~0
lJ"
24 JEi'"is
.1;1
j~1
rAjT
•
Sampling week
i{j---~~~-
-T ! I I I ITl:-TiTl ! I
•L
Fig. I Mean diametcl" of Proteocephrdflsjilicollis eeg [Inc! hexi'lc;;nth indifterent months. The letters S, F. A, M. J, lL srand. [nr Seplemhe,",Fcbru::lry, J\pril, May, !L1~e.July ar;.c [he number on the. right of tbe. k:l..:rindicates YCilf and sampling week. Bill'S repres~nt Sti.lIld"t:,J devlnlioll.",
S94 F95 A1 A2 M1 M2 M3 J2 J2 J3 J3 J4 J4 J4 J4 JU
30
20
10
0'
~~"g~u
'".:;~,1:1'~"@~:3R".~
88 Z. IQBAL & R WOODEN BIOLOGIA VOL, 54 (1) VARIATIONS IN OCCURRENCE OF P. FIUCOLLfS EGGS IN G ACULEATUS 89
Fig.4 Relationship between diameter of ProfeoceplwZus frlicollis egg- (~l1lFaild diameter of hexacanth (j1Jl1). These eggs were sampled inSeptember 1994, February, April. May, June, July 1995.
•
r.~~--!
4.5,
B.=;on00~~o ,•.... 3.5 _.-;lJE'"is
2.5L10
-_ - .._ ..~- .•..- -,/ ••••
///.. ///.. -/. : .)//1.~ •• I.--...J.' •. ~// .I' •. ...~.I: ... :-: ..
........ _ I''" -- .15 ~~""I'~'"
20
Diameter of hexacanth (Jlm)
Y = 1.08618 + 0.156228X
R.Squared = 0.646
~m, respectively) (Hunter, 1928; 1929); P. tumidocollius (oncosphere's diameter24-27~m) (Wagner, 1954); P. parallacticus (12 x 20 ~m and 35 x 32 to 42 x50~m, respectively) (Freeman, 1964); P. fluvitilis (egg diameter 57-97~m)(Fischer, 1968); P percae (47-50 jJm and 23-47 x 22.1 jJm, respectively)(Wootten, 1974); P. exiguous (40-50 jJm and 19.27 ~m, respectively) (Priemer,, 1987); P. neglectus (100 ~m and 2-32~m, ref~ectively) and P. torulosus (42-52~m and 23-32 ~m, respectively) (Scholz, 1991; 1993).
Thus, the difference in the size between eggs of P. filicollis described byMeggitt (1914) and Freze (1965) and present study seems considerable. Thevariation observed in the diameter of P filicollis egg during this study indicatesthat the winter eggs Were larger than those obtained in spring and summer. Inthe same egg samples (September 1994 to July 1995) the diameter of thehexacanth showed a similar trend compared to the egg diameter. The variationsIn egg diameter observed over three seasons may be due to adaptations todifferent environmental conditions in these seasons. The varibtions in thediameter of the egg may be due to differences in the swelling of outer floatmembrane in different seasons, since the diameter of the hexacanth did notshow much variation.
Smaller eggs were observed on lower infra-population size and larger eggsin higher infra-population size but the reason for this is not clear. This is clearly incontrast to the general principle of crowding effect as observed by Ghazal &Avery (1974), who reported that crowding decreased the linear dimension ofHymenolepis nana eggs, although the shape of the eggs was not affected.
There are not many evidences available at present to describe' the fate ofeggs produced in winter. These eggs experimentally infected A. robustuscopepod when maintained at 15-16'C. It seems possible that fish maintainworms in its intestine during the winter season and the eggs are not shed inwater. The survival of eggs does not seem possible during long cold winter. Thisaspect of biology of P. filicollis eggs needs further investigation.
REFERENCES
DISCUSSION
The maximum mean diameter of P. filicollis egg (40 ~m) was smaller ascompared to 58 ~m reported by Meggitt (1914). Whereas, the mean diameter ofP. filicollis egg was larger than of those eggs observed by Freze (1965) (i.e. 13~m)
The oncosphere's diameter measured by Meggitt (1914) was similarlylarger (23 ~m) compared to the oncosphere's diameter observed in the presentstudy (15.4 ~m). However, the oncosphere's diameters measured by Freze(1965) were smaller (1.7~m) as compared to the present study.
The maximum mean diameter of the egg (40 ~m) and hexacanth (16.5 ~m)embryo of P. filicollis observed in the present study falls in the range of thediameter of the egg and hexacanth embryo of other Proteocepha/us species,such as P. ambloplitis and P pingius (26-60 ~m and 20 ~m; 45.52 ~m and 20
Fischer, H., 1968. The life cycle of Proteocephalus fluviatilis Bangham (Cestoda)from small mouth bas Micropterus dolmieui Lacepede., Cana. J. Zool., 46:569-579.
Freeman, R. S., 1964. On the biology of Proteocephalus parallacticus Maclulich(Cestoda) in Algonquin Park, Canada, Cana. J. Zool, 42: 387-408.
Freze,' V., 1965. Essential of Cestodology. Vol. 5. Proteocephalata. Translatedfrom Russian. Israel Program for Scientific Translation, Jerusalem.
Ghazal, A. M. & Avery, R. A., 1974. Population dynamics of Hymenolepisdiminuta In mice: fecundity and the crowding effect. Parasitology, 69: 403pp.
Hunter, G W., 1928. Contribution to the life history of Proteocephalus ambloplitis(Leidy) Jour Parasit., 14: 229-242.
Hunter, G. W, 1929 Life history of Proteocephalus pinjuis (La Rue).Parasitology, 21: 487-496.
Ii
II
88 Z. IQBAL & R WOODEN BIOLOGIA VOL, 54 (1) VARIATIONS IN OCCURRENCE OF P. FIUCOLLfS EGGS IN G ACULEATUS 89
Fig.4 Relationship between diameter of ProfeoceplwZus frlicollis egg- (~l1lFaild diameter of hexacanth (j1Jl1). These eggs were sampled inSeptember 1994, February, April. May, June, July 1995.
•
r.~~--!
4.5,
B.=;on00~~o ,•.... 3.5 _.-;lJE'"is
2.5L10
-_ - .._ ..~- .•..- -,/ ••••
///.. ///.. -/. : .)//1.~ •• I.--...J.' •. ~// .I' •. ...~.I: ... :-: ..
........ _ I''" -- .15 ~~""I'~'"
20
Diameter of hexacanth (Jlm)
Y = 1.08618 + 0.156228X
R.Squared = 0.646
~m, respectively) (Hunter, 1928; 1929); P. tumidocollius (oncosphere's diameter24-27~m) (Wagner, 1954); P. parallacticus (12 x 20 ~m and 35 x 32 to 42 x50~m, respectively) (Freeman, 1964); P. fluvitilis (egg diameter 57-97~m)(Fischer, 1968); P percae (47-50 jJm and 23-47 x 22.1 jJm, respectively)(Wootten, 1974); P. exiguous (40-50 jJm and 19.27 ~m, respectively) (Priemer,, 1987); P. neglectus (100 ~m and 2-32~m, ref~ectively) and P. torulosus (42-52~m and 23-32 ~m, respectively) (Scholz, 1991; 1993).
Thus, the difference in the size between eggs of P. filicollis described byMeggitt (1914) and Freze (1965) and present study seems considerable. Thevariation observed in the diameter of P filicollis egg during this study indicatesthat the winter eggs Were larger than those obtained in spring and summer. Inthe same egg samples (September 1994 to July 1995) the diameter of thehexacanth showed a similar trend compared to the egg diameter. The variationsIn egg diameter observed over three seasons may be due to adaptations todifferent environmental conditions in these seasons. The varibtions in thediameter of the egg may be due to differences in the swelling of outer floatmembrane in different seasons, since the diameter of the hexacanth did notshow much variation.
Smaller eggs were observed on lower infra-population size and larger eggsin higher infra-population size but the reason for this is not clear. This is clearly incontrast to the general principle of crowding effect as observed by Ghazal &Avery (1974), who reported that crowding decreased the linear dimension ofHymenolepis nana eggs, although the shape of the eggs was not affected.
There are not many evidences available at present to describe' the fate ofeggs produced in winter. These eggs experimentally infected A. robustuscopepod when maintained at 15-16'C. It seems possible that fish maintainworms in its intestine during the winter season and the eggs are not shed inwater. The survival of eggs does not seem possible during long cold winter. Thisaspect of biology of P. filicollis eggs needs further investigation.
REFERENCES
DISCUSSION
The maximum mean diameter of P. filicollis egg (40 ~m) was smaller ascompared to 58 ~m reported by Meggitt (1914). Whereas, the mean diameter ofP. filicollis egg was larger than of those eggs observed by Freze (1965) (i.e. 13~m)
The oncosphere's diameter measured by Meggitt (1914) was similarlylarger (23 ~m) compared to the oncosphere's diameter observed in the presentstudy (15.4 ~m). However, the oncosphere's diameters measured by Freze(1965) were smaller (1.7~m) as compared to the present study.
The maximum mean diameter of the egg (40 ~m) and hexacanth (16.5 ~m)embryo of P. filicollis observed in the present study falls in the range of thediameter of the egg and hexacanth embryo of other Proteocepha/us species,such as P. ambloplitis and P pingius (26-60 ~m and 20 ~m; 45.52 ~m and 20
Fischer, H., 1968. The life cycle of Proteocephalus fluviatilis Bangham (Cestoda)from small mouth bas Micropterus dolmieui Lacepede., Cana. J. Zool., 46:569-579.
Freeman, R. S., 1964. On the biology of Proteocephalus parallacticus Maclulich(Cestoda) in Algonquin Park, Canada, Cana. J. Zool, 42: 387-408.
Freze,' V., 1965. Essential of Cestodology. Vol. 5. Proteocephalata. Translatedfrom Russian. Israel Program for Scientific Translation, Jerusalem.
Ghazal, A. M. & Avery, R. A., 1974. Population dynamics of Hymenolepisdiminuta In mice: fecundity and the crowding effect. Parasitology, 69: 403pp.
Hunter, G W., 1928. Contribution to the life history of Proteocephalus ambloplitis(Leidy) Jour Parasit., 14: 229-242.
Hunter, G. W, 1929 Life history of Proteocephalus pinjuis (La Rue).Parasitology, 21: 487-496.
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90 Z ..IQBAL & R. WOOTIEN BIOLOGIA BIOLOGIA{PAKISTAN) 2008,54 (1), 91-96PK ISSN 0006 - 3096
Iqbal, Z., 1998. Aspect of the biology of the cestode Proteocephalus fiticollis(Rudolophi) from Gasterosteus aculeatus L., PhD Thesis, University ofStirling, Scotland, U.K. pp282.
Iqbal. Z. & Wootten, R, 2001. Development. of Proteocepha/us filicol/is, acestode, In. the cope pod intermediate host under experimental conditions.Science International (Lahore), 13(1): 59-65.
Iqbal, Z. & Wootten, R., 2002. Ultrastructure of the embryonic envelopes inProteocephalus filicollis' (Cestode: Proteocephalidea), in the cope podintermediate host under experimental conditions. Pak. J. Zool .. 34( 1): 57-63.
Iqbal, Z. & Wootten, R., 2004. Biological.and Physicochemical features ofAirthary Loch, Scotland, U.K. Biologia-Pakistan, 50(2): 175-182.
Iqbal, Z. & Wootten, R, 2005. Infection of Proteocephalus filicol/is Rudolphi fromGasterosteus. aculeatus L., three-spined stickleback,in relation to sex andlength ot the host. Punjab University J. Zool., 20(1 ):15-23
Meggitt, F. J., 1914. The structure and the history of a tapeworm Ichthyotaeniafilicol/is (Rud.) parasitic in the stickleback. Proc. Zool. Soc. London. Part 1:113-138.
. Priemer, V. J., 1987. On the life cycle of Proteocepha/us exiguous (Cestoda)from Saimo gairdenri (Pisces). Helminthologia, 24: 75-85.
Scholz, T., 1991. Studies on the de~elopment of the cestode Proteocephalusneglectus La Rue, 1911 (Cestoda: Proteocephalidae) under experimentalconditions. Folia Parasitologica, 38: 39-55.
Scholz, T., 1993 .. Development of the Proteocephalus tumidocol/us in theintermediate host under experimental conditions. J. Helminthology, 67: 316-324.
Wagner, E. D., 1954. The life history of Proteocephalus tumidocol/us Wagner(Cestoda) in rainbow trout. Jour. Parasit., 40: 489-497.
Wootten, R, 1974. Studies on the life history and development ofProteocephalus percae (Muller) (Cestoda: Proteocephalidae). J.Helminthol.ogy, 48: 269-281 ..
~
Enumeration of total and fecal coliform bacteria in drinking waterof Khairpur city, Sindh, Pakistan
ABDUL HUSSAIN SHAR, YASMEEN KAZI, MIANDAD ZARDARI& IRSHAD HUSSAIN SOOMRO
Department of Microbiology, Shah Abdul Latif University, Khairpur,Sindh, Pakistan
ABSTRACT
Total coliform (TC) and fecal coliform (FC) bacteria were analyzed in drinkingwater of Khairpur city. Ninety samples were collected from main reservoir(source), distribution lines and consumer taps. The pH and residual chlorine ofwater samples were also determined. For pH analysis, pH PAL High AccuracyElectrochemistry Test Pen was used and residual chlorine was analyzed by usingHI 3831 Free Chlorine Test Kit. In coliform bacteriological analysis, inductivitymembrane filtration (MF) method was used for total (TC) and fecal (FC) coliformbacteria. All samples were found contaminated with total (TC) as well as fecalcoliform (FC) and the counts were higher than the maximum microbialcontaminant level (MMCL) recommended by World Health Organization (WHO). Itwas observed that pH was within limits of WHO standard while residual chlorinewas not detected in any sample of drinking water.
Key words: Colony forming unit (cfu), Coliform, Escherichia coli, Water quality,Contamination, Sanitation.
INTRODUCTION
Detection of microbial contaminants of fecal origin is a major priority in thecontrol of drinking water quality. The presence of fecal contamination is mostoften evaluated using members of coliform group (Gleeson & Gary, 1997). Waterfrom different sources, i.e., rivers, lakes, reservoirs and ground water aquifersare subjected to varying degrees of fecal pollution, and consequently freshwateris a vector of transmission of many pathogenic bacteria, viruses and protozoa(Feachman et al., 1983; Aneja, 1993 and Blumenthai et a/., 1999). Despite theworldwide efforts and modern technologies utilized for the production of safewater, the transmission of waterborne diseases is stiU a matter of major concern.For decades, the fecal coliform groups of bacteria have been used as indicator ofwater quality with respect to the presence of human pathogens, and due to rapidand reliable routine monitoring of the microbiological quality of water, it willremain fundamentally important in the control of waterborne diseases. Ideally theoccurrence and leveis of all human pathogens should be monitored (Clesceri etai,1998).
A lot of work has been done for the identification of contaminants ofdrinking water to control the waterborne diseases throughout the world
90 Z ..IQBAL & R. WOOTIEN BIOLOGIA BIOLOGIA{PAKISTAN) 2008,54 (1), 91-96PK ISSN 0006 - 3096
Iqbal, Z., 1998. Aspect of the biology of the cestode Proteocephalus fiticollis(Rudolophi) from Gasterosteus aculeatus L., PhD Thesis, University ofStirling, Scotland, U.K. pp282.
Iqbal. Z. & Wootten, R, 2001. Development. of Proteocepha/us filicol/is, acestode, In. the cope pod intermediate host under experimental conditions.Science International (Lahore), 13(1): 59-65.
Iqbal, Z. & Wootten, R., 2002. Ultrastructure of the embryonic envelopes inProteocephalus filicollis' (Cestode: Proteocephalidea), in the cope podintermediate host under experimental conditions. Pak. J. Zool .. 34( 1): 57-63.
Iqbal, Z. & Wootten, R., 2004. Biological.and Physicochemical features ofAirthary Loch, Scotland, U.K. Biologia-Pakistan, 50(2): 175-182.
Iqbal, Z. & Wootten, R, 2005. Infection of Proteocephalus filicol/is Rudolphi fromGasterosteus. aculeatus L., three-spined stickleback,in relation to sex andlength ot the host. Punjab University J. Zool., 20(1 ):15-23
Meggitt, F. J., 1914. The structure and the history of a tapeworm Ichthyotaeniafilicol/is (Rud.) parasitic in the stickleback. Proc. Zool. Soc. London. Part 1:113-138.
. Priemer, V. J., 1987. On the life cycle of Proteocepha/us exiguous (Cestoda)from Saimo gairdenri (Pisces). Helminthologia, 24: 75-85.
Scholz, T., 1991. Studies on the de~elopment of the cestode Proteocephalusneglectus La Rue, 1911 (Cestoda: Proteocephalidae) under experimentalconditions. Folia Parasitologica, 38: 39-55.
Scholz, T., 1993 .. Development of the Proteocephalus tumidocol/us in theintermediate host under experimental conditions. J. Helminthology, 67: 316-324.
Wagner, E. D., 1954. The life history of Proteocephalus tumidocol/us Wagner(Cestoda) in rainbow trout. Jour. Parasit., 40: 489-497.
Wootten, R, 1974. Studies on the life history and development ofProteocephalus percae (Muller) (Cestoda: Proteocephalidae). J.Helminthol.ogy, 48: 269-281 ..
~
Enumeration of total and fecal coliform bacteria in drinking waterof Khairpur city, Sindh, Pakistan
ABDUL HUSSAIN SHAR, YASMEEN KAZI, MIANDAD ZARDARI& IRSHAD HUSSAIN SOOMRO
Department of Microbiology, Shah Abdul Latif University, Khairpur,Sindh, Pakistan
ABSTRACT
Total coliform (TC) and fecal coliform (FC) bacteria were analyzed in drinkingwater of Khairpur city. Ninety samples were collected from main reservoir(source), distribution lines and consumer taps. The pH and residual chlorine ofwater samples were also determined. For pH analysis, pH PAL High AccuracyElectrochemistry Test Pen was used and residual chlorine was analyzed by usingHI 3831 Free Chlorine Test Kit. In coliform bacteriological analysis, inductivitymembrane filtration (MF) method was used for total (TC) and fecal (FC) coliformbacteria. All samples were found contaminated with total (TC) as well as fecalcoliform (FC) and the counts were higher than the maximum microbialcontaminant level (MMCL) recommended by World Health Organization (WHO). Itwas observed that pH was within limits of WHO standard while residual chlorinewas not detected in any sample of drinking water.
Key words: Colony forming unit (cfu), Coliform, Escherichia coli, Water quality,Contamination, Sanitation.
INTRODUCTION
Detection of microbial contaminants of fecal origin is a major priority in thecontrol of drinking water quality. The presence of fecal contamination is mostoften evaluated using members of coliform group (Gleeson & Gary, 1997). Waterfrom different sources, i.e., rivers, lakes, reservoirs and ground water aquifersare subjected to varying degrees of fecal pollution, and consequently freshwateris a vector of transmission of many pathogenic bacteria, viruses and protozoa(Feachman et al., 1983; Aneja, 1993 and Blumenthai et a/., 1999). Despite theworldwide efforts and modern technologies utilized for the production of safewater, the transmission of waterborne diseases is stiU a matter of major concern.For decades, the fecal coliform groups of bacteria have been used as indicator ofwater quality with respect to the presence of human pathogens, and due to rapidand reliable routine monitoring of the microbiological quality of water, it willremain fundamentally important in the control of waterborne diseases. Ideally theoccurrence and leveis of all human pathogens should be monitored (Clesceri etai,1998).
A lot of work has been done for the identification of contaminants ofdrinking water to control the waterborne diseases throughout the world
92 A. H. SHAR ET AL BIOLOGIA VOL. 54 (1) ENUMERATION OF TOTAL AND FECAL COLIFORM BACTERIA IN DRINKING WATER 93
."?
(Kaltenthaler et aI., 1996; Araujo et a/., 1997; Thurman et aI., 1998; Alnoso et a/.,1999; Ejaz & Ahmad, 2001; Kisteman et a/., 2002; Lee & Kim, 2003; Kirschner etal., 2004; Roslev et a/., 2004; Anderson et aI., 2005 and Leoni et a/., 2005)
In Pakistan, research work has been done on the drinking water in differentparts of country, but in the interior part of Sindh province such work on drinkingwater quality has not been done so far. Khairpur city was selected for presentwork in order to estimate the microbiological load, pH and chlorine (used fordisinfection) in drinking water supplied to the community. A large number ofpeople have moved into the city from rural areas and this city has the populationof 120,000. As the waterborne diseases are reported and people are unaware ofthe problems of drinking water contamination, therefore, this study is an attemptto asses the quality of drinking water at source point, and change in qualityduring distribution. This investigation also involves forecasting of drinking waterquality for people of the area. The data would be a useful tool for creatingawareness amongst the residents, planners and decision-makers for future watersupply schemes.
MATERIALS AND METHODS
Sampling techniques
An analysis was carried out for drinking water samples as per standards ofWHO (Anon., 1997). For sampling frequency, the whole city of Khairpur wasdivided into three sampling zones, i.e., main reservoir, distribution lines andconsumer taps Ninety samples in total, i.e. thirty samples from each site, werecollected; all sites were supplied from same water network which distributeswater, originating from surface water sources. Samples were collected, after aflow time of 5 minutes to eliminate any contamination present, in sterile screwcap 500 ml white glass flasks (Pyrex). In order to neutralize the residual freechlorine, a solution of 10% Sodium thiosulphate was added in sterile bottles(Mellado et al., 2006) after collection. Samples were placed in ice boxes andbrought to laboratory.
pH and residual chlorine analysis
Water pH and residual chlorine were determined at the time of collectionusing PAL High Accuracy Electrochemistry Test Pen and Free Chlorine Test Kitfor chlorine estimation, respectively.
Microbiological analysis
Samples were analyzed within 4 hours of collection by membrane filtrationmethod to determine the total coliform per 100 ml at 3rc and fecal coliform at44'C on Eosine Methylene Blue (EMS) agar after 24 hours incubation. Colonieswere counted and all distinct colony types were transferred from Eosine
. Methylene Slue (EMS) agar to Trypticase Soya agar (TSA) plates; isolatedcolonies from Trypticase Soya agar (TSA) plates were Gram stained and theoxidase test W2,S carried out All colonies tested were Gram negative, oxidasenegative rods,
RESULT:::
pH and residual chlorine
The pH of all samples was determined and found within the limits of WHOguidelines for drinking water, i.e., 6.5-8,5,Table 1 shows the m:nimum andmaximum values of pH in all water samples, i.e" the pH of main reservoir rangedfrom 6.31-7.41, that of distribution line the range was 6,78-7.81 and that ofconsumer taps was 6.87-7.5. The residual chlorine was not detected in allsamples collected from different locations, whereas WHO has recommended 0.2-05 mg/l for drinking water (Table 2).
Table 1: pH of water samples collected from different sites
Site n Minimum Maximum Average WHO limitsvalue value .
Mainreservoir 30 6,31 7.41 7.37 65-8,5
Distributionline 30 6.78 781 7.09 65-8.5
Consumertaps 30 6.87 7.5 7.01 6.5-85
Table 2: Residual Chlorine content in water samples collected fromdifferent sitesI
ISite n Minimum Maximum Average WHO limits
value valueMain
reservoir 30 0 0 0 0.2-0.5
Distributionline 30 0 0 0 0.2-0.5
Consumertaps 30 0 0 0 0.2-05
92 A. H. SHAR ET AL BIOLOGIA VOL. 54 (1) ENUMERATION OF TOTAL AND FECAL COLIFORM BACTERIA IN DRINKING WATER 93
."?
(Kaltenthaler et aI., 1996; Araujo et a/., 1997; Thurman et aI., 1998; Alnoso et a/.,1999; Ejaz & Ahmad, 2001; Kisteman et a/., 2002; Lee & Kim, 2003; Kirschner etal., 2004; Roslev et a/., 2004; Anderson et aI., 2005 and Leoni et a/., 2005)
In Pakistan, research work has been done on the drinking water in differentparts of country, but in the interior part of Sindh province such work on drinkingwater quality has not been done so far. Khairpur city was selected for presentwork in order to estimate the microbiological load, pH and chlorine (used fordisinfection) in drinking water supplied to the community. A large number ofpeople have moved into the city from rural areas and this city has the populationof 120,000. As the waterborne diseases are reported and people are unaware ofthe problems of drinking water contamination, therefore, this study is an attemptto asses the quality of drinking water at source point, and change in qualityduring distribution. This investigation also involves forecasting of drinking waterquality for people of the area. The data would be a useful tool for creatingawareness amongst the residents, planners and decision-makers for future watersupply schemes.
MATERIALS AND METHODS
Sampling techniques
An analysis was carried out for drinking water samples as per standards ofWHO (Anon., 1997). For sampling frequency, the whole city of Khairpur wasdivided into three sampling zones, i.e., main reservoir, distribution lines andconsumer taps Ninety samples in total, i.e. thirty samples from each site, werecollected; all sites were supplied from same water network which distributeswater, originating from surface water sources. Samples were collected, after aflow time of 5 minutes to eliminate any contamination present, in sterile screwcap 500 ml white glass flasks (Pyrex). In order to neutralize the residual freechlorine, a solution of 10% Sodium thiosulphate was added in sterile bottles(Mellado et al., 2006) after collection. Samples were placed in ice boxes andbrought to laboratory.
pH and residual chlorine analysis
Water pH and residual chlorine were determined at the time of collectionusing PAL High Accuracy Electrochemistry Test Pen and Free Chlorine Test Kitfor chlorine estimation, respectively.
Microbiological analysis
Samples were analyzed within 4 hours of collection by membrane filtrationmethod to determine the total coliform per 100 ml at 3rc and fecal coliform at44'C on Eosine Methylene Blue (EMS) agar after 24 hours incubation. Colonieswere counted and all distinct colony types were transferred from Eosine
. Methylene Slue (EMS) agar to Trypticase Soya agar (TSA) plates; isolatedcolonies from Trypticase Soya agar (TSA) plates were Gram stained and theoxidase test W2,S carried out All colonies tested were Gram negative, oxidasenegative rods,
RESULT:::
pH and residual chlorine
The pH of all samples was determined and found within the limits of WHOguidelines for drinking water, i.e., 6.5-8,5,Table 1 shows the m:nimum andmaximum values of pH in all water samples, i.e" the pH of main reservoir rangedfrom 6.31-7.41, that of distribution line the range was 6,78-7.81 and that ofconsumer taps was 6.87-7.5. The residual chlorine was not detected in allsamples collected from different locations, whereas WHO has recommended 0.2-05 mg/l for drinking water (Table 2).
Table 1: pH of water samples collected from different sites
Site n Minimum Maximum Average WHO limitsvalue value .
Mainreservoir 30 6,31 7.41 7.37 65-8,5
Distributionline 30 6.78 781 7.09 65-8.5
Consumertaps 30 6.87 7.5 7.01 6.5-85
Table 2: Residual Chlorine content in water samples collected fromdifferent sitesI
ISite n Minimum Maximum Average WHO limits
value valueMain
reservoir 30 0 0 0 0.2-0.5
Distributionline 30 0 0 0 0.2-0.5
Consumertaps 30 0 0 0 0.2-05
94 A. H. SHAR ET AL. BIOLOGIA VOL. 54 (1) ENUMERATION OF TOTAL AND FECAL COLIFORM BACTERIA ,IN DRINKING WATER 95
j4l: .
Microbiological analysis
Samples collected from different sites, viz: main reservoir, distribution lineand consumer taps, were used for quantitative and qualitative bacteriologicalanalysis. The total and fecal coliform bacteria were detected in all samples(100%) of drinking water of Khairpur city where the main source of supply wassurface water. The total coliforms range was found to be from 3.0 to 3.94 colonyforming units (log cfu), and fecal coliform (Escherichia calf) range was from 1.46-2.47 cfu per 100 ml. Table 3 shows the results of bacteriological parameters ofwater samples of different sites. There is variation in number of colony formingunits (clu) of different samples, i.e., main reservoir samples (3.00-368 cluj,distribution line (3.97-4.06) and consumer taps (400-4.15 cfu/100ml)
Table' 3: Microbiological analysis giving minimum, maximum and averageof total coliform and Escherichia coli
Total coliform (log cful Escherichia coli (log cfulSite N 100ml) 100ml)
Min. Max. Average Min. Max. Average
Mainreservoir 30 3.00 3.68 1.46 2.47 2.47 2.05
Distributionline 30 3.97 406 2.00 326 3.26 300
.
Consumertaps 30 4.00 4.15 2.50 3.53 353 2.99
..~
DISCUSSION
Total and fecal coliform wer: detected with a high freqUenCy't;~rinkingwater distribution network of Khairpur. All samples were found contaminated withtotal as .••well as fecal coliform. Different sites showed varying numbers ofbacteria."The highest occurrence was in consumer taps ranging from 1x 10' to 2x
. ",10', followed by distribution line which was rangin~ from 1.9x10' to 5x10'r ';'.followed by main reservoir that was ranging from 1x 10 to 8x 10' cfuJ 100ml, due
to contamination of water from the sewage due to, perhaps, the damageddistribution lines. Drinking water quality, in both urban and rural areas ofPakistan, is not being managed properly. Results from various investigationsprovide evidence that most of the' drinking water supplies are fecallycontaminated.
Cryptosporidium was isolated in drinking water of some residential areas ofLahore. Ihsanullah et al. (1999) isolated E. coli from drinking water of Risalpur,Pubbi, and Turnab. The present study also supports the above reports with. regard to the quality of drinking water in many parts of Pakistan. The detection oftotal coliform and E. coli in a large numbers (cfu count of 1x 10' to 8x lO' I100ml), denotes a health concern in drinking water. The WHO guideline valuesfor drinking water are zero cful 100ml for total as well as for fecal coliformbacteria. Keeping in view the above results, it can be said that the bacteriologicalquality of drinking water of Khairpur does not fulfill the standards of WHO. Theresidual chlorine was also not detected in any of drinking water samples whichshow absence of disinfection treatment of distribution system.
Further work is needed to assess the quality of drinking water of otherplaces where this type of work has not been carried out so far, so that somesuitable disinfective measures are taken to make the drinking water free of the.bacteria. The presence of any such contaminants in drinking water constitutes ahealth problem. A regular check of the distribution lines and removal of damagedlines to prevent fecal contamination from sewage line is also suggested.' .
REFERENCES
Alnoso. J. L., Soriano, S. & Amoros, O. S., 1999. Comparison and recovery ofEscherichia coli and thermotolerant coliforms in water with a chromogenicmedium incubated at41 and 44 °C J. App & Env. Mic:, 65: 3746-3749 .
Anderson, K. L., Whitlock, J. E. & Harwood, V. J., 2005. Persistence anddifferential survival of fecal indicator in subtropical waters and sediments. JAppl. & Env. Mic., 71: 3041-3048.
Aneja, K. R., 1993 Experiments in Microbiology: Plant Pathology Tissue cultureand Mushroom Cultivation. 2"' edition. Wishwa Pakshan., pp 288 and 301.
Anonymous, 1997. Guidelines For Drinking Water Quality Vol. 3 (2"' Edition).WHO, Geneva.
Araujo, R. M., Puig, A., Lasobras, L., Lucena, F. & Jofre, J., 1997. Phages ofenteric bacteria in freshwater with different levels of fecal pollution., J Appl.Mic, 82 (3) 281-286.
Blumenthal, U., Peasey, J, Ruiz-Palacios, G. & Mara, D. D., 1999. Guidelines forwastewater reuse in Agriculture and Aquaculture: Recommended revisionbased on new research evidence. WELL Study London School of Hygiene &Tropical Medicine and WEDC, London.
Clesceri. L.S., Grcenberg, A. & Eaton, A., (eds) 1998. Standrad Methods For The. Examination Of Water And Wastewater, 20th ed" American Public
Health Association, Washington.EJaz, M. & Ahmed, A., 2001. Physical, chemical and biological parameters in well
waters of Karachi and their health impacts. J. Chern Soc Pak .. 23: 263-267.
.!f':~~- , ';r
94 A. H. SHAR ET AL. BIOLOGIA VOL. 54 (1) ENUMERATION OF TOTAL AND FECAL COLIFORM BACTERIA ,IN DRINKING WATER 95
j4l: .
Microbiological analysis
Samples collected from different sites, viz: main reservoir, distribution lineand consumer taps, were used for quantitative and qualitative bacteriologicalanalysis. The total and fecal coliform bacteria were detected in all samples(100%) of drinking water of Khairpur city where the main source of supply wassurface water. The total coliforms range was found to be from 3.0 to 3.94 colonyforming units (log cfu), and fecal coliform (Escherichia calf) range was from 1.46-2.47 cfu per 100 ml. Table 3 shows the results of bacteriological parameters ofwater samples of different sites. There is variation in number of colony formingunits (clu) of different samples, i.e., main reservoir samples (3.00-368 cluj,distribution line (3.97-4.06) and consumer taps (400-4.15 cfu/100ml)
Table' 3: Microbiological analysis giving minimum, maximum and averageof total coliform and Escherichia coli
Total coliform (log cful Escherichia coli (log cfulSite N 100ml) 100ml)
Min. Max. Average Min. Max. Average
Mainreservoir 30 3.00 3.68 1.46 2.47 2.47 2.05
Distributionline 30 3.97 406 2.00 326 3.26 300
.
Consumertaps 30 4.00 4.15 2.50 3.53 353 2.99
..~
DISCUSSION
Total and fecal coliform wer: detected with a high freqUenCy't;~rinkingwater distribution network of Khairpur. All samples were found contaminated withtotal as .••well as fecal coliform. Different sites showed varying numbers ofbacteria."The highest occurrence was in consumer taps ranging from 1x 10' to 2x
. ",10', followed by distribution line which was rangin~ from 1.9x10' to 5x10'r ';'.followed by main reservoir that was ranging from 1x 10 to 8x 10' cfuJ 100ml, due
to contamination of water from the sewage due to, perhaps, the damageddistribution lines. Drinking water quality, in both urban and rural areas ofPakistan, is not being managed properly. Results from various investigationsprovide evidence that most of the' drinking water supplies are fecallycontaminated.
Cryptosporidium was isolated in drinking water of some residential areas ofLahore. Ihsanullah et al. (1999) isolated E. coli from drinking water of Risalpur,Pubbi, and Turnab. The present study also supports the above reports with. regard to the quality of drinking water in many parts of Pakistan. The detection oftotal coliform and E. coli in a large numbers (cfu count of 1x 10' to 8x lO' I100ml), denotes a health concern in drinking water. The WHO guideline valuesfor drinking water are zero cful 100ml for total as well as for fecal coliformbacteria. Keeping in view the above results, it can be said that the bacteriologicalquality of drinking water of Khairpur does not fulfill the standards of WHO. Theresidual chlorine was also not detected in any of drinking water samples whichshow absence of disinfection treatment of distribution system.
Further work is needed to assess the quality of drinking water of otherplaces where this type of work has not been carried out so far, so that somesuitable disinfective measures are taken to make the drinking water free of the.bacteria. The presence of any such contaminants in drinking water constitutes ahealth problem. A regular check of the distribution lines and removal of damagedlines to prevent fecal contamination from sewage line is also suggested.' .
REFERENCES
Alnoso. J. L., Soriano, S. & Amoros, O. S., 1999. Comparison and recovery ofEscherichia coli and thermotolerant coliforms in water with a chromogenicmedium incubated at41 and 44 °C J. App & Env. Mic:, 65: 3746-3749 .
Anderson, K. L., Whitlock, J. E. & Harwood, V. J., 2005. Persistence anddifferential survival of fecal indicator in subtropical waters and sediments. JAppl. & Env. Mic., 71: 3041-3048.
Aneja, K. R., 1993 Experiments in Microbiology: Plant Pathology Tissue cultureand Mushroom Cultivation. 2"' edition. Wishwa Pakshan., pp 288 and 301.
Anonymous, 1997. Guidelines For Drinking Water Quality Vol. 3 (2"' Edition).WHO, Geneva.
Araujo, R. M., Puig, A., Lasobras, L., Lucena, F. & Jofre, J., 1997. Phages ofenteric bacteria in freshwater with different levels of fecal pollution., J Appl.Mic, 82 (3) 281-286.
Blumenthal, U., Peasey, J, Ruiz-Palacios, G. & Mara, D. D., 1999. Guidelines forwastewater reuse in Agriculture and Aquaculture: Recommended revisionbased on new research evidence. WELL Study London School of Hygiene &Tropical Medicine and WEDC, London.
Clesceri. L.S., Grcenberg, A. & Eaton, A., (eds) 1998. Standrad Methods For The. Examination Of Water And Wastewater, 20th ed" American Public
Health Association, Washington.EJaz, M. & Ahmed, A., 2001. Physical, chemical and biological parameters in well
waters of Karachi and their health impacts. J. Chern Soc Pak .. 23: 263-267.
.!f':~~- , ';r
96 A. H. SHAR ET AL BIOLOGIA BIOLOGIA{PAKISTAN) 2008, 54 (1), 9T-103PK ISSN 0006 - 3096
Feachman, R G., Bradley, D. J., Garelick, H. & Dunean, M. .0., 1983. SanitationAnd Disease: Health Aspects Of Excreta And Wastewater Management.Published for World Bank by John Willey and Sons. New York. U S. A
Gleeson, C. & Gary, N., 1997. The Coliform Index And Waterborne DiseaseLondon. U. K.
Ihsanullah, M. K., Khattak, T. N. & Sattar, A, 1999. Determination of differentcontaminants in selective drinking water samples. The Nucleus, 36: 91-97.
Kaltenthaler, E. C., Drasar, F. B. & Potter, C. W., 1996. The Use Of MicrobiologyIn Study Of Hygiene Behavior. Published by the Faculty Press, Cambridge,Great Britain, 88: 35-43.
Kirschner T., Alexander K., Thomas, C. Zechmeister, G., Kavka, G, Christian,B., Alios, H., Mach, R L. & Farnleitner, H. R, 2004. Integral strategy forevaluation of fecal indicator performance in bird-influenced saline inlandwater. J App. & Env. Mic., 70: 7396-7403
Kisteman, T., ClaBen, T., Koch, C., Dangendorf, F., Fischeder, R., Gebel. J.,Vacata, V. & Exner. M., 2002. Microbial load of drinking water resesvoir
- tributaries during extreme rainfall and runoff. J App. & Env. Mic, 68: 2188-2197.
Lee, D. G. & Kim, S. J., 2003. Bacterial species in biofilms cultivated from theend of the Seoul water distribution system. J Appl. Mic., 95: 317-324.
Leoni, E., De Luca, G., Legnan, P.P., Sacchetti, R., Stampi, S. & Zanetti F.,2005. Legionella waterline colonization: detectiOfl of Legionella species in. domestic, hotel and hospital hot water systems. J Appl. Mic., 98: 373-379.
Mellado,' V., Yanez, M. A & Catalan, V., 2006. Evaluation of the Microfoss:Systemfor the analysis of Escherichia coli in water. J Micro. Res., 161: 20-24'.,
Roslev, P., Bjergbaek, L. A & Hesselsoe, M., 2004. Effect of oxygen on survivalcif fecal pollution indicators in drinking water. J Appl. Mic., 96: 938-945.
Thurman, R, Faulkner, B., Veal, D., Cramer, G. & Meiklejohn, M, 1998. Waterquality in Australia. J Appl. Micro, 84: 627-632.
Effects of chemical pretreatments of scales offish (Labeo rollita) onthe biosorption of lead from synthetic solution
MUllHAMAD ZAHID QURESHI, AYOUB RASHID, SYED ALI RAZA,MUHAMMAD FAHEEM ASIM, FAKHIRA RASHID & ARSHAD
Department q/Chemislry, GC U11l\;e,..\'i~)I,Lahore. Pakistan
ABSTRACT
The biosorption of heavy metals from aqueous solutions was investigated, usinga cheap and abundant dry biomass of fish (Labeo raMa) scales, The factorsinfluencing lead uptake were found to be contact time, pH, inITialconcentration, size andbiosorbant dose. The biosorption was solution pH dependent and the maximumadsorptionwas obtainedat a pH of 4. The maximummetaluptakevalues(qmax,mg/g)were found to be 87,79,The LangmuirModeland FreundlichEquationwere appliedtothe exp~rimental data. The Langmuir Model was found to be in better correlation with theexperimental data.
Key words: BiOSOl)Jlion,Lead,Labeo raMa, Scales
INTRODUCTION;
The discharge of heavy metals into aquatic ecosystems has become a matter ofconcern in Pakistan over the last few decades, These pollutants are introduced intothe aquatic systems significantly as a result of various industrial operations. Heavymetals have been used in a variety of ways for at least 2 millennia. Lead has been used inplumbing, paints and lead arsenate has been used to control insects in crops. Heavymetals, such as zinc, lead and chromium, have a number of applications in basicengineering works, paper and pulp industries, leather tanning, organochemicals,petrochemicals, fertlizers, etc. (Trivedi, 1989).
The human exposure to lead comes from mining operations, refining ores,sludge disposal, fly ash from incinerators, the processing of radioactive materials,metal plating, or the manufacture of electrical equipment, paints, alloys, batteries,pesticides or preseNatives. Major lead pcllution is through automobiles and batterymanufacturers. Over the few decades, several methods have been devised for thetreatment and removal of lead (Steve & Jennifer, 2005).
Lead is known to cause neurological damage in humans, with children beingmost susceptible to the toxic effects of lead ,Some of the lead that is present in theenvironment is from its use in batteries, gasoline, hunting ammunition, and paintNo biological function for lead has been identified. Lead concentrations up to 300 ppmwere found to stimulate growth in Escherichia coli, but higher lead concentrationsinhibited growth (Kumar & Upreti,2000),
96 A. H. SHAR ET AL BIOLOGIA BIOLOGIA{PAKISTAN) 2008, 54 (1), 9T-103PK ISSN 0006 - 3096
Feachman, R G., Bradley, D. J., Garelick, H. & Dunean, M. .0., 1983. SanitationAnd Disease: Health Aspects Of Excreta And Wastewater Management.Published for World Bank by John Willey and Sons. New York. U S. A
Gleeson, C. & Gary, N., 1997. The Coliform Index And Waterborne DiseaseLondon. U. K.
Ihsanullah, M. K., Khattak, T. N. & Sattar, A, 1999. Determination of differentcontaminants in selective drinking water samples. The Nucleus, 36: 91-97.
Kaltenthaler, E. C., Drasar, F. B. & Potter, C. W., 1996. The Use Of MicrobiologyIn Study Of Hygiene Behavior. Published by the Faculty Press, Cambridge,Great Britain, 88: 35-43.
Kirschner T., Alexander K., Thomas, C. Zechmeister, G., Kavka, G, Christian,B., Alios, H., Mach, R L. & Farnleitner, H. R, 2004. Integral strategy forevaluation of fecal indicator performance in bird-influenced saline inlandwater. J App. & Env. Mic., 70: 7396-7403
Kisteman, T., ClaBen, T., Koch, C., Dangendorf, F., Fischeder, R., Gebel. J.,Vacata, V. & Exner. M., 2002. Microbial load of drinking water resesvoir
- tributaries during extreme rainfall and runoff. J App. & Env. Mic, 68: 2188-2197.
Lee, D. G. & Kim, S. J., 2003. Bacterial species in biofilms cultivated from theend of the Seoul water distribution system. J Appl. Mic., 95: 317-324.
Leoni, E., De Luca, G., Legnan, P.P., Sacchetti, R., Stampi, S. & Zanetti F.,2005. Legionella waterline colonization: detectiOfl of Legionella species in. domestic, hotel and hospital hot water systems. J Appl. Mic., 98: 373-379.
Mellado,' V., Yanez, M. A & Catalan, V., 2006. Evaluation of the Microfoss:Systemfor the analysis of Escherichia coli in water. J Micro. Res., 161: 20-24'.,
Roslev, P., Bjergbaek, L. A & Hesselsoe, M., 2004. Effect of oxygen on survivalcif fecal pollution indicators in drinking water. J Appl. Mic., 96: 938-945.
Thurman, R, Faulkner, B., Veal, D., Cramer, G. & Meiklejohn, M, 1998. Waterquality in Australia. J Appl. Micro, 84: 627-632.
Effects of chemical pretreatments of scales offish (Labeo rollita) onthe biosorption of lead from synthetic solution
MUllHAMAD ZAHID QURESHI, AYOUB RASHID, SYED ALI RAZA,MUHAMMAD FAHEEM ASIM, FAKHIRA RASHID & ARSHAD
Department q/Chemislry, GC U11l\;e,..\'i~)I,Lahore. Pakistan
ABSTRACT
The biosorption of heavy metals from aqueous solutions was investigated, usinga cheap and abundant dry biomass of fish (Labeo raMa) scales, The factorsinfluencing lead uptake were found to be contact time, pH, inITialconcentration, size andbiosorbant dose. The biosorption was solution pH dependent and the maximumadsorptionwas obtainedat a pH of 4. The maximummetaluptakevalues(qmax,mg/g)were found to be 87,79,The LangmuirModeland FreundlichEquationwere appliedtothe exp~rimental data. The Langmuir Model was found to be in better correlation with theexperimental data.
Key words: BiOSOl)Jlion,Lead,Labeo raMa, Scales
INTRODUCTION;
The discharge of heavy metals into aquatic ecosystems has become a matter ofconcern in Pakistan over the last few decades, These pollutants are introduced intothe aquatic systems significantly as a result of various industrial operations. Heavymetals have been used in a variety of ways for at least 2 millennia. Lead has been used inplumbing, paints and lead arsenate has been used to control insects in crops. Heavymetals, such as zinc, lead and chromium, have a number of applications in basicengineering works, paper and pulp industries, leather tanning, organochemicals,petrochemicals, fertlizers, etc. (Trivedi, 1989).
The human exposure to lead comes from mining operations, refining ores,sludge disposal, fly ash from incinerators, the processing of radioactive materials,metal plating, or the manufacture of electrical equipment, paints, alloys, batteries,pesticides or preseNatives. Major lead pcllution is through automobiles and batterymanufacturers. Over the few decades, several methods have been devised for thetreatment and removal of lead (Steve & Jennifer, 2005).
Lead is known to cause neurological damage in humans, with children beingmost susceptible to the toxic effects of lead ,Some of the lead that is present in theenvironment is from its use in batteries, gasoline, hunting ammunition, and paintNo biological function for lead has been identified. Lead concentrations up to 300 ppmwere found to stimulate growth in Escherichia coli, but higher lead concentrationsinhibited growth (Kumar & Upreti,2000),
98 M. Z. QURESHI ET AL BIOlOGIA Val. 54 (1) EFFECTS OF CHEMICAL TREATMENT OF FISH SCALES ON BIOSORPTION 99
RE$UL TS AND DISCUSSION
Cf = Final Pb (II) concentration in solution, after the adsorption test (mg-r').M =Weight of biosorbent i.e. scales of fish (Labeo rohita)V = The solution volume (ml)
Effect of biosoment doseResults show that with increase in biosorbent dose, % removal of lead was
also increased. There was maximum adsorption capacity (53.86 mg/g) and % removal(89.61)at 0.15 biosorbent dose (Gadd et ai, 1988 and Fourest et ai, 1992)
6543PH
2
Fig. 1: Effect of pH
1
180160140120
~;100
~ 800' 60
402000
Effect of pH
Adsorption capacity was determined over the pH range 1-6 (Fig. 1). It wasobserved that Adsorption capacity increased with the increase in pH to a certainlimit and then decreased. There was maximum adsorption capacitY (q) (167.60mg/g) at pH 4 and at high pH hydroxy complexes were fomned(Sag et ai, 1995).
Heavy metal releases to the environment have been increasing continuously as aresult of activities and technological development, becoming a significant threat to theenvironment and public health because of their toxicity, accumulation in the food chainand persistenoe in nature. It is, therefore, important to develop new methods for metalremoval and recovery from dilute solution (1-100mg/I) and for the reduction of heavymetal ions to very low concentration. The use of conventional technologies, such asion exchange, chemical precipitation, reverse osmosis are insufficient or veryexpensive (Chong & Volesky, 1995 and Leusch etal, 1995)
The search for new technologies involving the removal of toxic metals trom wastewaters has directed attention to biosorption, based on metal binding capacities ofvarious biological materials.
The biosorption process involves a solid phase (sorbent or biosorbent; biologicalmaterial) and a liquid phase (solvent, normally water) containing a dissolved species tobe sorbed (sorbate, metal ions). Due to higher affinity of the sorbent for the sorbatespecies, the latter is attracted and bound there by different mechanisms. Theprocess continues till equilibrium is established between the amount of solid-boundsorbate species and its portion remaining in the solution. The degree of sorbentaffinity for the sorbate detemnines itsdistributionbetween the solid and liquidphases
Fish scales are composed of collagen which is the most important nutrient forbody metabolism. More than a third of the body's protein is collagen. It is a majorstructural protein constituting the major element of skin, bone, tendon, cartilage,blood vessels, teeth, and other connective tissue.
MATERIALS & METHODS
The biomass (scales of tish Labeo rohita) was washed with distilled water anddivided into three equal parts. One part was treated with 0.1M Benzene, other witho 1M Formaldehyde, and the third part with 0.1M Mercuric chloride for four hours. Aftertreatment biosorbent was washed with distilled water until a pH of 7 was obtained. Driedbiomass was grinded and screened through sieves to 'get three sizes, i.e., 40 micron, 80micronand 150micron.
The present research was carried out in following steps (Qaiser et ai, '999). In all theexperiments 100 ml of lead (II) solution was taken in each 250 ml conical flask, andone conical fiask as a control without adding biosorbent for each experiment pH wasadjusted by adding 0.1M NaOH and 0.1M H2S04. Dried dose of biosorbent was added toeach conical flask and the mouth was sealed with aluminum foil. The solutions wereagitated on orbital shaker incubator at 130:t2 rev/min for 24 hours at 30:t.2'C temperature.After 24 hours, samples were filtered and stored in sample bottles. The lead concentrationwas determined after 10 times diluting the solution, by Perkin Elmer Atomic AbsorptionSpectrophotometer.Uptake of Pb (illion was calculated from a mass balance equation
q = V (ei - CDM
where,q = Pb (II) uptake capacity (mg Pb (II) I g fish scales biomass)Ci = Initial Pb (II) concentration in solution, before the adsorption test (mg-r')
98 M. Z. QURESHI ET AL BIOlOGIA Val. 54 (1) EFFECTS OF CHEMICAL TREATMENT OF FISH SCALES ON BIOSORPTION 99
RE$UL TS AND DISCUSSION
Cf = Final Pb (II) concentration in solution, after the adsorption test (mg-r').M =Weight of biosorbent i.e. scales of fish (Labeo rohita)V = The solution volume (ml)
Effect of biosoment doseResults show that with increase in biosorbent dose, % removal of lead was
also increased. There was maximum adsorption capacity (53.86 mg/g) and % removal(89.61)at 0.15 biosorbent dose (Gadd et ai, 1988 and Fourest et ai, 1992)
6543PH
2
Fig. 1: Effect of pH
1
180160140120
~;100
~ 800' 60
402000
Effect of pH
Adsorption capacity was determined over the pH range 1-6 (Fig. 1). It wasobserved that Adsorption capacity increased with the increase in pH to a certainlimit and then decreased. There was maximum adsorption capacitY (q) (167.60mg/g) at pH 4 and at high pH hydroxy complexes were fomned(Sag et ai, 1995).
Heavy metal releases to the environment have been increasing continuously as aresult of activities and technological development, becoming a significant threat to theenvironment and public health because of their toxicity, accumulation in the food chainand persistenoe in nature. It is, therefore, important to develop new methods for metalremoval and recovery from dilute solution (1-100mg/I) and for the reduction of heavymetal ions to very low concentration. The use of conventional technologies, such asion exchange, chemical precipitation, reverse osmosis are insufficient or veryexpensive (Chong & Volesky, 1995 and Leusch etal, 1995)
The search for new technologies involving the removal of toxic metals trom wastewaters has directed attention to biosorption, based on metal binding capacities ofvarious biological materials.
The biosorption process involves a solid phase (sorbent or biosorbent; biologicalmaterial) and a liquid phase (solvent, normally water) containing a dissolved species tobe sorbed (sorbate, metal ions). Due to higher affinity of the sorbent for the sorbatespecies, the latter is attracted and bound there by different mechanisms. Theprocess continues till equilibrium is established between the amount of solid-boundsorbate species and its portion remaining in the solution. The degree of sorbentaffinity for the sorbate detemnines itsdistributionbetween the solid and liquidphases
Fish scales are composed of collagen which is the most important nutrient forbody metabolism. More than a third of the body's protein is collagen. It is a majorstructural protein constituting the major element of skin, bone, tendon, cartilage,blood vessels, teeth, and other connective tissue.
MATERIALS & METHODS
The biomass (scales of tish Labeo rohita) was washed with distilled water anddivided into three equal parts. One part was treated with 0.1M Benzene, other witho 1M Formaldehyde, and the third part with 0.1M Mercuric chloride for four hours. Aftertreatment biosorbent was washed with distilled water until a pH of 7 was obtained. Driedbiomass was grinded and screened through sieves to 'get three sizes, i.e., 40 micron, 80micronand 150micron.
The present research was carried out in following steps (Qaiser et ai, '999). In all theexperiments 100 ml of lead (II) solution was taken in each 250 ml conical flask, andone conical fiask as a control without adding biosorbent for each experiment pH wasadjusted by adding 0.1M NaOH and 0.1M H2S04. Dried dose of biosorbent was added toeach conical flask and the mouth was sealed with aluminum foil. The solutions wereagitated on orbital shaker incubator at 130:t2 rev/min for 24 hours at 30:t.2'C temperature.After 24 hours, samples were filtered and stored in sample bottles. The lead concentrationwas determined after 10 times diluting the solution, by Perkin Elmer Atomic AbsorptionSpectrophotometer.Uptake of Pb (illion was calculated from a mass balance equation
q = V (ei - CDM
where,q = Pb (II) uptake capacity (mg Pb (II) I g fish scales biomass)Ci = Initial Pb (II) concentration in solution, before the adsorption test (mg-r')
100 M. Z. QURESHI ET AL. BIOLOGIA VOL. 54 (1) EFFECTS OF CHEMICAL TREATMENT OF FISH SCALES ON BIOSORPTION 101
Effect of biosorbent sizeThe % removal for 150mic, 80mic, 40mic, size is 84.56,85.01,89,26 and q values51.26, 52.16, 54.75 as shown by Fi9. 2. Its reason is because by decreasing size ofbiosorbent, its surface area increases which provides more binding sites for adsorption(Raji et al., 1997)
Kinetic study revealed that biosorption took place in two steps, a rapidsurface adsorption within fifteen minutes and a slow intercellular adsorption up to theend time. This agrees with the previous results for the removal of heavy metalcontamination by bacteria and other microorganisms.
1000800600400
Initial metal concentration(mg/LI
200
Fig. 3: Effect of different initial metal concentrations
o
90~~IN~60o~W~~~~30~20
10o
200150100
Mesh size
50
55
54.5
54
Ci 5i5-Cl 53E1ij' 52.5
52
51.6
51
0
Fig. 2: Effect of biosorbent size
Effect of different initial metal concentrations 60
I ,
III
! Ix'
I i j ;1
f,
!, , I! !,
1 : I I !I , I..'!,
'7 I 1 ,I I,.{ , I9 j !
I ~ i rI
, l I I. I«, ! 1, ! . ,f;. , I I
15 30 60 120240 420 660 1440Time in minutes
Fig. 4: Kinetic study (Time during which maximum biosorption occurred)
o
10
~40~g30C' 20
60
II'
The increase in metal concentration increased the adsorption capacity andthe decrease of the % removal of lead (II). At 100mgn initial metal concentration therewas maximum (84.22 %) removal and adsorption capacity (q) (54.22m9/9) as shown byFi9. 3. As at higher initial metal concentration the ratio of initial number of moles oflead (II) to the available surface area is high, hence fractional biosorptionbecomes dependent on initial metal concentration (Anoop et al., 2003).
Kinetic study
In this experiment it was obselVed that maximum biosorption of lead (II)occurred mostly within fifteen minutes. Adsorption capacity (q) at different timeintelVals ranging from zero time to 24 hours was analyzed. The adsorption capacityand % removal till 15 minutes was 22.10 mg/g and 35.55, respectively. After 7h theequilibrium is established and it remains constant till 24h. Maximum adsorptioncapacity and % removal was 50.69 and 81.52,respectively(Fig 4).
II
IIII,
100 M. Z. QURESHI ET AL. BIOLOGIA VOL. 54 (1) EFFECTS OF CHEMICAL TREATMENT OF FISH SCALES ON BIOSORPTION 101
Effect of biosorbent sizeThe % removal for 150mic, 80mic, 40mic, size is 84.56,85.01,89,26 and q values51.26, 52.16, 54.75 as shown by Fi9. 2. Its reason is because by decreasing size ofbiosorbent, its surface area increases which provides more binding sites for adsorption(Raji et al., 1997)
Kinetic study revealed that biosorption took place in two steps, a rapidsurface adsorption within fifteen minutes and a slow intercellular adsorption up to theend time. This agrees with the previous results for the removal of heavy metalcontamination by bacteria and other microorganisms.
1000800600400
Initial metal concentration(mg/LI
200
Fig. 3: Effect of different initial metal concentrations
o
90~~IN~60o~W~~~~30~20
10o
200150100
Mesh size
50
55
54.5
54
Ci 5i5-Cl 53E1ij' 52.5
52
51.6
51
0
Fig. 2: Effect of biosorbent size
Effect of different initial metal concentrations 60
I ,
III
! Ix'
I i j ;1
f,
!, , I! !,
1 : I I !I , I..'!,
'7 I 1 ,I I,.{ , I9 j !
I ~ i rI
, l I I. I«, ! 1, ! . ,f;. , I I
15 30 60 120240 420 660 1440Time in minutes
Fig. 4: Kinetic study (Time during which maximum biosorption occurred)
o
10
~40~g30C' 20
60
II'
The increase in metal concentration increased the adsorption capacity andthe decrease of the % removal of lead (II). At 100mgn initial metal concentration therewas maximum (84.22 %) removal and adsorption capacity (q) (54.22m9/9) as shown byFi9. 3. As at higher initial metal concentration the ratio of initial number of moles oflead (II) to the available surface area is high, hence fractional biosorptionbecomes dependent on initial metal concentration (Anoop et al., 2003).
Kinetic study
In this experiment it was obselVed that maximum biosorption of lead (II)occurred mostly within fifteen minutes. Adsorption capacity (q) at different timeintelVals ranging from zero time to 24 hours was analyzed. The adsorption capacityand % removal till 15 minutes was 22.10 mg/g and 35.55, respectively. After 7h theequilibrium is established and it remains constant till 24h. Maximum adsorptioncapacity and % removal was 50.69 and 81.52,respectively(Fig 4).
II
IIII,
In order to understand the adsorption process, the Langmuir and FreundlichIsotherm were used to represent the equilibrium relationship for different initial lead (II)concentration experiment. Langmuir Equation transforms to the linearized form:
103EFFECTS OF CHEMICAL TREATMENT OF FISH SCALES ON BIOSORPTIONVOL. 54 (1)
Qaiser , M K, Roux, J. C, Khalid, Z. M. & Malik, K A, 1999. Application ofindustrial wastes biomass for chromium removal from tannery effluent. Biohor,2(1-4) 112-117.
Raji, C., Manju, G. N. & Anirudhan, T. S., 1997.Studies on Chromium (6) adsorption -desorptionusing immobilizedfungal biomass.Biore. Technol., 87: 17-26.
Trivedi, R K., 1989. Pollutionmanagement in industres. Environ, Technof., 13: 579-586.Sag, Y., Tatar, B. & Kutsal, T., 2003. Biosorption of Pb (II) and Cu (II) by activated
sludge in batch and oonijnuous-flow stirred reactors. Biore. Technof., 93(1): 27-33.Steve, T. & Jennifer, D., 2005. Sources of human exposure to lead. Biotechnol.,
14(8): 737-740.
BIOLOGIAM. Z. QURESHI ET AL
1/qe = 1/q max + II (b.q max) Ce
102
Adsorption isothenn
where,I/qe = interceptsCe= equilibriumooncentrationb = slopeq "'" = maximum adsorptioncapacity
The Langmuir Equation assumes that surface of the biosorbent consists ofadsorption sites; all adsorbed species interact with only a site and not with each otherand adsorption is limited to monolayer. It is then assumed that once a metal ionoccupies a site, no further sorption can take place. The capacity of fish scalesbiomass in binding with lead (II) was determined by plotting l/qe against VCe, using theLangmuir equation. The Freundlich Equation is an empirical relationship describing theadsoption of the solutes from a liquid to solid surface. Linearized form of FreundlichEquation is:
log q e = log K + 1/n log C ewhere,
K = interceptlin = slopeq e = equilibriumadsorption capacity.
REFERENCES
Anoop, K, Krishnan & Anirudhan, T. S., 2003. Removal of Cadmium (II) from aqueoussolution by steam activated sulphurised carbon prepared from sugar canebagasse pith:Kinefics and equilibrium sludies. Waler S.A. 29: 56-59.
Chong, K. H. & Voiesky, B., 1995. Description of two metal biosorption equilibria byLangmuir- Types models. Biotechnof. Bioeng., 47: 1 -10.
Fourest, E & Roux, C. J., 1992. Heavy metal biosorption by fungal mycial byproductmechanism and influence of pH. Appf. Microb. and Biotech, 37(3) 399-403.
Gadd, G. M., Norris, P. R. & Kelly, D. P., 1988. Heavy metal and radio nuclide byfungi and yeasts Bio Hydrometaflurgy, (ed.A Rowe) Chippenham,Wilt., U K.
Kumar, M.S. & Upreti,' S. 2000. Removal of metal ions using an industria withreference to environmental oontrol. Int. J. Miner Proce., 53: 107 -120.
Leusch, L, Holn, Z. R, & Volesky, 6., 1995. Biosorption of heavy metals(Cd,Cu,Ni,Pb,Zn) by chemically reinforced biomass of marine algae. J.Chern Techno/. Biotec, 62(3): 279-288.
I
1
'1
I'
I:
i'
In order to understand the adsorption process, the Langmuir and FreundlichIsotherm were used to represent the equilibrium relationship for different initial lead (II)concentration experiment. Langmuir Equation transforms to the linearized form:
103EFFECTS OF CHEMICAL TREATMENT OF FISH SCALES ON BIOSORPTIONVOL. 54 (1)
Qaiser , M K, Roux, J. C, Khalid, Z. M. & Malik, K A, 1999. Application ofindustrial wastes biomass for chromium removal from tannery effluent. Biohor,2(1-4) 112-117.
Raji, C., Manju, G. N. & Anirudhan, T. S., 1997.Studies on Chromium (6) adsorption -desorptionusing immobilizedfungal biomass.Biore. Technol., 87: 17-26.
Trivedi, R K., 1989. Pollutionmanagement in industres. Environ, Technof., 13: 579-586.Sag, Y., Tatar, B. & Kutsal, T., 2003. Biosorption of Pb (II) and Cu (II) by activated
sludge in batch and oonijnuous-flow stirred reactors. Biore. Technof., 93(1): 27-33.Steve, T. & Jennifer, D., 2005. Sources of human exposure to lead. Biotechnol.,
14(8): 737-740.
BIOLOGIAM. Z. QURESHI ET AL
1/qe = 1/q max + II (b.q max) Ce
102
Adsorption isothenn
where,I/qe = interceptsCe= equilibriumooncentrationb = slopeq "'" = maximum adsorptioncapacity
The Langmuir Equation assumes that surface of the biosorbent consists ofadsorption sites; all adsorbed species interact with only a site and not with each otherand adsorption is limited to monolayer. It is then assumed that once a metal ionoccupies a site, no further sorption can take place. The capacity of fish scalesbiomass in binding with lead (II) was determined by plotting l/qe against VCe, using theLangmuir equation. The Freundlich Equation is an empirical relationship describing theadsoption of the solutes from a liquid to solid surface. Linearized form of FreundlichEquation is:
log q e = log K + 1/n log C ewhere,
K = interceptlin = slopeq e = equilibriumadsorption capacity.
REFERENCES
Anoop, K, Krishnan & Anirudhan, T. S., 2003. Removal of Cadmium (II) from aqueoussolution by steam activated sulphurised carbon prepared from sugar canebagasse pith:Kinefics and equilibrium sludies. Waler S.A. 29: 56-59.
Chong, K. H. & Voiesky, B., 1995. Description of two metal biosorption equilibria byLangmuir- Types models. Biotechnof. Bioeng., 47: 1 -10.
Fourest, E & Roux, C. J., 1992. Heavy metal biosorption by fungal mycial byproductmechanism and influence of pH. Appf. Microb. and Biotech, 37(3) 399-403.
Gadd, G. M., Norris, P. R. & Kelly, D. P., 1988. Heavy metal and radio nuclide byfungi and yeasts Bio Hydrometaflurgy, (ed.A Rowe) Chippenham,Wilt., U K.
Kumar, M.S. & Upreti,' S. 2000. Removal of metal ions using an industria withreference to environmental oontrol. Int. J. Miner Proce., 53: 107 -120.
Leusch, L, Holn, Z. R, & Volesky, 6., 1995. Biosorption of heavy metals(Cd,Cu,Ni,Pb,Zn) by chemically reinforced biomass of marine algae. J.Chern Techno/. Biotec, 62(3): 279-288.
I
1
'1
I'
I:
i'
BIOLOGIA(PAKISTAN) 2008,54 (1),105-116PK ISSN 0006 - 3096
Survival analysis of infants: case study concerning gender andbirth weights
SAIlIA ASfiKAF, MlIfifiAMAD KfiALID J>EKVAIZ, MlIEEN-lIll-DIN AZAIl,HINA KfiAN & NIGHAT ZAHRA
/)epOI'IlIIel1l (~lSfall~\'lics.GC University, Lahore, Pakislan
ABSTRACT
A survival analysis, was carried out on birth weight data collected fromChildren Hospital, Lahore, Pakistan. The Kaplan-Meier Survival Estimates andNelson-Aalen Cumulative Hazard Estimates were obtained for babies withdifferent gender and birth weights. The statistical tests of significance for equalityof survival functions were carried out, which showed that the survival functionswere slightly different for male and female babies but significantly different as faras birth weights were concerned. These Survival Function Tests also showed thatnormal birth weight babies had higher probability of survival as compared with thelow birth weight babieS. The probability of survival in babies with normal birthweight after 6 months was 0.49, while for low birth weight babies this probabilitywas 0.12. Tr,e survival time for low birth weight babies was 24% of normal birthweight babies
Key words: Survival rate, Hazard rate, Kaplan-Meier Estimate, Nelson-AalenCumulative Hazard Estimate, Logrank Test, Life Table
INTRODUCTION
In any community, babies constitute a priority group. In sheer numbers, theycomprise approximately 10 percent of the population of the developing countries.Mothers and babies not only constitute a large group, but they are also a"vulnerable" or special risk group. The risk is connected with childbearing in caseof women and growth and survival in case of infants and babies.
The babies up to one year of age are known as infants. The age group of 0-12 months in babies is the most important age group in all societies, not becauseit constitutes a large number of total populations but because.there is a renewedawareness that the determinants of chronic diseases in later life and healthbehavior are laid down in this stage. Survival of an infant depends upon manyfactors such as the age and education of mother, place of living etc. Certainspecific biological and psychological needs must be met to ensure the survivaland healthy development of the child and the future adult.
Measurement of weight and rate of gain in weight are the best parametersfor assessing physical growth. A single weight record only indicates the child'sweight at the moment, it does not give any information about whether a child'sweight is increasing, stationary or declining. This is because normal variation inweight at a specific age is wide. By comparing the measurements with reference
,I'IIII!I
Ii!'I,
II
III
IO() S. ASHRAF. ET AL. BIOLOGIA VOL. 54 (1) SURVIVAL ANALYSIS OF INFANTS Ill7
to standard weight of babies of the same age, the idea can be developed forincrease, stationary or decline in weights.
In the analysis and research theory, eminent statisticians have used manystatistical tools. A brief account is given below:
Parzen & Lipsitz (1999) developed a global goodness of fit test for CoxRegression Model. The statistics developed by them has an approximate ChiSquare distribution under correctly specified model. Fleming & Lin (1999), in theirstudies, mentioned notable references such as Kaplan & Meier (1958), Cox(1972) and Aalen (1975) Goggins el al. (1999) developed a methodology forestimation of the effect of a binary time-varying covariate on failure times whenthe change time of the covariate is interval censored, whereas, Freidlin el aJ.(1999) worked on the selection of a single method of analysis when the datahave been generated by one of several possible models. Li & Ryan (1999) useda group sequential test for survival trials. It is an alternative to rank-basedprocedures.
Barber & Jennison (1999) developed Symmetric Tests and ConfidenceIntervals for Survival Probabilities and Quintiles of Censored Survival Data. Songel aJ. (2002) employed a Semi Parametric Likelihood Approach to joint modelingof longitudinal and time-to-event data. Maples el aJ. (2002) developed Two-LevelProportional Hazards Models. They extended the proportional Hazards Model toa two-level model with a random intercept term and random coefficients.Whereas, Peto &. Peto (1972) gave a nonparametric generalized maximum-likelihood estimate of the survival function for interval-censored data.
Shen & Cai (2001) formulated maximum of the weighted Kaplan-MeierTests with the application to cancer prevention and screening trials. A class 01maximum weighted Kaplan-Meier Test statistics was described where the weightfunctions were chosen from a family of smooth functions. Lunceford el al. (2002)proposed estimate of survival distributions of treatment policies in two-stagerandomization designs in clinical trials.
MATERIALS AND METHODS
In the modern medical research, the statistical techniques are widelyused. In order to carry out the analysis, the data was collected for the babieswithin the age of '0' to '12' months from Children Hospital, Lahore, Pakistan, OutQf 1287 babies, 598 were female ba.bies. The number of babies with low birthweight was 198. The Survival Functions Estimates were carried out, by usingKaplan-Meier Product Limit, Nelson-Aalen Cumulative Hazard Estimation andLife Table Estimation methods. To check out the significance of the survivalfunction, the Logrank-Wilcoxon (Breslow) and Peto-Peto Tests have been used.
In practice, three-survival functions can be used to illustrate differentaspects of the data. A basic problem in survival data analysis is to estimate fromthe sampled data, one or more of these three functions and to draw inferencesabout the survival pattern in the population, In this research, SurvivorshipFunction and Hazard Function have been used, These are elaborated below:
The survival function denoted by S (t), is defined as the probability that anindividual survives longer than t:
S (t) = P (T > t)
where 'T' denotes the survival time. The graph of S(I) is called the survivalcurve. The Kaplan-Meier (Product Limit) method is used to estimate the SurvivorFunction. A statistical technique useful for censored data called fOllow-up LifeTable, is also used to estimate the survivor function.
The hazard function h(l) of survival time 'T' gives the conditional failurerate. This is defined as the probability of failure during a very small interval"assuming that the individual has survived to the beginning of the interval"(I) = lim [Plan individual of age I fails in the time interval (t, 1 + /1,,1 )}I /1,,(
\( 4(1
The Nelson-Aalen cumulative hazard estimate was used to estimate thehazard estimate. The main focus of interest in survival analysis is comparing thesurvival patterns of different groups, In survival analysis we avoid the assumptionthat the hazards of the event of interest are constant over the study period.
. Instead, we assume that the ratio of the hazards in the two groups remainsconstant over time, even if the underlying hazards change. In other words, weassume that at all times t:
", (I) = constant"0 (I)where h, (t) is the hazard in the exposed group at time t and ho(t) is the
hazard in the unexposed group at time I. This important assumption is known asthe proportional hazards assumption.
The Logrank test is used to check out the equality of two differentsurvivorship' functions. Researchers may wish to statistically compare thesurvival curves of two different groups, While the Kaplan-Meier Product Limitmethod may be used to plot estimates of the survival curve for each of the twogroups of patients The Log-Rank Test is used to determine whether there is astatistically significant difference between the two survival curves.
RESULTS AND DISCUSSION
The survival estimates are calculated by using Kaplan-Meier and LifeTable methods. The Cumulative Hazard Estimates are calculated by using thetechnique of Nelson-Aalen.
1. Estimated Survivor Functions
The Kaplan-Meier Estimated Survivor Function, standard errors andconfidence intervals were calculated for 1287 babies within the age of '0' to '12'
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IO() S. ASHRAF. ET AL. BIOLOGIA VOL. 54 (1) SURVIVAL ANALYSIS OF INFANTS Ill7
to standard weight of babies of the same age, the idea can be developed forincrease, stationary or decline in weights.
In the analysis and research theory, eminent statisticians have used manystatistical tools. A brief account is given below:
Parzen & Lipsitz (1999) developed a global goodness of fit test for CoxRegression Model. The statistics developed by them has an approximate ChiSquare distribution under correctly specified model. Fleming & Lin (1999), in theirstudies, mentioned notable references such as Kaplan & Meier (1958), Cox(1972) and Aalen (1975) Goggins el al. (1999) developed a methodology forestimation of the effect of a binary time-varying covariate on failure times whenthe change time of the covariate is interval censored, whereas, Freidlin el aJ.(1999) worked on the selection of a single method of analysis when the datahave been generated by one of several possible models. Li & Ryan (1999) useda group sequential test for survival trials. It is an alternative to rank-basedprocedures.
Barber & Jennison (1999) developed Symmetric Tests and ConfidenceIntervals for Survival Probabilities and Quintiles of Censored Survival Data. Songel aJ. (2002) employed a Semi Parametric Likelihood Approach to joint modelingof longitudinal and time-to-event data. Maples el aJ. (2002) developed Two-LevelProportional Hazards Models. They extended the proportional Hazards Model toa two-level model with a random intercept term and random coefficients.Whereas, Peto &. Peto (1972) gave a nonparametric generalized maximum-likelihood estimate of the survival function for interval-censored data.
Shen & Cai (2001) formulated maximum of the weighted Kaplan-MeierTests with the application to cancer prevention and screening trials. A class 01maximum weighted Kaplan-Meier Test statistics was described where the weightfunctions were chosen from a family of smooth functions. Lunceford el al. (2002)proposed estimate of survival distributions of treatment policies in two-stagerandomization designs in clinical trials.
MATERIALS AND METHODS
In the modern medical research, the statistical techniques are widelyused. In order to carry out the analysis, the data was collected for the babieswithin the age of '0' to '12' months from Children Hospital, Lahore, Pakistan, OutQf 1287 babies, 598 were female ba.bies. The number of babies with low birthweight was 198. The Survival Functions Estimates were carried out, by usingKaplan-Meier Product Limit, Nelson-Aalen Cumulative Hazard Estimation andLife Table Estimation methods. To check out the significance of the survivalfunction, the Logrank-Wilcoxon (Breslow) and Peto-Peto Tests have been used.
In practice, three-survival functions can be used to illustrate differentaspects of the data. A basic problem in survival data analysis is to estimate fromthe sampled data, one or more of these three functions and to draw inferencesabout the survival pattern in the population, In this research, SurvivorshipFunction and Hazard Function have been used, These are elaborated below:
The survival function denoted by S (t), is defined as the probability that anindividual survives longer than t:
S (t) = P (T > t)
where 'T' denotes the survival time. The graph of S(I) is called the survivalcurve. The Kaplan-Meier (Product Limit) method is used to estimate the SurvivorFunction. A statistical technique useful for censored data called fOllow-up LifeTable, is also used to estimate the survivor function.
The hazard function h(l) of survival time 'T' gives the conditional failurerate. This is defined as the probability of failure during a very small interval"assuming that the individual has survived to the beginning of the interval"(I) = lim [Plan individual of age I fails in the time interval (t, 1 + /1,,1 )}I /1,,(
\( 4(1
The Nelson-Aalen cumulative hazard estimate was used to estimate thehazard estimate. The main focus of interest in survival analysis is comparing thesurvival patterns of different groups, In survival analysis we avoid the assumptionthat the hazards of the event of interest are constant over the study period.
. Instead, we assume that the ratio of the hazards in the two groups remainsconstant over time, even if the underlying hazards change. In other words, weassume that at all times t:
", (I) = constant"0 (I)where h, (t) is the hazard in the exposed group at time t and ho(t) is the
hazard in the unexposed group at time I. This important assumption is known asthe proportional hazards assumption.
The Logrank test is used to check out the equality of two differentsurvivorship' functions. Researchers may wish to statistically compare thesurvival curves of two different groups, While the Kaplan-Meier Product Limitmethod may be used to plot estimates of the survival curve for each of the twogroups of patients The Log-Rank Test is used to determine whether there is astatistically significant difference between the two survival curves.
RESULTS AND DISCUSSION
The survival estimates are calculated by using Kaplan-Meier and LifeTable methods. The Cumulative Hazard Estimates are calculated by using thetechnique of Nelson-Aalen.
1. Estimated Survivor Functions
The Kaplan-Meier Estimated Survivor Function, standard errors andconfidence intervals were calculated for 1287 babies within the age of '0' to '12'
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IlIN S. ASHRAF. ET AL BIOLOGIA VOL 54 (1) SURVIVAL ANALYSIS OF INFANTS IU9
months. These are given in Table 1. These babies started their life from age 0,out of which 8 babies died before reaching the age of 0.0298 months (about aday) The probability that a baby will survive after 0.0298 months was 0 9938with standard error 0.0022. Using this Estimated Survivor Function and standarderror, the 95% confidence interval was 0.9876 to 0.9969.
The 193 babies lost their lives between the time intervals of 02281 to02282 months. The probability that a baby will survive after 0.2281 months (7,days) was 09913 with standard error 0.0026. Using this Estimated SurvivorFunction and standard error, the 95% confidence interval was 09843 to 09952.The 219 babies survived up to the age of 3 months. Only 25 babies out of 1287
Table 1: Kaplan-Meier Survival Estimate
95 % Confide'neeInterval
0.9876 0.9969---~9876 0996~109866 0.996409866 09964
0.9866 0.99640.9855 099580.9843 0.99520.9843 0.9952- -_._-09763 .09912-~ •..... ~--------09480 09772 Io 9252t6:9632 10.9041 094940.8868 0.938808693 0.927508311 0-90311
0.7911 '-1587121-_.- _._---- --07250 0.833706836 08063
0.6618 0.791406029 07502
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Table 2: Life Table Estimate
babies survived up to the age of 12 months. At this age the probability that ababy will survive was 004667 with standard error 0.0533. Probability that a babywill survive after 1200 months was between 0.3597-0.5667. It became clear thatby increasing the time the Estimated Survival Function goes on decreasing whilethe standard error goes on increasing and the confidence interval also increases.
The shape of the Kaplan-Meier Survival Estimate with respect to the agein months is shown in Figure 1 where it can be observed that at age of 0 theKaplan-Meier Survival Estimate was maximum, which went on decreasinggradually with the passage of time. At the age of 9 months, the survival estimatediminished rapidly and then again decreased gradually. . .
The relation between Nelson-Aalen Cumulative Hazard Estimates withrespect to age in months can be visualized from Fig. 2 where it can be seen thatat age zero months the Nelson-Aalen Cumulative Hazard Estimate' wasminimum, which went on increasing gradually with the passage of time. It can beseen that the hazard estimate increased from 0.00 to 0.20 in 5.80 months andapproximately up to the age of 9-10 months, it increased to 0040. Hazardestimate increased rapidly and up to the age of 12 months it reached' the. maximum point
To look at the Survival Probabilities within an interval, Life Table'wasdeveloped. The Life Table Estimated Survival Function, standard .errors"i3ridconfidence intervals were also calculated for 1287 babies within the age of '0' to'12' months. These are given in Table 2. The probability thata baby will surviveafter one month was 0.9702, with standard error 0.0058. The probabilityihat ababy will survive after 6 months was 0.7910 and after 12 months was 004967. Itis obvious that at age of '0' the life table survival function was maximum, .whichwent on decreasing gradually with the passage of time. At the age of 10 months,the survival estimate reduced fast and then again decreased gradually. It can bevisualized from Figure 3.
BeginningInterval Total Deaths Lost Survival Std. Error 95% Cont. Int.
.
0-1 1287 26 830 0.9702 00058 0.9565 0.97961,2 431 15 130 0.9304 0.0115 0.9041 0.94972,3 286 8 59 0.9014 0.0151 0.8671 0.9269I 3 ' 4 219 7 38 08698 0.0186 0.8271 0.90144 ' 5 174 6 29 0.8371 0.0224 0.7866 0.87505 ' 6 139 7 24 0.7910. 0.0270 07309 083766 ' 7 108 3 15 0.7674 0.0294 0.7024 0.81857 ' 8 90 3 7 0.7408 00322 0.6703 07969. 8 ' 9 80 4 9 0.7015 0036 06237 076489 ' 10 67 9 11 0.5989 00441 05063 0.678310,11 47 3 2 0.5598 0.0466 0.4630 0644611 ,12 42 4 13 0.4967 I 0.0509 03931 0590812 ' 13 25 7 18 0.2794 00679 01566 0.4154
0.67190.6492
,-----
05667
0.49770.468603597
StandardError
0.00220.0022
000230.0023
00023000250.00260.0026000370.0073000950.01140.01310.01470.01820.02180.0276
0.031200330
00376
004460.04630.0533
0.9938 .
099380993009930
0.9930099220.99130.99130.9855
096560.94750.93020.91660.90240.87170.8393078520.7511
0732906829
0.59050.56430.4667
SurvivorFunction
NetLost
o1227252433451931
4
1
151
4
1
oooooo1
o
8ooooo1
oo5ooooo1
1
2
1
2
1o2
Deathsj---rT;;;e-[' BeginningI (Months) Total; I
rOOOOO 1287:00298 1279i ...0~0694---1265,
1
1 ~0992 --1-238
0.1289 121301686 118601983 115302281 1107
._----- ---0.4760 663
I-6.99iT- ----~-
.-. ---_._._------- _._-_._-
~
1.4980 39119890 3012.5090 2593.0000 219
1-4~61io---'17114'"9920-- --- 140
[6050~__ . 1087.0740 90
------80990 809.0250 67
10.0800 47~_.- -. 11.1200 42
1_~~ooiJo=:'25
IlIN S. ASHRAF. ET AL BIOLOGIA VOL 54 (1) SURVIVAL ANALYSIS OF INFANTS IU9
months. These are given in Table 1. These babies started their life from age 0,out of which 8 babies died before reaching the age of 0.0298 months (about aday) The probability that a baby will survive after 0.0298 months was 0 9938with standard error 0.0022. Using this Estimated Survivor Function and standarderror, the 95% confidence interval was 0.9876 to 0.9969.
The 193 babies lost their lives between the time intervals of 02281 to02282 months. The probability that a baby will survive after 0.2281 months (7,days) was 09913 with standard error 0.0026. Using this Estimated SurvivorFunction and standard error, the 95% confidence interval was 09843 to 09952.The 219 babies survived up to the age of 3 months. Only 25 babies out of 1287
Table 1: Kaplan-Meier Survival Estimate
95 % Confide'neeInterval
0.9876 0.9969---~9876 0996~109866 0.996409866 09964
0.9866 0.99640.9855 099580.9843 0.99520.9843 0.9952- -_._-09763 .09912-~ •..... ~--------09480 09772 Io 9252t6:9632 10.9041 094940.8868 0.938808693 0.927508311 0-90311
0.7911 '-1587121-_.- _._---- --07250 0.833706836 08063
0.6618 0.791406029 07502
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Table 2: Life Table Estimate
babies survived up to the age of 12 months. At this age the probability that ababy will survive was 004667 with standard error 0.0533. Probability that a babywill survive after 1200 months was between 0.3597-0.5667. It became clear thatby increasing the time the Estimated Survival Function goes on decreasing whilethe standard error goes on increasing and the confidence interval also increases.
The shape of the Kaplan-Meier Survival Estimate with respect to the agein months is shown in Figure 1 where it can be observed that at age of 0 theKaplan-Meier Survival Estimate was maximum, which went on decreasinggradually with the passage of time. At the age of 9 months, the survival estimatediminished rapidly and then again decreased gradually. . .
The relation between Nelson-Aalen Cumulative Hazard Estimates withrespect to age in months can be visualized from Fig. 2 where it can be seen thatat age zero months the Nelson-Aalen Cumulative Hazard Estimate' wasminimum, which went on increasing gradually with the passage of time. It can beseen that the hazard estimate increased from 0.00 to 0.20 in 5.80 months andapproximately up to the age of 9-10 months, it increased to 0040. Hazardestimate increased rapidly and up to the age of 12 months it reached' the. maximum point
To look at the Survival Probabilities within an interval, Life Table'wasdeveloped. The Life Table Estimated Survival Function, standard .errors"i3ridconfidence intervals were also calculated for 1287 babies within the age of '0' to'12' months. These are given in Table 2. The probability thata baby will surviveafter one month was 0.9702, with standard error 0.0058. The probabilityihat ababy will survive after 6 months was 0.7910 and after 12 months was 004967. Itis obvious that at age of '0' the life table survival function was maximum, .whichwent on decreasing gradually with the passage of time. At the age of 10 months,the survival estimate reduced fast and then again decreased gradually. It can bevisualized from Figure 3.
BeginningInterval Total Deaths Lost Survival Std. Error 95% Cont. Int.
.
0-1 1287 26 830 0.9702 00058 0.9565 0.97961,2 431 15 130 0.9304 0.0115 0.9041 0.94972,3 286 8 59 0.9014 0.0151 0.8671 0.9269I 3 ' 4 219 7 38 08698 0.0186 0.8271 0.90144 ' 5 174 6 29 0.8371 0.0224 0.7866 0.87505 ' 6 139 7 24 0.7910. 0.0270 07309 083766 ' 7 108 3 15 0.7674 0.0294 0.7024 0.81857 ' 8 90 3 7 0.7408 00322 0.6703 07969. 8 ' 9 80 4 9 0.7015 0036 06237 076489 ' 10 67 9 11 0.5989 00441 05063 0.678310,11 47 3 2 0.5598 0.0466 0.4630 0644611 ,12 42 4 13 0.4967 I 0.0509 03931 0590812 ' 13 25 7 18 0.2794 00679 01566 0.4154
0.67190.6492
,-----
05667
0.49770.468603597
StandardError
0.00220.0022
000230.0023
00023000250.00260.0026000370.0073000950.01140.01310.01470.01820.02180.0276
0.031200330
00376
004460.04630.0533
0.9938 .
099380993009930
0.9930099220.99130.99130.9855
096560.94750.93020.91660.90240.87170.8393078520.7511
0732906829
0.59050.56430.4667
SurvivorFunction
NetLost
o1227252433451931
4
1
151
4
1
oooooo1
o
8ooooo1
oo5ooooo1
1
2
1
2
1o2
Deathsj---rT;;;e-[' BeginningI (Months) Total; I
rOOOOO 1287:00298 1279i ...0~0694---1265,
1
1 ~0992 --1-238
0.1289 121301686 118601983 115302281 1107
._----- ---0.4760 663
I-6.99iT- ----~-
.-. ---_._._------- _._-_._-
~
1.4980 39119890 3012.5090 2593.0000 219
1-4~61io---'17114'"9920-- --- 140
[6050~__ . 1087.0740 90
------80990 809.0250 67
10.0800 47~_.- -. 11.1200 42
1_~~ooiJo=:'25
110 S. ASHRAF. ET AL. BIOLOGIAVOL 54 (l) SURVIVAL ANALYSIS OF INFANTS "'
2. Comparison of estimated sUlVival functions by gender and birth weight
The survival comparison by gender and birth weight was performed. Forthe analysis to be carried out. the Kaplan-Meier Survival Estimate for gender andbirth weight was compared.
The relationship between Kaplan-Meier Survival Estimate and age in.months according to the gender can be visualized from Figure 4 and it can beseen that for male and female babies the survival estimate went on decreasingwith almost same trend from 0 to 9 months. But Kaplan-Meier survival estimatedecreased with significantly different trend in the interval of 9 to 12 months. Theprobability of survival in male babies after 10 months was 0.80 while in femalebabies this probability was 0.67. i.e. the survival time for female babies was 84%of male babies. The month-wise summary is given in Table 3. .:"1'
T.,.ble 3: Kaplan-Meier SUlVival Estimate for gender
Age Male Female Percentage(Months)1 0.96 0.96 1002 0.94 0.92 98. 3 0.91 0.89 984 0.87 0.87 100,5 085 0.85 1006 , 0.84 080 957 0.84 0.80 958 0.84 080 959 0.84 0.77 9210 080 0.67 8411 078 065 8312 0.74 0.59 80
The relation between Kaplan-Meier Survival Estimate and age in monthsaccording to the birth weights can be envisaged from Fig. 5.lt can be seen thatfor the babies born with normal birth weight and the babies born with low birthweight the Kaplan-Meier Survival Estimate went on decreasing with 21most sametrend from 0 to 1.7 months. But after this, Kaplan-Meier Survival Estimatedecreased with significantly different trend up to 11.75 months. It is interesting tonote that the Kaplan-Meier Survival Estimate for the babies born with low birthweight remained constant'in the interval of 5.80 to 11.75 months, and afterreaching this point both types of babies diminished with same trend. Theprobability of survival in babies with normal birth weight after 6 months was 0.49while for low birth weight babies this probability was 0.12. That is the survivaltime for low birth weight babies was 24% of normal birth weight babies. The. month-wise summary is given in Table 4.
Table 4: Kaplan-Meier SUlVival Estimate for birth weight
Age Normal Birth Low Birth Percentage(Months) Weillht Weint.:
1 0.76 076 1002 073 0.60 823 0.73 0.46 634 063 032 515 058 0.32 556 0.49 012 247 0.49 012 248 0.49 0.12 249 0.46 0.12 2610 0.32 012 3711 0.24 0.12 5012 0.11 0.03 27
3. Testing the equality of SUlVival Functions by gender and birth weight
To test the equality of survival functions by gender and birth weight,Log-Rank test was used. Data in Table 5 was used for the comparison of Kaplan-Meier Survival Function by gender using Log-Rank Test This Table contains theresults of X' statistics alongwith its p-value. From the results of this Table it canbe readily seen that the survival function for both genders are not statisticallydifferent as the result of X' statistics is not significant (p = 0.2843). Although thegraph of Kaplan-Meier Survival Function shows some difference for survivalfunction at particular age. but the overall survival functions proved identical,irrespective of the gender.
Table 5: Log-Rank Test for gender
EventsGender ObselVed ExpectedMale 20 2333
Female 20 16.67
Total 40 4000
Chi' (1) =115Pr.>chi' = 0.2843
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110 S. ASHRAF. ET AL. BIOLOGIAVOL 54 (l) SURVIVAL ANALYSIS OF INFANTS "'
2. Comparison of estimated sUlVival functions by gender and birth weight
The survival comparison by gender and birth weight was performed. Forthe analysis to be carried out. the Kaplan-Meier Survival Estimate for gender andbirth weight was compared.
The relationship between Kaplan-Meier Survival Estimate and age in.months according to the gender can be visualized from Figure 4 and it can beseen that for male and female babies the survival estimate went on decreasingwith almost same trend from 0 to 9 months. But Kaplan-Meier survival estimatedecreased with significantly different trend in the interval of 9 to 12 months. Theprobability of survival in male babies after 10 months was 0.80 while in femalebabies this probability was 0.67. i.e. the survival time for female babies was 84%of male babies. The month-wise summary is given in Table 3. .:"1'
T.,.ble 3: Kaplan-Meier SUlVival Estimate for gender
Age Male Female Percentage(Months)1 0.96 0.96 1002 0.94 0.92 98. 3 0.91 0.89 984 0.87 0.87 100,5 085 0.85 1006 , 0.84 080 957 0.84 0.80 958 0.84 080 959 0.84 0.77 9210 080 0.67 8411 078 065 8312 0.74 0.59 80
The relation between Kaplan-Meier Survival Estimate and age in monthsaccording to the birth weights can be envisaged from Fig. 5.lt can be seen thatfor the babies born with normal birth weight and the babies born with low birthweight the Kaplan-Meier Survival Estimate went on decreasing with 21most sametrend from 0 to 1.7 months. But after this, Kaplan-Meier Survival Estimatedecreased with significantly different trend up to 11.75 months. It is interesting tonote that the Kaplan-Meier Survival Estimate for the babies born with low birthweight remained constant'in the interval of 5.80 to 11.75 months, and afterreaching this point both types of babies diminished with same trend. Theprobability of survival in babies with normal birth weight after 6 months was 0.49while for low birth weight babies this probability was 0.12. That is the survivaltime for low birth weight babies was 24% of normal birth weight babies. The. month-wise summary is given in Table 4.
Table 4: Kaplan-Meier SUlVival Estimate for birth weight
Age Normal Birth Low Birth Percentage(Months) Weillht Weint.:
1 0.76 076 1002 073 0.60 823 0.73 0.46 634 063 032 515 058 0.32 556 0.49 012 247 0.49 012 248 0.49 0.12 249 0.46 0.12 2610 0.32 012 3711 0.24 0.12 5012 0.11 0.03 27
3. Testing the equality of SUlVival Functions by gender and birth weight
To test the equality of survival functions by gender and birth weight,Log-Rank test was used. Data in Table 5 was used for the comparison of Kaplan-Meier Survival Function by gender using Log-Rank Test This Table contains theresults of X' statistics alongwith its p-value. From the results of this Table it canbe readily seen that the survival function for both genders are not statisticallydifferent as the result of X' statistics is not significant (p = 0.2843). Although thegraph of Kaplan-Meier Survival Function shows some difference for survivalfunction at particular age. but the overall survival functions proved identical,irrespective of the gender.
Table 5: Log-Rank Test for gender
EventsGender ObselVed ExpectedMale 20 2333
Female 20 16.67
Total 40 4000
Chi' (1) =115Pr.>chi' = 0.2843
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II~ S. ASHRAF. ETAL. BIOLOGIA VOL. 54 (1) SURVIVAL ANALYSIS OF INFANTS 113
Table 6 indicates the comparison of Kaplan-Meier Survival Function bydjfferent birth weights using log-Rank Test. This Table contains the results of
'statistics alongwith its p-value for the comparison of survival function bydifferent birth weights. From the results of this Table it can be readily seen thatt~e survival function for b,oth groups are statistically different as the result of
x statistics is significant (p = 0.0411). The graph of Kaplan-Meier SurvivalFunction revealed some difference for survival function at particular age, and theoverall survival functions are significantly different for both groups of babies withdifferent weights.
Table 6: log-Rank Test for birth weight
EventsWeight Observed E".I'ected
Normal Birth 21 2569Weight
low Birth 12 731WeightTotal 33 33.00
.--
1.2
1.0,
"C 0.8~'"~I 06E=>U 0'
02
00L,
0 1 2 3 4 5 6 7 8
Ag e in LI ontl1$9 10 11 12
Chi2 (1) = 4.17Pr.>chi2 = 0.0411
Fig. 2: Nelson-Aalen Cumulative Hazard Estimate
1.0
0.8
~ 0.6
OJE80.4
0.2
0.0
o 1 2 3 4 5 6 7 B 9 10 11 12
Age in Months
Fig. 1: Kaplan-Meier Survival Estimate
-
"
";;.>,o",coEou
"
",
Life Table Survival Function
"Age in Months
Fig. 3: Life Table Survival Function
" "
II~ S. ASHRAF. ETAL. BIOLOGIA VOL. 54 (1) SURVIVAL ANALYSIS OF INFANTS 113
Table 6 indicates the comparison of Kaplan-Meier Survival Function bydjfferent birth weights using log-Rank Test. This Table contains the results of
'statistics alongwith its p-value for the comparison of survival function bydifferent birth weights. From the results of this Table it can be readily seen thatt~e survival function for b,oth groups are statistically different as the result of
x statistics is significant (p = 0.0411). The graph of Kaplan-Meier SurvivalFunction revealed some difference for survival function at particular age, and theoverall survival functions are significantly different for both groups of babies withdifferent weights.
Table 6: log-Rank Test for birth weight
EventsWeight Observed E".I'ected
Normal Birth 21 2569Weight
low Birth 12 731WeightTotal 33 33.00
.--
1.2
1.0,
"C 0.8~'"~I 06E=>U 0'
02
00L,
0 1 2 3 4 5 6 7 8
Ag e in LI ontl1$9 10 11 12
Chi2 (1) = 4.17Pr.>chi2 = 0.0411
Fig. 2: Nelson-Aalen Cumulative Hazard Estimate
1.0
0.8
~ 0.6
OJE80.4
0.2
0.0
o 1 2 3 4 5 6 7 B 9 10 11 12
Age in Months
Fig. 1: Kaplan-Meier Survival Estimate
-
"
";;.>,o",coEou
"
",
Life Table Survival Function
"Age in Months
Fig. 3: Life Table Survival Function
" "
'" S. ASHRAF. ET AL. BIOLOGIAVOL. 54 (1) SURVIVAL ANALYSIS OF INFANTS 115
Fig. 4: Kaplan-Meier Survival Estimates, by sex
; Kapian-MeiersurVIvafeslim ates, by sex•~~J~.::::::::~'--_:":":"_~~_~.~-''''''''~~''''''-''.. --,-._""..~- ~.".~w..~. --.This study has been initiated by obtaining the survival function of babies
of various age groups, The survival functions had been obtained for each gender .The survival functions for low and normal birth weight babies were also obtained.The life table is constructed for one month age interval. From above, it is foundthat:
• The survival probability decreases with increase in the age.• The survival function has a decreasing trend as suggested by~the medical
theory.• The male babies have slightly higher probability of survival as compared with
female babies, but the differences were not statistically significant.• Normal birth weight babies have higher probability of survival as compared
with the low birth weight babies as suggested by the medical theory. Further,these survival functions are significantly different from each other on thebas.is of various tests of comparing two survival functions.
• The probability of survival in male babies after 10 months was 0.80 while infemale babies this probability was 0,67, i.e" the survival time for femalebabies was 84% of male babies.
• The probability of survival in babies with normal birth weights afler6 monthswas 0.49 while for low birth weight babies this probability was 0,12. That is,the survival time for low birth weight babies was 24% of normal birth weightbabies.
Male
Female
6 "i 'li
i~M~~ths
114
,",}r-..:d::t
•:lillei
~:t
Kaplan-Meier survival estimates .. ;','.,' ,. ",.',=",", .. "'.';"", __ .;"'"";''',";_;_~'X'='',_' :.,oq
:'1.0'•...to~-,
:::il,c,
~ci
8ci .
" '"~ ,'\
in Months
-(
1I, Normal Birth
Weight (NBW)
Low BirthWeight (LBW)
REFERENCES
Aalen, N., 1975. Statistical inference for a family of counting process. Ph.D.Dissertation, University of California, Berkeley.
Barber, S, & Jennison, C" 1999. Symmetric tests and confidence intervals forsurvival probabilities and quantiles of censored survival data. Biometrics,55(2): 430-436
Cox, D. R., 1972, Regression models and life tables. Jour Roy. Stat, Soc.,Series-B, 34: 187-220
Fleming, T, R. & Lin, D. Y" 1999. Survival analysis in clinical trials pastdevelopments and future directions. Biometrics, 55(4): 971-983.
Freidlin, B, Podgor, M, J, & Gastwirth, J, L., 1999. Efficiency robust tests forsurvival or ordered categorical data. Biometrics, 55 (3): 883-886.
Goggins. W, B, Finkelstein, D, M, & Zaslavsky, A. M" 1999. Applying the coxproportional hazards model when the change time of a binary time-varyingcovariate is interval censored. Biometrics, 55 (2): 445-451.
Fill. 5: Kaplan-Meier Survival Estimates, bv weillht (lbs)
'" S. ASHRAF. ET AL. BIOLOGIAVOL. 54 (1) SURVIVAL ANALYSIS OF INFANTS 115
Fig. 4: Kaplan-Meier Survival Estimates, by sex
; Kapian-MeiersurVIvafeslim ates, by sex•~~J~.::::::::~'--_:":":"_~~_~.~-''''''''~~''''''-''.. --,-._""..~- ~.".~w..~. --.This study has been initiated by obtaining the survival function of babies
of various age groups, The survival functions had been obtained for each gender .The survival functions for low and normal birth weight babies were also obtained.The life table is constructed for one month age interval. From above, it is foundthat:
• The survival probability decreases with increase in the age.• The survival function has a decreasing trend as suggested by~the medical
theory.• The male babies have slightly higher probability of survival as compared with
female babies, but the differences were not statistically significant.• Normal birth weight babies have higher probability of survival as compared
with the low birth weight babies as suggested by the medical theory. Further,these survival functions are significantly different from each other on thebas.is of various tests of comparing two survival functions.
• The probability of survival in male babies after 10 months was 0.80 while infemale babies this probability was 0,67, i.e" the survival time for femalebabies was 84% of male babies.
• The probability of survival in babies with normal birth weights afler6 monthswas 0.49 while for low birth weight babies this probability was 0,12. That is,the survival time for low birth weight babies was 24% of normal birth weightbabies.
Male
Female
6 "i 'li
i~M~~ths
114
,",}r-..:d::t
•:lillei
~:t
Kaplan-Meier survival estimates .. ;','.,' ,. ",.',=",", .. "'.';"", __ .;"'"";''',";_;_~'X'='',_' :.,oq
:'1.0'•...to~-,
:::il,c,
~ci
8ci .
" '"~ ,'\
in Months
-(
1I, Normal Birth
Weight (NBW)
Low BirthWeight (LBW)
REFERENCES
Aalen, N., 1975. Statistical inference for a family of counting process. Ph.D.Dissertation, University of California, Berkeley.
Barber, S, & Jennison, C" 1999. Symmetric tests and confidence intervals forsurvival probabilities and quantiles of censored survival data. Biometrics,55(2): 430-436
Cox, D. R., 1972, Regression models and life tables. Jour Roy. Stat, Soc.,Series-B, 34: 187-220
Fleming, T, R. & Lin, D. Y" 1999. Survival analysis in clinical trials pastdevelopments and future directions. Biometrics, 55(4): 971-983.
Freidlin, B, Podgor, M, J, & Gastwirth, J, L., 1999. Efficiency robust tests forsurvival or ordered categorical data. Biometrics, 55 (3): 883-886.
Goggins. W, B, Finkelstein, D, M, & Zaslavsky, A. M" 1999. Applying the coxproportional hazards model when the change time of a binary time-varyingcovariate is interval censored. Biometrics, 55 (2): 445-451.
Fill. 5: Kaplan-Meier Survival Estimates, bv weillht (lbs)
Kaplan, E. L. & Meier, P., 1958. Nonparametric estimation from incompleteobservations. Jour. Amer. Stat. Assoc., 53: 457-481.
Li, Y. & Ryan, L., 1999. Modeling spatial survival data using semi-parametricfrailty Models. Biometrics, 55(2): 287-297.
Lunceford, J. K., Davidian, M. & TSiatis, A A, 2002. Estimation of survivaldistributions of treatment policies in two-stage randomization designs inclinical trials. Biometrics, 58(1): 48-57.
Maples, J. J., Murphy, S. A & Axinn, W. G., 2002. Two-level proportionalhazards models. Biometrics, 58(4): 754-763.
Parzen, M. & Lipsitz, S. R., 1999. A global goodness-of-fit statistic for coxregression models. Biometrics, 55(2): 580-584.
Peto, R. & Peto, J., 1972. Asymptotically efficient rank invariant procedure.JourRoy. Stat. Soc., Series A, 135: 185-207
Shen. Y & Cai, J., 2001. Maximum of the weighted Kaplan-Meier tests withapplication to cancer prevention and screening trials. Biometrics, 57(3):837-843.
Song, X., Davidian, M. & Tsiatis, A A, 2002. A semiparametric likelihoodapproach to joint modeling of longitudinal and time-to-event data.Biometrics., 58(4): 742-753.
116 S. ASHRAF. ET AL. BIOLOGIA BIOLOGICAL SOCIETY OF PAKISTANBiological Laboratories, GC University, Lahore, Pakistan
www.biosoc.org.pk
INSTRUCTIONS TO THE CONTRIBUTORS IN BIOLOGIA- PAKISTAN
1. (a) Manuscripts should be computer typed (in double spacing on one side of the paperonly). Two hard copies and a soft copy should be submitted, followin9 the format ofthe latest volumes of Biologia-Pakistan. However, the articles can also be submittedonline at www.biosoc.org.pk
(b) Nothing in the text should be underlined, except the scientific names which should bewritten in italics or underlined.
(c) The manuscripts should be typed according to the format of Biologia-Pakistan, thatincludes (i) Complete title of the paper wilh the authorls namel names andaddresses, (ii) Abstract, (iii) Key words, (iv) Introduction, (v) Materials and Methods,(vi) Results, (vii) Discussion. (The authorls hasl have the option to give results anddiscussion under a combined heading i.e. Results and Discussion), (viii) References .
. 2. The manuscripts will be charged @ Rs. 200 per page. However, for members of theSociety, first two pages will be free and the subsequent pages will be charged at theusual rates.
3. The abstract should not exceed 200 words. It should be printed in small font.4. The author! s may suggest a running title not exceeding four words, for their
papers. It should be indicated on the titte page.5. (a) Tables should be typed on separate sheets. Footnotes should be avoided as far as
possible.(b) More than two Tables shall be charged as per composing and printing rates per page.
6. (a) Diagrams should be either original photographs, or if hand drawn, should be inkedwith black Indian ink.
(b) The authorls namel s and the number of the Figure should be written on the back ofeach drawing.
(c) The size of an illustration, after reduction, should not exceed 4.5 x 6.5 inches.7. (a) There will be no extra charges for black and white photographs.
(b) Colored photographs will be charged @ aclual cost of the positives, plates, printingetc. involved.
8. (a) References should be cited in the text by giving the author's name followed by theyear of publication. The abbreviations of Journals or books should be in italics. Incase of journal, the volume number should be more bold than the other parts of thereference.
Baker, E. w., 1949. A review of the mile family Cheyletidae in the United StatesNational Museum. Proc. U. S. Nat. Mus., 99: 267-320.
David, L. & Weiser, J., 1994. Role of hemocytes in the propagation of amicrosporidian infection in larvae of Galleria mellonella. J. Invel1ebr. Pathol., 63:212-213.
Wiegand, M. D., 1992. Vitellogenesis in fish. In: Proc. Internal. Symp. Reprod.Physiol. Fish. (eds Riether, C. J. J. and Goose, H. J.), pp. 136-146.
(b) The names of the joumals and books should be abbreviated according to the latestedition of the World List of Scientific Periodicals.
9. (a) Page-proofs will be sent to the authorl s for correction, which should be returnedwithin a week of their receipt.
(b) Authorl s may be required to pay for alterations on proofs other than those forcorrections of printer's error.
10. Author! S Will receive 5 offprints gratis.
Kaplan, E. L. & Meier, P., 1958. Nonparametric estimation from incompleteobservations. Jour. Amer. Stat. Assoc., 53: 457-481.
Li, Y. & Ryan, L., 1999. Modeling spatial survival data using semi-parametricfrailty Models. Biometrics, 55(2): 287-297.
Lunceford, J. K., Davidian, M. & TSiatis, A A, 2002. Estimation of survivaldistributions of treatment policies in two-stage randomization designs inclinical trials. Biometrics, 58(1): 48-57.
Maples, J. J., Murphy, S. A & Axinn, W. G., 2002. Two-level proportionalhazards models. Biometrics, 58(4): 754-763.
Parzen, M. & Lipsitz, S. R., 1999. A global goodness-of-fit statistic for coxregression models. Biometrics, 55(2): 580-584.
Peto, R. & Peto, J., 1972. Asymptotically efficient rank invariant procedure.JourRoy. Stat. Soc., Series A, 135: 185-207
Shen. Y & Cai, J., 2001. Maximum of the weighted Kaplan-Meier tests withapplication to cancer prevention and screening trials. Biometrics, 57(3):837-843.
Song, X., Davidian, M. & Tsiatis, A A, 2002. A semiparametric likelihoodapproach to joint modeling of longitudinal and time-to-event data.Biometrics., 58(4): 742-753.
116 S. ASHRAF. ET AL. BIOLOGIA BIOLOGICAL SOCIETY OF PAKISTANBiological Laboratories, GC University, Lahore, Pakistan
www.biosoc.org.pk
INSTRUCTIONS TO THE CONTRIBUTORS IN BIOLOGIA- PAKISTAN
1. (a) Manuscripts should be computer typed (in double spacing on one side of the paperonly). Two hard copies and a soft copy should be submitted, followin9 the format ofthe latest volumes of Biologia-Pakistan. However, the articles can also be submittedonline at www.biosoc.org.pk
(b) Nothing in the text should be underlined, except the scientific names which should bewritten in italics or underlined.
(c) The manuscripts should be typed according to the format of Biologia-Pakistan, thatincludes (i) Complete title of the paper wilh the authorls namel names andaddresses, (ii) Abstract, (iii) Key words, (iv) Introduction, (v) Materials and Methods,(vi) Results, (vii) Discussion. (The authorls hasl have the option to give results anddiscussion under a combined heading i.e. Results and Discussion), (viii) References .
. 2. The manuscripts will be charged @ Rs. 200 per page. However, for members of theSociety, first two pages will be free and the subsequent pages will be charged at theusual rates.
3. The abstract should not exceed 200 words. It should be printed in small font.4. The author! s may suggest a running title not exceeding four words, for their
papers. It should be indicated on the titte page.5. (a) Tables should be typed on separate sheets. Footnotes should be avoided as far as
possible.(b) More than two Tables shall be charged as per composing and printing rates per page.
6. (a) Diagrams should be either original photographs, or if hand drawn, should be inkedwith black Indian ink.
(b) The authorls namel s and the number of the Figure should be written on the back ofeach drawing.
(c) The size of an illustration, after reduction, should not exceed 4.5 x 6.5 inches.7. (a) There will be no extra charges for black and white photographs.
(b) Colored photographs will be charged @ aclual cost of the positives, plates, printingetc. involved.
8. (a) References should be cited in the text by giving the author's name followed by theyear of publication. The abbreviations of Journals or books should be in italics. Incase of journal, the volume number should be more bold than the other parts of thereference.
Baker, E. w., 1949. A review of the mile family Cheyletidae in the United StatesNational Museum. Proc. U. S. Nat. Mus., 99: 267-320.
David, L. & Weiser, J., 1994. Role of hemocytes in the propagation of amicrosporidian infection in larvae of Galleria mellonella. J. Invel1ebr. Pathol., 63:212-213.
Wiegand, M. D., 1992. Vitellogenesis in fish. In: Proc. Internal. Symp. Reprod.Physiol. Fish. (eds Riether, C. J. J. and Goose, H. J.), pp. 136-146.
(b) The names of the joumals and books should be abbreviated according to the latestedition of the World List of Scientific Periodicals.
9. (a) Page-proofs will be sent to the authorl s for correction, which should be returnedwithin a week of their receipt.
(b) Authorl s may be required to pay for alterations on proofs other than those forcorrections of printer's error.
10. Author! S Will receive 5 offprints gratis.