microscopy author(s): ana c. bahia, nágila f. c. secundino ... · morphology,systematics,evolution...

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Ultrastructural Comparison of External Morphology ofImmature Stages of Lutzomyia (Nyssomyia) intermedia andLutzomyia (Nyssomyia) whitmani (Diptera Psychodidae)Vectors of Cutaneous Leishmaniasis by Scanning ElectronMicroscopyAuthor(s) Ana C Bahia Naacutegila F C Secundino Joseacute C Miranda Deboraci BPrates Ana P A Souza Fernando F Fernandes Aldin Barral and Paulo F PPimentaSource Journal of Medical Entomology 44(6)903-914 2007Published By Entomological Society of AmericaDOI httpdxdoiorg1016030022-2585(2007)44[903UCOEMO]20CO2URL httpwwwbiooneorgdoifull1016030022-258528200729445B9033AUCOEMO5D20CO3B2

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MORPHOLOGY SYSTEMATICS EVOLUTION

Ultrastructural Comparison of External Morphology of Immature Stagesof Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia)

whitmani (Diptera Psychodidae) Vectors of CutaneousLeishmaniasis by Scanning Electron Microscopy

ANA C BAHIA1 NAGILA F C SECUNDINO1 JOSE C MIRANDA2 DEBORACI B PRATES2

ANA P A SOUZA2 FERNANDO F FERNANDES3 ALDINA BARRAL2

AND PAULO F P PIMENTA14

J Med Entomol 44(6) 903ETH914 (2007)

ABSTRACT Lutzomyia (Nyssomyia) intermedia (Lutz amp Neiva 1912) and Lutzomyia (Nys-somyia) whitmani (Antunes amp Coutinho 1939) (Diptera Psychodidae) are vectors of Americancutaneous leishmaniasis in several endemic regions of Brazil We analyzed the external morpho-logical aspects of the immature stages of these two vectors by using scanning electron microscopyIn general the larval stages of the two species are morphologically similar although somedifferences were noted Detailed examination of the eggs of both species revealed similarexchorionic ornamentations of unconnected parallel ridges The larval head capsules are welldeTHORNned heavily sclerotized and bear prominent chewing mouthparts The abdominal segmentsare easily recognized by the presence of prolegs on their ventral surfaces The morphology of theanal lobe on the terminal abdominal segment differs between the two species We found thefollowing three types of sensillae inserted on the antennae 1) clavate basiconic 2) small bluntcoeloconic and 3) multipourous clavate coleoconic In addition THORNve subtypes of trichoid sensillaewere found on the larval body 1) long 2) short 3) curved long 4) brush-like and 5) weaklybrush-like The caudal THORNlaments located on the last abdominal segment were recognized as longtrichoid sensillae We observed pores on the surface of the clavate coelonic sensillae and on thecaudal THORNlaments that presumably function as chemoreceptors The larvae of the two species showsimilarities in the lobular-form antennae of L1 larvae which changes to digitiform in second instar(L2) L3 and L4 This study demonstrated that the external surface of the eggs and larvae of Luintermedia and Lu whitmani are morphologically similar but they can be distinguished by detailsin the microanatomy observed by scanning electron microscopy

KEY WORDS Lutzomyi intermedia Lutzomyi whitmani immature stages scanning electron mi-croscopy

Lutzomyia (Nyssomyia) intermedia (Lutz amp Neiva1912 and Lutzomyia (Nyssomyia) whitmani (Antunesamp Coutinho 1939) are South American phlebotominesand szligies and proven vectors of Leishmania brazilien-sis (Vianna 1911) the causative agent of Americancutaneous leishmaniasis in Brazil and other SouthAmerican countries (Vexenat et al 1986 Jones et al1987 Rangel et al 1990 Hashiguchi et al 1992Salomon et al 1995 2001a b Brito et al 2002 Souza

et al 2002) These two closely related species (Maz-zoni et al 2002) are sympatric in some endemic areas(Oliveira dos Santos et al 1993 Miranda et al 2002)

Sand szligies are holometabolous insects with four lifecycle developmental stages egg larva pupa and adultPioneering taxonomic studies of immature stages ofphlebotomines used optical microscopy as a commontool (Barreto 1941 Guitton and Sherlock 1969 Ward1972) Later Ward and Ready (1975) introducedscanning electron microscopy (SEM) to describe de-tails of the exchorionic ornamentation of eggs of 13species of Brazilian phlebotomines Thereafter otherstudies used SEM to reveal ultrastructural details ofvarious immature stages of Phlebotomus tobbi (Adleramp Theodor 1930) (Killick-Kendrick et al 1989) andpupae and THORNrst and fourth (L1 and L4) instars ofLutzomyia longipalpis (Lutz amp Neiva 1912) (Leite etal 1991 Leite and William 1996 1997) Secundino and

1 Laboratory of Medical Entomology Centro de Pesquisas ReneRachou FIOCRUZ-MG Av Augusto de Lima 1715 CEP 30190-002Belo Horizonte MG Brazil

2 Laboratory of Immunoparasitology Centro de Pesquisas GoncaloMoniz Fundacao Oswaldo Cruz FIOCRUZ-BA Rua Waldemar Fal-cao 121 Candeal CEP 40245-001 Salvador BA Brazil

3 Laboratory of Medical and Veterinary Arthropodology Institutode Patologia Tropical e Saude Publica Universidade Federal de GoiasCEP 74605-050 Goiania GO Brazil

4 Corresponding author e-mail pimentacpqrrTHORNocruzbr

0022-2585070903ETH0914$04000 2007 Entomological Society of America

Pimenta (1999) described morphological characteris-tics of all developmental stages of Lu longipalpisthrough SEM Until now only optical microscopicobservations have been made on the external mor-phology of Lu intermedia and Lu whitmani (Barreto1941) This is the THORNrst time SEM has been used tocompare ultrastructural features on the external sur-faces of immature stages of these two sympatric vectorsand szligies

Materials and Methods

Sand Flies Sand szligies were captured live with CDClight traps (Sudia and Chamberlain 1962) at Corte dePedra (13 32 S 39 25 W) in the State of BahiaBrazil and transferred to a colony cage (30 cm3)where they were blood fed on hamsters and provideda 50 sugar solution ad libitum After blood feedingthe szligies were held in the colony cages at 26C and 60RH to allow for mating and digestion of the bloodmealThree or 4 d postfeeding the bloodfed females weretransferred to oviposition containers (Modi and Tesh1983) where they laid their eggs Upon hatching thelarvae were fed the appropriate diet (Secundino andPimenta 1999) to complete their life cycle Twentyspecimens of each developmental stage except pupawere collected (eggs and the four instars of larvae)washed in phosphate-buffered saline and immedi-ately THORNxed overnight in 25 glutaraldehyde in 01Mcacodylate buffer pH 72 at room temperatureScanningElectronMicroscopyFixed samples were

processed for SEM as described previously (Secun-dino and Pimenta 1999) Brieszligy samples were post-THORNxed in 1 OsO4 plus 08 potassium ferricyanide in01 M cacodylate buffer pH 72 and dehydrated inethanol at increasing concentrations of 50 70 90 and100 After that the samples were critical point-driedunder CO2 They were then coated with a 20-nm layerof gold particles and examined under SEM (JSM5600JEOL Tokyo Japan)

Results and Discussion

Several ultrastructural details of the external sur-face of the immature stages of Lu intermedia and Luwhitmani were revealed by SEM allowing us to com-pare the microanatomy of these two important vec-tors

The eggs of Lu intermedia and Lu whitmani areabout the same size (length 0308 0016 mm andwidth 0074 0003 mm for Lu intermedia length0307 0010 mm and width 0077 0002 mm for Luwhitmani) having a long oval shape and ornamenta-tions that consist of parallel ridges covering the entireexchorion (Figs 1ETH4) Barreto (1941) described theseridges as ldquoconnected ridgesrdquo in these same species byusing optical microscopy However our SEM obser-vations revealed that most of these ridges are uncon-nected (Figs 1 and 2) Similar observations of uncon-nected ridges were reported by Almeida et al (2004)on Lu intermedia eggs High SEM magniTHORNcation re-vealed that these ridges are formed by single lines of

rounded palisade units (diameter 0576 0074 m forLu intermedia and 0775 0095 m forLuwhitmani)that project from the surface of eggshell (high 0454 0040 m for Lu intermedia and 0597 0043 mfor Lu whitmani) (Figs 3 and 4) In contrast Lulongipalpis egg exchorions bear unconnected ridgesformed by lines of double-rounded palisade units(Secundino and Pimenta 1999)

Some authors have speculated that exchorionic or-namentations are adaptive structures that facilitateoviposition in distinct environments (Ward and Ready1975 Enrique Perez and Ogusuku 1997) In thepresent work observations of the egg surfaces showedno differences in the exchorion ornamentations of thetwo sympatric sand szligies In another study Rogo et al(1992) showed intraspeciTHORNc differences in the excho-rions of Phlebotomus aculeatus (Lewis Minter amp Ash-ford 1974) and Phlebotomusmartini (Parrot 1936) Yetanother report showed that the egg exchorions ofPhlebotomus pedifer (Lewis Mutinga amp Ashford) Lulongipalpis and Lutzomyia diabolica (Hall 1936) havesimilar patterns of unconnected ridges (Endris et al1987 Rogo et al 1992 Secundino and Pimenta 1999)even though these species occur in widely separatedgeographic areas Others suggest based on SEM ex-amination of 23 sand szligy species that exchorionic or-namentations reszligect phylogenetic relationships be-tween the species (Endris et al 1987 Feliciangeli et al1993 Enrique Perez and Ogusuku 1997 Fausto et al2001) Because the exchorionic ornamentation is notnecessarily species speciTHORNc it is unlikely that it wouldbe useful as a taxonomic tool

Sand szligy larvae escape from the egg by breaking theshell in amanner similar tootherDipteraTheTHORNrst signof hatching is the appearance of a fracture toward theanterior end of the eggshell which opens as a door-like szligap through which the larvaOtildes head emerges (Figs5ETH7) revealing the egg burster (Fig 8) Apparently asthe THORNrst instar moves its head inside the eggshell thisspine located on the apex of the head capsule scoresthe inside of the shell in an arc Subsequent pressureappliedby the larvacauses thedoor-like fracturealongthearcAs theheadreachesoutside the shell thedistalends of the two caudal THORNlaments also can be seen (Figs7ETH9) These sequential events allow the larva to escape(Figs 9 and 10) A similar mechanism of egg breakingand larval escape was described for Lu longipalpis(Leite and William 1997 Secundino and Pimenta1999)

The breaking of the eggshell in several Diptera isdue to the action of similar structures called egg burst-ers (synonyms hatching spine egg-breaker or burst-ing teeth) SEM showed the egg burster on the apexof the head of Lu intermedia and Lu whitmani Theegg burster exists only on the dorsal surface of the THORNrstinstar head and has been used to differentiate the THORNrstinstar from the second instar in Diptera along withother taxonomic characters (Breland 1959 Alvan-Aguilar and Hamada 2003) Details of the microanat-omy of the sand szligy egg burster can be seen on the apexof head capsule when the larval head is completely outof the eggshell (Fig 19) The egg bursters of these two

904 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 6

sand szligies are similar structures with small differencesin their dimensions (length 0027 0003 mm andwidth 0018 0001 mm for Lu intermedia and length0023 0002 mm and width 0019 0001 mm for Luwhitmani) The egg burster is a cuticular formationthat rises from the top of the larvaOtildes head like a min-iature volcanic cone (Fig 19) Leite and William(1997) and Secundino and Pimenta (1999) observeda similar egg burster in Lu longipalpis

The general microanatomy revealed by SEM of thelarvae of both sand szligy species is similar The bodies ofboth larvae are long and divided into 12 segmentsthree thoracic and nine abdominal segments (Figs11ETH14) In both species the larval integument isadorned dorsally and laterally with numerous setae(Figs 11ETH14) except on the anal lobe (Figs 15 and16) on the head base and on the mouthparts (Figs17ETH19) The abdominal segments have prolegs (pro-truding elliptical structures) ventrally that are used for

locomotion and are not present on the THORNrst threesegments nor on the anal lobe (Figs 13 and 14 insets)These anatomical aspects are similar in Lu longipalpisand Lutzomyia bahiensis (Mangabeira amp Sherlock1964) larvae (Sherlock and Carneiro 1963 Leite andWilliam 1996 1997 Secundino and Pimenta 1999)Luintermedia and Lu whitmani prolegs have small dif-ferences in size (length 0068 0001 mm and width0030 0004 mm for Lu intermedia length 0108 0005 mm and width 0057 0006 mm for Lu whit-mani) In addition the morphology of the anal lobeis different between the species (Figs 15 and 16)The anal lobe of Lu whitmani is more wrinkled andshorter than that of Lu intermedia (length 0114 0002 mm and width 0123 0003 mm for Luintermedia length 0067 0006 mm and width0076 0006 mm for Lu whitmani) These charac-teristics may comprise distinguishing features thatmight be useful to conTHORNrm the identiTHORNcation of these

Figs 1ndash4 Egg surfaces of Lu intermedia (1 and 3) and Lu whitmani (2 and 4) showing exchorionic ornamentations ofunconnected parallel ridges (arrowheads) High magniTHORNcation images (3 and 4) show that these ridges are formed by singlelines of round palisade units (asterisks) MagniTHORNcations 300 (1 and 2) 4000 (3) and 3000 (4)

November 2007 BAHIA ET AL EXTERNAL MORPHOLOGY OF IMMATURE Lutzomyia SPP 905

Figs 5ndash10 Sequential events of egg hatching of Lu intermedia and Lu whitmani Figures 5 and 6 show a door-like szligapat the anterior end of the eggshell (arrow) where the larval head THORNrst emerges The entire head (h) is exposed outside theeggshell in 7 and 8 In 8 it is possible to see the egg burster (arrowhead) on the larvaOtildes head and the distal ends of the caudalTHORNlaments (cf) 9 and 10 show respectively two larvae one larva halfway and larva almost completely emerged from theeggshell Note the egg burster (arrowhead) on the larvaOtildes head MagniTHORNcations 270 (5) 330 (6) 230 (7 and 9) 550(8) and 190 (10)


Figs 11ndash14 External morphology of Lu intermedia (11 and 13) and Lu whitmani larvae (12 and 14) Lateral views ofthe sand szligy larvae (11 and 12) show the heads (h) and the caudal THORNlaments (arrowheads) Ventral views (13 and 14) showthe larval bodies divided into 12 segments three thoracic and nine abdominal segments (white asterisks on second third andTHORNfth abdominal segments) between the head and the anal lobule (black asterisks) Note the prolegs (white asterisks in 13and 14) are only present on the abdominal segments Details of the prolegs and curved long trichoid sensillae (circle) areseen in the two insets The brush-like trichoid sensillae (arrows) are located in dorsal and lateral parts of larval segmentsMagniTHORNcation 220 (11) 60 (12) 160 (13 and inset 500) and 35 and inset 250)


species under SEM further supporting ordinaryadult taxonomy

The larvae of both species have well-deTHORNned roundand sclerotized head capsules (Figs 11ETH14 and 17ETH19)The mouth apparatus consisting of clypeus labrummandibles maxillae and mentum located ventrally onthe head measures 0147 0025 mm in length and0135 0025 mm in width in Lu intermedia and0092 0032 mm and 0091 0027 mm in Lu whit-mani (Figs 17ETH18) This ldquomasticatorrdquo-type mouthpartapparatus was described by Leite and William (19961997) and Mukhopadhyay and Ghosh (2000)

Antennal morphology is similar in Lu intermediaand Lu whitmani and changes between the L1 andsubsequent instars (L2 L3 and L4) The L1 antennaeare two-segmented with a basal conical segment fusedwith a short ovoid distal segment that is almost as longas the basal segment (Figs 19ETH21) In the L2 throughL4 the antennae are also two-segmented but thedistal segment is greatly elongated (digitform) to 3times the length of the conical basal segment (Figs22ETH23) This observation differs from the initial THORNnd-ings of Barreto (1941) who observed digitiform an-tennae on all larval instars of both species using opticalmicroscopy Recently Pessoa et al (2001) describedsimilar digitiform antennae on the L4s of several sandszligy species

We analyzed in detail all the sensilla types presenton the immature stages of Lu intermedia and Luwhitmani categorizing them according to the classi-THORNcation scheme of Hallberg and Hansson (1999) andMitchell et al (1999) There are THORNve subtypes of tri-choid sensillae 1) long 2) short 3) curved long 4)brush-like and 5) weakly brush-like Long trichoidsensillae (0066 0011 mm in length for Lu interme-dia and 0068 0022 mm in length for Lu whitmani)

are located on the forehead in a horizontal row in frontof the weakly brush-like trichoid sensillae (Figs 17ETH19 28 and 29) Short trichoid sensillae (0021 0010mm for Lu intermedia and 0028 0012 mm for Luwhitmani) were observed on the mouthparts (Figs 17and 18 and 28) Long and short trichoid sensillae ringthe anal lobes in both species (Figs 15 and 16) Thistype of sensillum was not found in Lu longipalpis andLu bahiensis (Sherlock and Carneiro 1963 Leite andWilliam 1996 1997 Secundino and Pimenta 1999)The curved long trichoid sensillae which exhibit vary-ing degrees of curvature are located on the lateralsides of the prolegs in both species (Figs 13 and 14insets 27 and 28) These were not observed by Sher-lock and Carneiro (1963) in Lu bahiensis or by Leiteand William (1996 1997) and Secundino and Pimenta(1999) in Lu longipalpis Straight and curved forms oflong trichoid sensillae and short trichoid sensillaewere observed previously by Fernandes and Linardi(2002) in segment II of the mouthparts ofDermatobiahominis (L Jr 1781) (Diptera Oestridae) Mech-anosensory sensillae with similar morphology havebeen found in the maxillary palps ofDrosophila mela-nogaster Meigen by Riesgo-Escovar et al (1997)Brush-like trichoid sensillae are localized along thedorsal and lateral surfaces of the larval body but theyare absent on the ventral surfaces and behind theantennae on the top of the head (Figs 11ETH14 1924ETH25) Weakly brush-like trichoid sensillae are foundin the middle of the head in front of the antennae(Figs 19 and 26)

Detailed examination of the larval antennae re-vealed three types of sensillae on the ventral andapical surfaces of the terminal segment present in allinstars regardless of the changes in antennal morphol-ogy from L1 to L2ETHL4 (Fig 30) These sensillae are

Figs 15ndash16 Details of the larvaeOtildes anal lobe In Luwhitmani (15) this structure is more wrinkled and shorter than in Luintermedia (16) Note the crowns of short and long trichoid sensillae (arrows) surrounding the base of the anal lobes Somesensillae were accidentally removed during sampling process and it is possible to see their insertions sites (arrowheads)MagniTHORNcations 500 (15) and 400 (16)


Figs 17ndash19 External morphology of the larval heads of Lu intermedia (17 and 19) and Luwhitmani (18) Ventral viewsof the heads show the mouth apparatus consisting of clypeus (c) labrum (asterisks) mandibles (ma) maxillas (mx) andmemtum (me) Note trichoid sensillae on the apex of the head (arrowheads) and the short trichoid sensillae on the mouthapparatus (arrows) Dorsal view of the head (19) shows the egg burster (asterisk) as a ldquovolcanic conerdquo structure A row ofbrush-like trichoid sensillae (arrowheads) is located in front of the egg burster (asterisk) On the forehead are two weaklybrush-like trichoid sensillae (small arrows) inserted slightly forward and between the antennae (ant) and long trichoidsensillae (large arrows) are inserted further down toward the mouth MagniTHORNcations 350 (17) 300 (18) and 1000 (19)


important sensory organs that function as hygrore-ceptors contact chemoreceptors tactile receptorsand olfactory receptors and temperature receptorsenabling the insects to detect changes in the surround-ing environment Other sensillae are found on theantennae of Lu whitmani and Lu intermedia larvae

The THORNrst of these is a clavate-basiconic sensillum lo-cated at the apex of the segment (Fig 30) At theproximal end of this antennal segment are three shortblunt coeloconic sensillae inserted at the base of asingle large multiporous clavate-coeloconic sensil-lum (Figs 30 and 31) Olfactory sensillae with bul-

Figs 20ndash23 Antennal morphology in larvae of Lu intermedia (20 and 22) and Lu whitmani (21 and 23) The antennaeof L1 are two-segmented with a basal conical segment fused with a short ovoid distal segment (asterisks) (20 and 21) Theantennae in L2s through L4s are also two-segmented but the distal segment is greatly elongated (digitiform) (c) (22 and23) Note the sensillae inserted on the superior portion of the antennae (arrows) MagniTHORNcations 2000 (20 and 21) 1000(22 and 23)


bous porous apical dilations (clavate sensillae) sim-ilar to the multiporous clavate-coeloconic sensilladescribed here were observed on the antennal clavaeof Trichogramma galloi (Zucchi 1988) and Tricho-gramma pretiosum (Riley 1879) (Hymenoptera Tri-chogrammatidae) on the maxillary palps ofCulicoides hollensis (Melander amp Brues 1903) andCulicoides melleus (Coquillett 1901) (DipteraCeratopogonidae) and on the antennal funiculusof Cochliomyia hominivorax (Coquerel 1858) (DipteraCalliphoridae) (Consoli et al 1999 Kline and Axtell1999 Fernandes et al 2004)

Another long type of trichoid sensillum with wallpores is located on the last larval segment (Fig 32) Insand szligies sensillae of this type are called caudal THORNla-ments There are two caudal THORNlaments on the L1 andfour on the L2 through L4 Different patterns of pores

exist on the caudal THORNlaments of the two species stud-ied Lu whitmani has pores situated inside the wallgrooves (Fig 34) In Lu intermedia the grooves arenot as deep as in Lu whitmani and the pores occur tobe on the surface (Fig 33) Pessoa et al (2001) foundpores on caudal THORNlaments of larvae of Lu intermediaLu whitmani Lutzomyia lenti (Mangabeira 1938) Lulongipalpis and Lutzomyia evandroi (Costa Lima ampAntunes 1936) but they did not THORNnd any differencesin the pattern of pores between the THORNrst two speciesZacharuk (1985) suggests that the absence of pores onthe sensorial cuticle does not allow chemical sensitiv-ity and that the presence of a single pore limits thesensitivity to taste whereas several pores allow thesense of olfaction According to Zacharuk the caudalTHORNlaments and the multiporous clavate coeloconic sen-sillae of the THORNrst instar of Lu intermedia and Lu whit-

Figs 24ndash29 Microanatomy of the sensillae of Lu intermedia and Luwhitmani larvae showing brush-like trichoid sensillapresent on the head (24) and on the lateral and dorsal aspects of the body segments (25) weakly brush-like trichoid sensillaon the middle of the head (26) curved long trichoid sensillae with different degrees of bending on the lateral sites of theprolegs (27 and 28) and short trichoid sensillae (arrowhead) and long trichoid sensillae (arrow) on the top of the larvaOtildeshead (29) MagniTHORNcations 1000 (24 and 25) 2000 (26 27 29) and 3000 (28)


mani respond to olfactory molecules thus they areconsidered chemoreceptors

In this study we found mainly trichoid sensillae onthe larval segments of both sand szligy species studiedGreen and Hartenstein (1997) analyzed the sensillarpatterns on the thoracic and abdominal segments ofseveral insect orders and described hairs and bristles

(trichoid and chaetic sensillae) of varied sizes andnumber in primitive insects and peg-like sensillae (ba-siconic styloconic and coeloconic) and papilla sen-sillae in Diptera These authors suggested that thetrichoid sensillae represent mechanoreceptors thatare stimulated by hair deszligection However Zacharuk(1985) claims that trichoid sensillae can have various

Figs 30ndash34 Microanatomy of the antennae and caudal THORNlaments ofLu intermedia (30 and 31 32 and 33) andLuwhitmani(30 and 31 and 34) larvae Image 30 shows a general view of the antenna of Lu intermedia L1 larva (similar to Luwhitimani)Note THORNve sensillae one clavate basiconic sensilla (cb) on the apex one multiporous clavate coeloconic sensilla (asterisk) onthe base and three short blunt coeloconic sensillae (bc) on the proximal region Observe the pores on the clavate coeloconicsensillumatahighermagniTHORNcation(31) Image32 shows thegeneral viewof thecaudalTHORNlaments(cf)(ie long typeof trichoidsensillae) of Lu intermedia L1 which are similar in Lu whitmani (not shown) at low magniTHORNcation Pores in the caudalTHORNlaments are only seen at high magniTHORNcations In Lu whitimani the pores are inside wall grooves (34) whereas in Luintermedia (33) the pores are revealed in the surface because the grooves are not so deep MagniTHORNcations 9000 (30) 21600(31) 150 (32) 6000 (33) and 8000 (34)


functions including mechanical chemical and ther-mal reception that enable the larvae to discernchanges in their environment Future ultrastructuraland electrophysiological studies will be necessary todetermine the exact function of these sensillae in sandszligies Added to the results of the present work thesestudies may supply information about the behaviorand physiology of these important vectors that will beuseful in the development of vector control strategies

In summary our comparative study showed that theexternal morphology of the eggs and larvae of Luintermedia and Lu whitmani have similar features butare distinguishable by details in their microanatomyThe similarities found between these two species mostlikely reszligect their close phylogenetic relationship

Acknowledgments

We thank Tereza Magalhaes and Fernando Monteiro forreading and commenting this article This work was par-tially supported by the following Brazilian agencies Con-selho Nacional de Desenvolvimento CientotildeTHORNco e Tecno-logico (CNPq) Fundacao de Amparo a Pesquisa do Estadode Minas Gerais (FAPEMIG-PRONEX) and Fundacao Os-waldo Cruz (FIOCRUZ) This study is part of the MSthesis of ACB Centro de Pesquisas Rene RachouFiocruz-MG

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Received 20 September 2005 accepted 19 December 2006


MORPHOLOGY SYSTEMATICS EVOLUTION

Ultrastructural Comparison of External Morphology of Immature Stagesof Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia)

whitmani (Diptera Psychodidae) Vectors of CutaneousLeishmaniasis by Scanning Electron Microscopy

ANA C BAHIA1 NAGILA F C SECUNDINO1 JOSE C MIRANDA2 DEBORACI B PRATES2

ANA P A SOUZA2 FERNANDO F FERNANDES3 ALDINA BARRAL2

AND PAULO F P PIMENTA14

J Med Entomol 44(6) 903ETH914 (2007)

ABSTRACT Lutzomyia (Nyssomyia) intermedia (Lutz amp Neiva 1912) and Lutzomyia (Nys-somyia) whitmani (Antunes amp Coutinho 1939) (Diptera Psychodidae) are vectors of Americancutaneous leishmaniasis in several endemic regions of Brazil We analyzed the external morpho-logical aspects of the immature stages of these two vectors by using scanning electron microscopyIn general the larval stages of the two species are morphologically similar although somedifferences were noted Detailed examination of the eggs of both species revealed similarexchorionic ornamentations of unconnected parallel ridges The larval head capsules are welldeTHORNned heavily sclerotized and bear prominent chewing mouthparts The abdominal segmentsare easily recognized by the presence of prolegs on their ventral surfaces The morphology of theanal lobe on the terminal abdominal segment differs between the two species We found thefollowing three types of sensillae inserted on the antennae 1) clavate basiconic 2) small bluntcoeloconic and 3) multipourous clavate coleoconic In addition THORNve subtypes of trichoid sensillaewere found on the larval body 1) long 2) short 3) curved long 4) brush-like and 5) weaklybrush-like The caudal THORNlaments located on the last abdominal segment were recognized as longtrichoid sensillae We observed pores on the surface of the clavate coelonic sensillae and on thecaudal THORNlaments that presumably function as chemoreceptors The larvae of the two species showsimilarities in the lobular-form antennae of L1 larvae which changes to digitiform in second instar(L2) L3 and L4 This study demonstrated that the external surface of the eggs and larvae of Luintermedia and Lu whitmani are morphologically similar but they can be distinguished by detailsin the microanatomy observed by scanning electron microscopy

KEY WORDS Lutzomyi intermedia Lutzomyi whitmani immature stages scanning electron mi-croscopy

Lutzomyia (Nyssomyia) intermedia (Lutz amp Neiva1912 and Lutzomyia (Nyssomyia) whitmani (Antunesamp Coutinho 1939) are South American phlebotominesand szligies and proven vectors of Leishmania brazilien-sis (Vianna 1911) the causative agent of Americancutaneous leishmaniasis in Brazil and other SouthAmerican countries (Vexenat et al 1986 Jones et al1987 Rangel et al 1990 Hashiguchi et al 1992Salomon et al 1995 2001a b Brito et al 2002 Souza

et al 2002) These two closely related species (Maz-zoni et al 2002) are sympatric in some endemic areas(Oliveira dos Santos et al 1993 Miranda et al 2002)

Sand szligies are holometabolous insects with four lifecycle developmental stages egg larva pupa and adultPioneering taxonomic studies of immature stages ofphlebotomines used optical microscopy as a commontool (Barreto 1941 Guitton and Sherlock 1969 Ward1972) Later Ward and Ready (1975) introducedscanning electron microscopy (SEM) to describe de-tails of the exchorionic ornamentation of eggs of 13species of Brazilian phlebotomines Thereafter otherstudies used SEM to reveal ultrastructural details ofvarious immature stages of Phlebotomus tobbi (Adleramp Theodor 1930) (Killick-Kendrick et al 1989) andpupae and THORNrst and fourth (L1 and L4) instars ofLutzomyia longipalpis (Lutz amp Neiva 1912) (Leite etal 1991 Leite and William 1996 1997) Secundino and

1 Laboratory of Medical Entomology Centro de Pesquisas ReneRachou FIOCRUZ-MG Av Augusto de Lima 1715 CEP 30190-002Belo Horizonte MG Brazil

2 Laboratory of Immunoparasitology Centro de Pesquisas GoncaloMoniz Fundacao Oswaldo Cruz FIOCRUZ-BA Rua Waldemar Fal-cao 121 Candeal CEP 40245-001 Salvador BA Brazil

3 Laboratory of Medical and Veterinary Arthropodology Institutode Patologia Tropical e Saude Publica Universidade Federal de GoiasCEP 74605-050 Goiania GO Brazil

4 Corresponding author e-mail pimentacpqrrTHORNocruzbr

0022-2585070903ETH0914$04000 2007 Entomological Society of America
















































Acknowledgments


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Acknowledgments


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microscopy author(s): ana c. bahia, nágila f. c. secundino ... · morphology,systematics,evolution...

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