THE ROLE OF AGRRESSIVE AND NATURAL DEFENSE BEHAVIORS IN CONTROLLING VARROA MITE IN HONEY

BEE COLONIES (APIS MELLIFERA L.)

SALLY F. ALLAM, MAHMOUD E. ZAKARIA* AND MAHMOUD E. NOUR **

Dept. of Agric. Zoology and Nematology, Fac. of Agric. Cairo Univ., Giza, Egypt. *Dept. of Apiculture, Plant Protection Research Institute, Agriculture Research Center,

Dokki, Cairo, Egypt .**Dept. of Economic Entomology and Pesticides, Fac. of Agric. Cairo

Univ., Giza, Egypt.

ABSTRACT The aggressive and natural defense behaviors (grooming and cleaning behaviors) of honey bee Apis mellifera L. for controlling varroa mites were investigated in autumn and winter seasons. The results indicated clear differences between the tested races and hybrids of bees (Aggressive and non- aggressive) in grooming and cleaning behaviors against varroa mites. The results also revealed clear relationship between the natural and aggressive defense behaviors and types of the sensilla organs in the tested antennae.

INTRODUCTION

The use of chemicals or natural products for controlling varroa mites, the most dangerous parasite to honey bee colonies, is not quite enough. The options for controlling varroa mites by using colony management or biotechnical measures alone are limited ( Dung et al., 1997). So the priority should be references to the field of genetic improvement of the natural defense mechanisms against varroa mites (Rinderer et al., 1997 and Salem et al.,2001). Some bee races could resist varroa infestation without any treatment and this was mainly due to a high percentage of infertile female mites in worker brood cells. Several characters of honey bee can affecting resistance / tolerant to varroa mites as grooming and brood removal behaviors (hygienic behavior). The grooming is possible factor limiting mite success. Grooming would lead to low numbers of offspring (Flottum, 1997). Besides the grooming activity of the bees, the damage rate under field conditions is certainly influenced by several factors such as mite mortality inside the brood cells and other potential mite predators ( Hoffman, 1993 and Boecking,1994). The hygienic behavior is considered the primary natural defense against some diseases is believed to be controlled by two independently assorting, recessive genes: one for uncapping and another for removing disease brood from the nest. If a colony removes all of these pupae within 48h. the colony probably be resistant to disease (Mayer,1996 and Abd El Wahab et al.,2006). Mite population growth in individual colonies was negatively correlated with reduced infestation after 40-h. of brood exposure and with reduced mite fertility after one week (Villa et al., 2009). The antennae sensilla organs of worker bees play an important role in the natural defense behaviors against varroa mites, because they responsive to the stimuli from a mite infested workers as well as brood cells. There is a relationship between the

natural defense behaviors and number and sort of the sensilla organs in the honey bees infested and non – infested with varroa mites (Stort and Rebustini, 1998). This study aimed to clarify the role of aggressive and natural defense behaviors in relation to antennal sensilla organs in controlling varroa mite in honey bee colonies (Apis mellifera L.).

MATERIALS AND METHODS

This work was carried out in an private apiary in Ckerdasa region, Giza governorate, Egypt, during autumn and winter seasons, 2008.

Twelve honey bees colonies were used for this study. The tested colonies of Egyptian race (Apis mellifera lamarkii), Egyptian hybrid and aggressive and non- aggressive of Carniolian race (Apis mellifera carnica), were subjected for this study (Three colonies for each race and egyptian hybrid). The tested bee colonies were not received any chemical treatments against varroa mites during the experiment period. І – Evaluation of the aggressive behavior in tested colonies

For evaluating aggressiveness of bees, a modified technique of Stort, (1974) and El-Sarrag,(1977) was used. A strong perfume of plant origin was grayed on the black leather ball before swinging the ball in front of the entrance hole of the hive to provoke even the quiet colonies. Half an hour between every two testes was convenient to settle down the parlous temper before the next provocation.

ІІ-Determine the nature defense behaviors in the tested colonies: 1- Grooming behavior Fallen dead and alive varroa mites on the sheet of white paper which placed on the bottom board of the bee colonies were collected every 2 hours intervals/ 3 days /week from 10.am to 4pm. during the heavy infestation levels of varroa mites in autumn and winter seasons (De Jong, 1984). The collected mites were clearly by Nesbitts solution and mounted in Hoyer’s medium. The morphological specifications were detailed using light microscope. The damage occurred of each part of the body mites by the grooming behavior was detected and classified into 5 main cutting groups as follows; 1- Gnathsoma and some segments of the legs.2 - Gnathsoma and all segments of legs except coxae.3- Gnathsoma , some segments of the legs and all segments of legs except coxae 4-Gnathsoma , legs from coxae and parts of the abdomen . 5-The dorsal shield.

2 – Cleaning (Hygienic) behavior

Sealed worker brood from each tested bee colony was divided into pieces of squares (2 inch square) and frozen for 48 h., then placed into brood combs of heavy

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infested bee colonies. The removal of dead brood cells by the worker bees were estimated at 24, 48 h., week and 2 weeks, respectively. Score each colony ; 1- Didn’t clean. 2- Poor (clean in two weeks). 3- Good (cleaned in 48 h.) – 4- Excellent ( Cleaned in 24 h.). The colonies which removed the frozen killed brood during the first 24 h. relished by the high degree of brood removal behavior (hygienic behavior) (Kefuss and Taber 1996).

ІІІ- Determination of some biological activities of tested bee colonies The sealed brood area (Inch2) and the number of combs covered with bees were

recorded every 12 days intervals during the experimental period.

ІV- Study the antennal sensilla organs using Scanning Electron Microscopic studies (SEM) The Scanning Electron Microscopic technique (SEM) (JEOL GM 4200) was used at the Applied Center for Entomonematodes (ACE), Fac. of Agric., Cairo University to investigate the types and numbers of the sensilla organs on the antennae of the bee workers represented the tested bee races and hybrids. The antennae were dissected out from the bees then dried to the critical point using co2., mounted and coated with the gold according to method of Fashing et al., (2000). The sensilla organs were determined and recorded on the 10 flagellomeres, photographed, counted/ flagellomere and expressed as mean numbers / unit area (125 x 83.3 µm).

RESULTS AND DISCUSSIONS

І- Determine of the nature defense behaviors 1-Grooming behavior

As shown in Table (1) all the tested colonies ( Aggressive and non-aggressive behaviors) indicated the grooming behavior against varroa mites. Also there were clear differences in this behavior according to the bee race and hybrid. The total No. of fallen varroa mites according to the grooming behavior was clear in aggressive Carniolian race 302 mites (44.8%) followed with the nature defense behavior of the Egyptian race 180 mites (26.7%), while non aggressive of Carniolian and Egyptian hybrid recorded the lowest values of the fallen dead varroa mites 103 mites(15.28%) and 89(13.2%), respectively. The microscopic studies of the damaged varroa fallen due to the Grooming behavior, showed several forms of damage varroa mite (Fig.1) as follows; a- Gnathsoma and segments of the legs. b- Gnathsoma and portions of the legs from coxae. c- Gnathsoma and portion of legs as segment and from coxae. d -Gnathsoma , legs from coxae and parts of the abdomen. e-The dorsal shield. The results also indicated that the damage to varroa bodies represented the highest percentage in the adjective (a) 14.56%, from the total, while the lowest one was 1.32% for non-aggressive and aggressive Carniolian race, respectively. Non- aggressive Carniolian race recoded the highest percentage for the adjective (b) (9.7%), while the lowest percentage was with Aggressive Carniolian race(1.65%). The adjective (c) showed higher percentage (4.49%) with the Egyptian hybrid, whereas the lower registration (0%) was in aggressive Carniolian race. The Egyptian

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race distinguished by decisive percentage of the adjective (d) (18.33%) among other tested bee races and hybrids. Biting the dorsal shield (adj. e) showed lowest percentage in and between all adjectives of the cutting varroa body.

2-The hygienic behavior

The Egyptian bee race savor by the higher and faster hygienic behavior (removal dead frozen varroa mites during the 1st 24hrs.) ( 20%) than other tested bee races and hybrid aggressive and non-aggressive which not increased than 5% on more (Table 2) which consider poor in the cleaning behavior. The grooming and hygienic behaviors in honey bee colonies may be not related to the aggressive behaviors of tested colonies, that possible too due the race and hybrid of bee colonies. The tested colonies showed different forms of damage to the varroa mites, this may be due to the origin genetic of the bee race and hybrid. It could be recommended to utilize from the obtained results in the improvement hereditary program for naturally controlling varroa mite.

ІІ-The biological activities

Results presented in Table (2) showed that sealed worker brood area were higher with the Egyptian hybrid followed with Carniolian and Egyptian races. While the aggressive of the Carniolian race showed the lowest values, while the number of covered combs with bees were higher in Egyptian race and its hybrid, whereas the lowest value was recorded too with the aggressive of the Carniolian bees. These results were reflected the effect of the natural defense behavior against varroa mites on the development of the biological activities of tested bee colonies.

ІІІ-The antennal sensilla organs Higher percentage of the Trichodea type (B) (97.14%) was recorded with Egyptian race followed with the aggressive Carniolian race (79.58%), while the lowest percentage was recoded with the Egyptian hybrid (38.70% ) (Table 3 and Fig.2). The long Trichodea type (B) (54. µm long) (Fig.3) were related with the aggressive of the Carniolian race (12.88%) in comparison with the other bee races which recorded the lower percentage. Solitude the Egyptian hybrid by presence the Trichodea type (C) by the lower percentage (1.15%) (Fig.4). From the obtained results it could be concluded clearly relationship between the natural and aggressive defense behaviors and types of the sensilla organs in the tested antennae particularly Trichodea type (B) in Egyptian and aggressive Carniolian bee races(Table 4).

The results obtained are in agreement with the finding of Abd El Wahab,(2001) who reported that the grooming behavior is more clear in tolerant the Egyptian race followed with Carniolian race from Manzala region of egypt. The African honey bees Apis mellifera lamarkii , are more active in grooming and cleaning hygienic behaviors than European bees twice as susceptible to infestation as Africanized bees (Aumeier et al.,1996 and Guzman et al. 1996). Other idea including variation among different colonies at different times of the year and different weather and

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humidity conditions (Rinderer, 1986). The highly aggressive behavior may have evolved in response to greater levels of predation (Schneider and Nally 1992). The defensive response consists of several behavior patterns of worker bees; stinging, guarding and pursuing. The positive feedback on guarding behavior could be explained by either releaser or primer effects of alarm pheromone or perhaps the act of guarding causes release of neurohormones (neuropeptides or biogenic amines) that reinforce the behavior. Over 40 aliphatic and aromatic compounds have been identified in the alarm pheromone produced primarily in the Koshewnikov gland associated with the sting apparatus (Wager and Breed, 2000). Neuromodulatory systems associated with aggression in vertebrates include the biogenic amines serotonin (5-HT), dopamine (DA), and the neurotransmitter, g-aminobutyric acid (GABA) (Miczek et al., 2002). Significant variation in (JH) titer among different bee colonies. The correlation between (JH) and levels of aggressiveness within a colony suggests a regulatory role for (JH), but variation among colonies involves factors other than (JH) (Pearce, et al., 2001). Stimulation of olfactory receptor neurons in the presence of (IAA) as measured by electroantennograms can be reduced by the smoking (Visscher et al., 1995). The aggressive tested Carniolian honey bee are more aggressive than other bees and less active in their biological activities inside bee colonies as the Africanized honey bees “killer bees.” which attack in larger groups, make less honey, and less wax than European honey bees. There were reducing significant in the average No. of sensilla Trichodea type A and B of antennae among the infested and healthy newly emerged honey bee workers particularly in the deformed worker bees. Significant lowers of mean length, width and mean surface area of antennal flagellomer were found in the heavily infested and deformed worker and drone bees(Abd El Wahab et al.,2006). That may be clarify the role of these sense organs particularly in the aggressive bee colonies. Egyptian bee venom and the hybrid bee venom have strong similarities in their biochemical composition (Zalat et al., 2002).

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Table 1. Grooming behavior of different bee races and hybrids against Varroa mite in honeybee colonies.

Race of bees

Date Groo_

ming bev.

(fallen mites)

Adjectives of the grooming behavior

a b c d e Total

Egyptian race

1/10 0 - - - -1/11 0 - - - -1/12 0 - - - -1/1 104 13(12.5%) 3(2.88%) 2(1.92%) 20(19.2%)1/2 50 6(12%) 2(4%) - 9(18%)1/3 26 0 4(15..38%) 1(3.84%) 4(15.38%)

Total180 (26.7%)

10.55% 5% 1.66% 18.33% 35.55%

Egyptian hybrid

1/10 0 - - - -1/11 2 - - - -1/12 5 - - - -1/1 20 - - - -1/2 37 2(5.40%) 2(5.40%) 3(8.10%) 3(8.10%)1/3 25 1(4%) 2(8%) 1(4%) 1(4%)

Total89 (13.2%)

3.37% 4.49% 4.49% 4.49% 16.85%

Car

niol

ian

race

Nor

mal

1/10 0 - - - -1/11 0 - - - -1/12 3 - - - -1/1 30 2(6.66%) 3(10%) 2(6.66%) 2(6.66%) 1(3.3%)1/2 50 12(24% 5(10%) - 1(2%)1/3 20 1(5%) 2(10%) - -

Total103 (15.28%)

14.56% 9.7% 1.94% 1.94%% 1.94% 30.08%

Agg

ress

iven

ess

1/10 2 - - - -1/11 5 - - -1/12 20 - - - -1/1 50 - - - -1/2 124 4(3.22%) 3(2.41%) 6(4.83%) 2(1.16%)1/3 101 - 2(1.98%) 4(3.96%) 1(0.99%)

Total302(44.8%)

1.32 % 1.65 % 0.0% 3.31 % 0.99% 7.28%

Total 674*a) Severed Gnathsoma and some segments of the legs; b) Severed Gnathsoma and all segments of legs except coxae. c) Severed Gnathsoma, some segments of the legs and all segments of legs except coxae. d) Severed Gnathsoma, legs from coxae and parts of the abdomen, e) Severed dorsal shield.

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Table 2. The hygienic behavior and some biological activities ofdifferent honeybee races and hybrids. Bee race Hygienic

behavior %Sealed worker

brood area (inch2)No. of combs

covered with bees

Native Egyptian 20 100 10Egyptian hybrid 5 255 9Carniolian N 4 230 8

A 5 90 7N: Normal; A: Aggressive

Table 3. Mean numbers and percentages of sensilla organs on theantennae of honeybee workers in different bee races and hybrids.

Bee race

Antennae sensilla organsTrichodea Basiconica Placodea Campaniformea Coeloconica

Type A Type B Type C Long TrichodeaType A Type B

Native Egyptian

0.33(0.83%)

38.4(97.14%)

0.0(0.0%)

0.0(0.0%)

0.0(0.0%)

0.70(1.77%)

0.10(0.26%)

0.0(0.00%)

0.0(0.0%)

Egyptian Hybrid

11.11(21.28%)

20. 238.70%

0.60 (1.15%)

0.0(0.0%)

0.20(0.39%)

0.0(0.0%)

19.5 (37.34%)

0.20(0.38%)

0.40(0.76%)

Car

linio

n ra

ce

N 12..3(19.46%)

35..9(56.80%)

0.0(0.0%)

0.6(0.94%)

0.7(1.11%)

0.30(0.47%)

11..50(18.20%)

1.0(1.59%)

0.90(1.43%)

A 1.64(3.62%)

36.14(79.58%)

0.0(0.0%)

1.64(3.61%)

5.85(12.88%)

0.14(0.31%)

0.0(0.0%)

0.0(0.0%)

0.0(0.0%)

N: Normal; A: Aggressive

Table 4. Comparison between normal and aggressive defense behaviors in Egyptian and Carniolian bee races.

Defense behavior Normal Aggressive

Native Egyptian race Agricultural Carniolian race

Grooming behavior% 26.7 44.8

Severing Varroa body% 18.33 3.31

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Hygienic behavior% 20 5

Trichodea sensilla(organ type B)%

97.14 79.58

a bc d

Fig. (1): Different damages of varroa mite due to the grooming behavior.

a – Biting of Gnathosoma and some segments of the legs. b- Biting of Gnathsoma and all segments of legs except coxae c- Biting of Gnathsoma and, some segments of the legs and all segments of legs except coxae d- Biting of Gnathsoma and legs from coxae and parts of the abdomen( The white arrow pointed to Gnathosoma position, wile the black one pointed to the site of the legs cutting Coxa or segment).

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Fig. (2): Predominate of the Trichodea type (B) in the Egyptian bee race (54. µm long & 97.%) .

Fig. (3) : Solitude of the long Trichodea type (B) in the aggressive Carniolian race (12.63 µm. long &12.88%).

Fig. (4) : Solitude Egyptian hybrid by presence the Trichodea type (C) in the lower percentage (1.15%) (white arrow).

REFERENCES

Abd El- Wahab, T.E., 2001. Physiological and morphological studies on the natural defense behavior in honey bee colonies against varroa mites. Ph.D. Thesis, Fac. of Agric. Cairo, Univ., 154. Abd El- Wahab, T.E., M. E. Zakaria and M. E. Nour,2006.Influence of the Infestation by varroa mite Varroa destructor on some antennal sense organs of the worker and drone honey bees Apis mellifera L . Journal of Applied Sciences Research 2(2): 80-85.

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Aumeier, P., P. Rosenkranz and L.s. Goncalves , 1996. Defense mechanisms of honey bees against varroasis and brood disease: comparison between Apis mellifera carnica and Africanized bees in Brazil. Apidologie, 27 (4): 286-288.Boecking,O.1994.The removal behavior of Apis mellifera towards mite infested brood cells as a defense mechanism against the ectoparasitic mite Varroa jacobsoni. Inaurgural –Dissertation zur Erlangung des Grades,Rheinischen Friedrich-Wilhems Universität zu Bonn,Germany,131pp.(Apic./Abst.663/1995).De Jong, D., 1984. Current knowledge and open questions concerning reproduction in the honey bee mite Varroa jacobsoni. Adv. Inv. Repord., 3: 547-552.Dung, N., N. Tan, L. HAUN and W. Boot ,1997. Control of honey bee mites in Vietnam without the user chemicals. Bee Word.,78(2): 78-83. El-Sarrag, M.S.,1977. Morphometrical and biological studies on Sudanese honey bees Apis mellifera L. (Hymenoptera: Apidae). Ph.D. Fac. of Agric. Cairo. Univ. 207.Fashing, N.J., B.M. Oconnor and R.L. Kitching, 2000. Lamingtona carus, a new genus of Algophagidae (Acari: Astigmata) from water filled treeholes in Queensland, Australia. Invertebrate Taxonomy, 14: 591-606.Flottum,K., 1997. 21st century Apiculture: A review of the east lasing symposium. Bee Culture.125, 8: 24-26.Guzman, E.N., A. Sanchez, R.E. Jr. Page and T. Garcia, 1996. Susceptibility of European and Africanized honeybees (Apis mellifera L.) and their hybrids to Varroa jacobsoni Oud. Apidologie, 27 : 93-103.

Hoffmann,S.,1993. The occurrence of damaged mites in cage test and under field conditions in hybrids of different Carniolian lines. Apidologie,24:493-495.Kefuss, J. and S. Taber, 1996. A practical method to test for disease resistance in honeybees. Am. Bee J., 136: 31-32.Mayer,M.,1996. Testing for super hygienic bees. Bee Culture,124:517-519.Miczek, K.A., E.W. Fish, J.F. de Bold and R .M.M. de Almeida, 2002.Social and neural determinants of aggressive behavior: pharmacotherapeutictargets at serotonin, dopamine and g-aminobutyric acidsystems. Psychopharmacology 163, 434–458.Pearce, A.N. , Z.Y. Huang and M.D. Breed, 2001. Juvenile hormone and aggression in honey bees. Journal of Insect Physiology, 47 :1243–1247Rinderer, T. E., 1986. Africanized Bees: An Overview. Amer.Bee. J., 126:98-100:128-129. Rinderer T. E., N. V. Kuznetsov, R. G. Danka and G. T. Delatte, 1997: An importation of potentially varroa resistant honey bees from Far Eastern Russia. Amer. Bee. J.,137(11): 787-789.Salem, M.S., M.E. Nour, N.E. Dimetry and T.E. Abd El Wahab, 2001. Scanning Electron Microscopic studies of some antennal receptors of the worker honey bees tolerant to varroa mite. Integrated pest management. Proceedings of the 1st congress Cairo Univ. Fac. of Agric. Dept. of Economic Entomology and Pesticides , 22-23 April.Schneider, S. S. and L. C. Mc Nally, 1992. Colony defense in the African honey bee in Africa. Environmental Entomology. 21: 1362-1370. Stort,A.C.,1974. Genetical study of aggressiveness of two subspecies of Apis mellifera in Brazil. 1. Some testes to measure aggressiveness. J. Apic. Res.,13,1:33-38. Stort A. C. and M. E. Rebustini ,1998. Differences in the number of some antennal sensilla of four honeybee (Apis mellifera) types and comparisons with the defensive behaviour. J. Apic. Res., 37(1): 3-10.

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Villa ,J. D., R.G. Danka and J.W. Harris, 2009. Simplified methods of evaluating colonies for levels of varroa sensitive hygiene (VSH). J. Apic. Res., 48 (3): 162 – 167.Visscher, P.K., R.S, Vetter and G.E. Robinson, 1995. Alarm pheromoneperception in honey bees is decreased by smoke (Hymenoptera:Apidae). Journal of Insect Behavior, 8: 11–18.Wager, B.R. andBreed, M.D., 2000. Does honey bee sting alarm pheromonegive orientation information to defensive bees? Annals of theEntomological Society of America 93, 1329–1332.Zalat, S., A. Abouzeid, A. Ibrahim and M. Abd El-Aal ,2002. Protein pattern of the honey bee venoms of Egypt. Egyptian Journal of Biology , 4: 142-146.

العربي الملخص

سلو و دور الشراسة فى كيات الطبيعي مكافحةالدفاع العسل نحل طوائف فى الفاروا طفيل

– * نور – السيد محمود زكريا عزت محمود عالم فاروق **ساليمصر – – – – الجيزة القاهرة جامعة الزراعة كلية والنيماتودا الزراعي الحيوان .قسم

مصر – – – - الدقى الزراعية البحوث مركز النباتات وقاية بحوث معهد النحل *.قسم – - مصر – – الجيزة القاهرة جامعة الزراعة كلية والمبيدات االقتصادية الحشرات **.قسم

دراسة م تمت فى الشراسة وسلوك الطبيعي الدفاع فى كافحةسلوك الفاروا طفيلالعسل طوائف والشتاء –الكرنيولى –المصري نحل الخريف موسمي فى المصري .الهجين

وغير ( أن النتائج أوضحت شرسة المختبرة النحل وهجن سالالت بين واضحة اختالفات هناك . هناك) أن أيضا الدراسة أوضحت الفاروا لطفيل والتنظيف التقطيع سلوكي من كل فى شرسة

شغاالت استشعار قرون فى الحس أعضاء ونوع الدفاعي السلوك نشاط بين واضحة عالقةالنحل .

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