IMPACTS OF SUB-LETHAL DOSES OF AMITRAZ AND TAU- FLUVALINATE ON SOME PARAMETERS OF HONEY BEE WORKERS AND DRONES

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1 516 Mun. Ent. Zool. Vol. 12, No. 2, June 2017 IMPACTS OF SUB-LETHAL DOSES OF AMITRAZ AND TAU- FLUVALINATE ON SOME PARAMETERS OF HONEY BEE WORKERS AND DRONES Hossm F. Abou-Shr*, **, Mrtin Stron* nd Ttin Čermáková* * Animl Production Reserch Centre Nitr, Institute of Apiculture Liptovský Hrádok, SLOVAKIA. ** Deprtment of Plnt Protection, Fculty of Agriculture, Dmnhour University, Dmnhour, 22516, EGYPT. E-mil: hossm.frg@gr.dmu.edu.eg [Abou-Shr, H. F., Stron, M. & Čermáková, T Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on some prmeters of honey bee workers nd drones. Munis Entomology & Zoology, 12 (2): ] ABSTRACT: There re vrious chemicls vilble for the control of Vrro mites from honey bee colonies including mitrz nd tu-fluvlinte. The optimum dose of these chemicls should give the highest Vrro control efficcy with miniml negtive impcts on honey bees. The high doses re expected to hve destructive impcts on honey bees. However, the impcts of the sub-lethl doses of these chemicls on honey bees re not well known. Therefore, the potentil effects of the sub-lethl doses of these two chemicls on the survivl of honey bee workers nd drones, nd on some body prmeters were investigted. The study showed tht the exposure of honey bee workers nd drones to sub-lethl doses of chemicl A (mitrz 125 mg/ml) or chemicl B (tu-fluvlinte 240 mg/ml) hd no cler negtive impcts on their survivl rtes. Only tu-fluvlinte nd not mitrz showed dverse impcts on mesured body prmeters of workers nd drones, suggesting impcts on bee physiology. More insights into the potentil impcts of tu-fluvlinte t different doses including the optimum one on bee physiology re dvisble. KEY WORDS: Honey bees, Vrro, mitrz, tu-fluvlinte Honey bee colonies re impcted by mny fctors including biotic nd biotic ones. Vrro mites, Vrro destructor, re mong the biotic fctors which strongly impct honey bees in pssive wy worldwide. These mites were considered s one fctor responsible for colony collpse disorder (CCD) s mentioned by Rngel, Trpy (2016). Mny nturl, mechnicl or chemicl methods to control Vrro hve been developed nd tested s reviewed by Abou- Shr (2014). However, the use of chemicl mterils (i.e. cricides) is lmost the most effective wy. One of the problems of using cricides is the high residue level of them in bee products, for exmple mitrz in nlyzed honey smples by Çobnoğlu, Tüze (2008). The chemicls pproved for Vrro control re different mong countries but chemicls contin either mitrz or tu-fluvlinte mong the common ones. For exmple, chemicls contin tu-fluvlinte is common in North Americ (Frost et l. 2013) while those contin mitrz is used in some Africn nd Europen countries. The resistnce to these two cricides by Vrro mites hs been found to be different ccording to piry loction (Kmler et l. 2016). Applied cricides within beehives hve effects on Vrro mites nd on honey bee s well. Unfortuntely, evlution the efficcy of cricides ginst Vrro mites (Škerl et l. 2011; Semkiw et l. 2013) or other mites within colonies (Vndenberg & Shimnuki, 1990) gined more studies over evlution of their effects on honey bees. Therefore, evluting the potentil impcts of cricides on honey bees is essentil.

2 Mun. Ent. Zool. Vol. 12, No. 2, June Amitrz nd fluvlinte hve reltively high efficcy ginst Vrro mites if compred with other Vrro control options. The efficcy hs been found to be from 90.6% to 94.6% for mitrz (Krus & Berg, 1994) nd bout 69.21% for fluvlinte strips (Ahmd et l., 2013). However, previous studies hve shown some pssive impcts of mitrz nd fluvlinte on honey bees. Strcheck et l. (2012) hve found negtive impcts of mitrz on the cuticle proteolytic system of honey bee workers, where hydrophobic protein concentrtions were reduced in treted bees. Škerl et l. (2010) hve detected cricides in treted lrve nd dult of worker bees including fluvlinte which suggesting negtive impct on treted bees. Shouky et l. (2013) hve found low sperm count in drones treted with fluvlinte or mitrz. Most studies hve concentrted on using lethl or higher doses of the cricides. Thus, investigting the potentil effects of the sublethl doses on honey bees is strongly required especilly honey bees cn frequently be exposed to sub-lethl doses. Therefore, the study imed to investigte the potentil impcts of the sub-lethl doses of two common cricides (mitrz nd tu-fluvlinte) on the survivl nd some body prmeters of honey bee workers nd drones. MATERIALS AND METHODS 1. The experimentl conditions. Metl cges with glss sides nd perforted bottoms were used in this study (Fig. 1). During the experiments ll cges were left t room temperture bout (18±1 C) t the lbortory of the Institute of Apiculture, VÚŽV, Slovki. The cged bees were supplied with 1 ml of sugr syrup in perforted Eppendorf tubes (Fig. 1) on dily bsis. The bees used in the experiments were collected from Crnioln honey bee colonies t the piry of the Institute of Apiculture. All the bees were pproximtely with ge less thn 21 dys. 2. Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on honey bee workers. Two commercil chemicls contin mitrz or tu-fluvlinte were used in the experiments; 1) chemicl A (mitrz 125 mg/ml) nd 2) chemicl B (tufluvlinte 240 mg/ml). These two chemicls were pplied in similr wy to their use under field conditions. The stndrd methods to use chemicl A, for one beehive box with bees nd without brood, is by dding two drops of it on smll pper sheet (bout 2.5 X 10 cm, WXL) nd then burn it nd close the beehive up to 30 minutes. In the experiment, only 1 µl, 2 µl or 3 µl of chemicl A (herefter mitrz) were dded to smll pper sheet (2.5 X 3 cm, WXL) nd ws burnt inside glss box with dimensions (25X 25X40 cm, L X H XW) s shown in Fig. 2 for 10 minutes. The pplied mounts were less thn the optimum one to ensure tht bees exposed to the sub-lethl doses of this chemicl. For chemicl B (herefter tu-fluvlinte), it is used by mixing 50 ml of wter with 5 drops of it, nd then piece of pper (15X15 cm pproximtely) is immersed in the solution nd plced inside the beehive. In the experiment, only 1, 2 or 3 drops were mixed with 50 ml of distilled wter nd then smll pper (2X10 cm, WXL) ws immersed inside the mixture nd then ws plced in the cge. Thus, in this experiment 6 tretments were used (3 for mitrz nd 3 for tu-fluvlinte) beside one control group with sugr syrup only nd without ny chemicls. For ech tretment, 3 cges were used nd in ech cge 10 bees were dded with totl of 30 bees per tretment (group). Then, some prmeters were mesured to treted bees.

3 518 Mun. Ent. Zool. Vol. 12, No. 2, June Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on honey bee drones. The sme experiment done with worker bees ws repeted in the sme wy with drones. For ech tretment, three cges were used nd in ech cge group of 10 workers nd 5 drones were dded. Thus, for ech tretment 15 drones were tested. The workers were dded only to feed bee drones nd to mke the necessry thermoregultion within cges. 4. Mesured prmeters. For workers nd drones, dily survivl rte ws recorded up to one week fter the tretment. The ded bees were removed from the cges dily. Also, hed weight ws recorded for one worker or drone per cge (three per tretment) using sensitive blnce (KERN ABJ 220/4NM, Germny). Moreover, fresh weight (W1) ws determined nd then bees were plced in n oven t 100 C for 48 hours to record the dry weight (W2) in line with Abou-Shr (2015) method. Then, the body wter content ws determined s: (W1-W2/W1) X 100. For workers only, the developmentl degree of the hypophryngel glnds ws recorded using Hess (1942) scle, considering undeveloped glnds s degree 1 while fully developed glnds s degree Sttisticl nlysis. Three replictes (or cges) were ssigned per ech tretment in complete rndom design (CRD). Mens nd their stndrd errors (S.E.) were clculted for mesured prmeters. The rcsine trnsformtion ws utilized to trnsfer % into degrees before the sttisticl nlysis. The mens were compred using Duncn s multiple rnge test t 5% degree of probbility using SAS (Version ,2004, SAS Institute. Cry. NC, USA). Due to some conditions, redings of dy 5 nd 6 were not recorded. RESULTS 1. Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on honey bee workers. The % of surviving bees declined over one week s shown in Fig. 3. The% of surviving bees declined from 100% t dy 1 to 16.66, 53.33, 66.66, 26.66, 26.66, 3.33, nd 60% t dy 7 for tretments of 3µl of mitrz, 2µl of mitrz, 1 µl of mitrz, 1 drop of tu-fluvlinte, 2 drops of tu-fluvlinte, 3 drops of tufluvlinte, nd control group, in respect (Fig. 1). Tretments hd no significnt impct on % of surviving bees (df= 6, F= 0.69, P= > 0.05). At dy 7 only, tretments of 3µl of mitrz nd 3 drops of tu-fluvlinte hd the less surviving % with significnt differences (P<0.05) thn the other tretments. All tretments did not differ significntly thn the control group over the experiment period, except the forementioned tretments t dy 7. It is pprent tht incresing the mount of the chemicl mteril cused high decrese in % of surviving bees. It could be sid tht the sub-lethl doses showed no pssive impcts on the survivl of worker bees. Most of the mesured prmeters showed insignificnt difference (P>0.05) thn the control group s presented in Tble 1. Only tu-fluvlinte tretments differed significntly less thn control group in body dry weight. Moreover, bee group treted with 3 drops of tu-fluvlinte were less significnt in hed weight thn the control group. It seems tht the impct of tested tretments of mitrz hd no dverse impcts on mesured body prmeters of honey bees while tufluvlinte showed some dverse impcts.

4 Mun. Ent. Zool. Vol. 12, No. 2, June Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on honey bee drones. Drones either in the control group or in the other tretment groups were not ble to survive for long period (only up to 6 dys). Fig. 4 compres mong survivl rtes of the drones t different dys (2, 3 nd 4 dys fter the tretments). Tretments, in generl, impcted survivl rtes of bee drones significntly (df=20, F=2.35 nd P= <0.05). All drones of the control group hd died by dy 3 while those of the other groups survived during dy 3 nd 4 with significnt differences (P<0.05) thn the control group. The figure clerly highlights tht the highest survivl rtes (13.33%) were to groups of 1 µl of mitrz nd 1 drop of tufluvlinte while the other groups impcted the survivl rtes in similr wy (6.67% for dy 3 nd 4). According to these results, tretments of mitrz or tufluvlinte hve no negtive impct on the survivl of bee drones. Mesured body prmeters showed no significnt differences (P> 0.05) mong tretments, except tretments of 3 drop of tu-fluvlinte nd 2µl of mitrz which were less significntly (P< 0.05) thn the control group in fresh body weight. Also, tretments of 1 drop of tu-fluvlinte nd 3 drop of tufluvlinte were less significntly (P<0.05) thn the control group in dry body weight nd body wter %, respectively (Tble 2). The tu-fluvlinte showed some dverse impcts on mesured body prmeters of honey bee drones, in wy similr to this found for the workers. DISCUSSION 1. Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on honey bee workers. The tested doses of mitrz or tu-fluvlinte showed no significnt impct on the survivl of honey bee workers thn the control group. It could be sid the sublethl doses hve no destructive impct on worker bees. These results re supported by the work of Rngel, Trpy (2016), they found tht colonies provided with combs contminted with cricides hd significntly more ctivities (i.e. comb building nd food storge) thn untreted colonies. Thus, tretments sometimes hve no negtive impct on honey bees, but on the contrry their impcts could be positive. Incresing the mount of the chemicl mteril either of mitrz or tu-fluvlinte cused higher decrese in % of surviving bees. This trend is expected nd could be considered s norml becuse incresing the mount of chemicl mteril could hve some physiologicl impcts leding to impct the survivl bility. In similr wy, Vndenberg nd Shimnuki (1990) found higher bee mortlity when high doses of mitrz (bout 0.01 g) were pplied on worker bees in smll miling cges. Amitrz tretments showed no dverse impcts on mesured prmeters of bee workers. Tretments of tu-fluvlinte hd significntly negtive impcts on mesured prmeters of bee workers thn the control group. It is expected tht tu-fluvlinte hd pssive impcts on the physiology of honey bees. Similrly, Škerl et l. (2010) detected high quntities of fluvlinte in worker bee heds nd in worker lrve fter 8 dys (105 ng/g) nd 4 dys (110 ng/g) from tretment, respectively suggesting negtive impcts while very few quntities of mitrz were detected. Moreover, negtive effects of fluvlinte were found by Frost et l. (2013) on lerning, memory, responsiveness to sucrose, nd survivl of honey bee. This supports the ide tht tu-fluvlinte hs negtive impcts on the physiology of honey bees.

5 520 Mun. Ent. Zool. Vol. 12, No. 2, June Impcts of sub-lethl doses of mitrz nd tu-fluvlinte on honey bee drones. Treted drones or those of the control group were not ble to survive for long period or t lest for one week. This could be explined by the experimentl conditions where the study ws done t bout 18 C. It is known tht temperture of honey bee colonies is round 34.5 C (Jones et l. 2005). Therefore, the experimentl temperture is lower thn the optimum one which could impct the bility of drones to survive for long time pssively regrdless of tretments. In ccordnce with this, Neves et l. (2011) found no drones left the colony t tempertures of 26.5, 28.3, or even 31.3 C, suggesting high impct of low temperture of bee drones. Treted drones with either fluvlinte or mitrz were ble to survive more thn control group during dy 3 nd 4. This result is somewht resemble to those obtined by Rngel nd Trpy (2016), they found treted colonies hd significntly better ctivities thn untreted ones. This supports the ide tht tretments could hve positive impct on honey bees especilly when sub-lethl doses re used. Here, tu-fluvlinte lso showed some negtive impcts on mesured prmeters of honey bee drones. This result is in line with results obtined for worker bees. Moreover, the results of Shouky et l. (2013) re in greement with the present study, they found tht the lest body weight of gm ws to drones treted with fluvlinte. They lso found tht treted drones with fluvlinte or mitrz hd significntly low sperm number thn untreted ones. The present study showed the bsence of dverse impcts of mitrz or tufluvlinte on honey bee workers nd drones except tu-fluvlinte which could hs some physiologicl impcts on honey bees. Also, it could be expected tht mitrz is better thn tu-fluvlinte. Especilly, the efficcy of mitrz is somewht stble over long period of ppliction s found by Semkiw et l. (2013). Similrly, Abou-Shr (2014) concluded tht mitrz is best cricides ginst Vrro. CONCLUSION The study showed tht the exposure of honey bee workers nd drones to sublethl doses of chemicl A (mitrz 125 mg/ml) or chemicl B (tu-fluvlinte 240 mg/ml) hd no cler pssive impcts on their survivl rtes. Also, mitrz showed no dverse impcts on mesured body prmeters of workers or drones while tufluvlinte showed the vice vers. It could be expected tht tu-fluvlinte hs some impcts on bee physiology. More insights into the potentil impcts of tufluvlinte t different doses including the optimum one on bee physiology re recommended. Also, investigting the impcts of the sub-lethl doses on Vrro mites is lso dvisble. ACKNOWLEDGEMENTS Thnks re given to the Ntionl Scholrship Progrm (NSP) of Slovki for covering the costs of the sty of Dr. Hossm Abou-shr t the Institute of Apiculture. LITERATURE CITED Abou-Shr, H. F Continuous mngement of Vrro mite in honey bee, Apis mellifer, colonies. Acrin, 22 (2): Abou-Shr, H. F Therml tolernce chrcteristics of two honey bee rces. Journl of Agriculturl nd Urbn Entomology, 31 (1): 1-8.

6 Mun. Ent. Zool. Vol. 12, No. 2, June Ahmd, K. J., Rzzq, A., Abbsi, K. H., Shfiq, M., Sleem, M. & Arshdullh, M Thymol s control gent of mites (Vrro destructor) on honeybees (Apis mellifer). Pkistn Journl of Agriculturl Reserch, 26 (4): Çobnoğlu, S. & Tüze, S Determintion of mitrz (Vrroset) residue in honey by high performnce liquid chromtogrphy (HPLC). Trim Bilimleri Dergisi, 14 (2) Frost, E. H., Shutler, D. & Hillier, N. K Effects of fluvlinte on honey bee lerning, memory, responsiveness to sucrose, nd survivl. Journl of Experimentl Biology, 216: Hess, G Über den Einfluß derweisellosigkeit und des Fruchtbrkeitsvitmins E uf die Ovrien der Bienenrbeiterin. Beihefte zur Schweizerischen Bienen-Zeitung, 1 (2): (In Germn). Jones, J. C., Helliwell, P., Beekmn, M., Mleszk, R. & Oldroyd, B. P The effects of rering temperture on developmentl stbility nd lerning nd memory in the honey bee, Apis mellifer. Journl of Comprtive Physiology A, 191: Kmler, M., Nesvorn, M., Str, J., Erbn, T. & Hubert, J Comprison of tu-fluvlinte, crinthrin, nd mitrz effects on susceptible nd resistnt popultions of Vrro destructor in vil test. Experimentl nd Applied Acrology, 69 (1): 1-9. Krus, B. & Berg, S Effect of lctic cid tretment during winter in temperte climte upon Vrro jcobsoni Oud. nd the bee (Apis mellifer L.) colony. Experimentl nd Applied Acrology, 18: Neves, E. F., Fit, M. R., Gi, L. D. O., Júnior, V. V. A. & Antonilli-Junior, W. F Influence of climte fctors on flight ctivity of drones of Apis mellifer (Hymenopter: Apide). Sociobiology, 57 (1): Rngel, J. & Trpy, D. R In-Hive miticides nd their effect on queen supersedure nd colony growth in the honey bee (Apis mellifer). Journl of Environmentl nd Anlyticl Toxicology, 6: 377. doi: / Semkiw, P., Skubid, P. & Pohoreck, K The mitrz strips efficcy in control of Vrro destructor fter mny yers ppliction of mitrz in piries. Journl of Apiculturl Science, 57 (1): Semkiw, P., Skubid, P. & Pohoreck, K The mitrz strips efficcy in control of Vrro destructor fter mny yers ppliction of mitrz in piries. Journl of Apiculturl Science, 57 (1): Shouky, R. S., Khttby, A. M., El-Shekh, A. A., Abo-Ghli, A. H. & Elbnn, S. M Effect of some mterils for controlling Vrro mite on the honeybee drones (Apis mellifer L.). Egyptin Journl of Agriculturl Reserch, 91 (3): Škerl, M. I. S., Nkrst, M., Žvokelj, L. & Gregorc, A The cricidl effect of flumethrin, oxlic cid nd mitrz ginst Vrro destructor in honey bee (Apis mellifer crnic) colonies. Act Veterinri Brno, 80: Škerl, M. I. S., Nkrst, M., Žvokelj, L. & Gregorc, A Exposure to pesticides t sublethl level nd their distribution within honey bee (Apis mellifer) colony. Bulletin of Environmentl Contmintion nd Toxicology, 85 (2): Strcheck, A., Pleolog, J., Olszewski, K. & Borsuk, G Influence of mitrz nd oxlic cid on the cuticle proteolytic system of Apis mellifer L. workers. Insects, 3: Vndenberg, J. D. & Shimnuki, H Effect of mitrz tretments on honey bees nd on the honey bee trchel mite. Apidologie, 21 (3): Tble 1. Mens ± SE. of some prmeters mesured for bee workers exposed to different mounts of mitrz nd tu-fluvlinte.*: Mens followed by the sme letter within the sme column re not significntly vried ccording to Duncn s multiple rnge test0.05. Tretment Fresh weight Dry weight Mens ± S.E.* Body wter (%) Hed weight Glnds develop ment (degree) Control 0.10± µl of mitrz 0.11± µl of mitrz 0.09±0.01 3µl of mitrz 0.10± drop of tu- 0.08±0.003 fluvlinte 2 drops of tufluvlinte 3 drops of tufluvlinte 0.09± ± ± A 0.026±0.003 Abc 0.03±0.00 Ab 0.026± Abc 0.02±0.00 C 0.02±0.00 C 0.02±0.003 Bc 64± ± ± ± ± ± ±4.33 b 0.008± b 0.006±0.00 2b 2.66± 0.33

7 522 Mun. Ent. Zool. Vol. 12, No. 2, June 2017 Tble 2. Mens ± SE. of some prmeters mesured for bee drones exposed to different mounts of mitrz nd tu-fluvlinte.*: Mens followed by the sme letter within the sme column re not significntly vried ccording to Duncn s multiple rnge test0.05. Tretment Fresh weight Dry weight Mens ± S.E.* Body wter (%) Hed weight Control 0.24± µl of mitrz 0.23±0.003 b 2µl of mitrz 0.20±0.02 b 3µl of mitrz 0.22±0.007 b 1 drop of tufluvlinte 0.21±0.01 b 2 drops of tufluvlinte 0.21±0.01 b 3 drops of tufluvlinte 0.19±0.01 b 0.06±0.001 b 0.06±0.001 b 0.062±0.001 b 0.06±0.001 b 0.06± b 0.06± b 0.065± ± ± ±3.28 b 70.33±1.20 b 71.00±2.00 b 69.33±1.66 b 66.00±1.52 b 0.016± ± ±0.002 Figure 1. Cges used in the experiments, left: dimensions of the cges, nd right: cged bees. Figure 2. The glss box used during treting cged bees with Amitrz.

8 Mun. Ent. Zool. Vol. 12, No. 2, June Figure 3. Men % of surviving bees over one week fter the tretments. A: 3µl of mitrz, B: 2µl of mitrz, C: 1 µl of mitrz, D: 1 drop of tu-fluvlinte, E: 2 drops of tu-fluvlinte, F: 3 drops of tu-fluvlinte, nd G: control group. Figure 4. Men % of surviving bee drones t dy 2, 3 nd 4 fter the tretments. A: 3µl of mitrz, B: 2µl of mitrz, C: 1 µl of mitrz, D: 1 drop of tu-fluvlinte, E: 2 drops of tufluvlinte, F: 3 drops of tu-fluvlinte, nd G: control group.