Sperm parameters of honeybee drones exposed to imidacloprid

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1 Sperm prmeters of honeybee drones exposed to imidcloprid Andrzej Ciereszko, Jerzy Wilde, Grzegorz J. Dietrich, Mciej Siud, Bet Bąk, Sylwi Judyck, Hlin Krol To cite this version: Andrzej Ciereszko, Jerzy Wilde, Grzegorz J. Dietrich, Mciej Siud, Bet Bąk, et l.. Sperm prmeters of honeybee drones exposed to imidcloprid. Apidologie, Springer Verlg, 217, 48 (2), pp <1.17/s >. <hl > HAL Id: hl Submitted on 21 Sep 217 HAL is multi-disciplinry open ccess rchive for the deposit nd dissemintion of scientific reserch documents, whether they re published or not. The documents my come from teching nd reserch institutions in Frnce or brod, or from public or privte reserch centers. L rchive ouverte pluridisciplinire HAL, est destinée u dépôt et à l diffusion de documents scientifiques de niveu recherche, publiés ou non, émnnt des étblissements d enseignement et de recherche frnçis ou étrngers, des lbortoires publics ou privés.

2 Apidologie (217) 48: * The Author(s), 216. This rticle is published with open ccess t Springerlink.com DOI: 1.17/s Originl rticle Sperm prmeters of honeybee drones exposed to imidcloprid Andrzej CIERESZKO 1, Jerzy WILDE 2, Grzegorz J. DIETRICH 1, Mciej SIUDA 2, Bet BĄK 2, Sylwi JUDYCKA 1, Hlin KAROL 1 1 Deprtment of Gmete nd Embryo Biology, Institute of Animl Reproduction nd Food Reserch, Polish Acdemy of Sciences in Olsztyn, Tuwim 1, 1-748, Olsztyn, Polnd 2 Deprtment of Apiculture, Fculty of Animl Bioengineering, University of Wrmi nd Mzury in Olsztyn, Słoneczn 48, 1-957, Olsztyn, Polnd Received 28 Februry 216 Revised 1 July 216 Accepted 5 August 216 Abstrct The objective of this study ws to evlute the effects of chronicl exposure of honeybee drones to environmentl (5 ppb) nd non-environmentl concentrtion (2 ppb) of imidcloprid (IMD) on sperm concentrtion, motility, vibility, nd mitochondril membrne potentil mesured in semen obtined from 18 drones originting from 18 colonies. The results demonstrte tht IMD exposure did not ffect sperm concentrtion; however, there were significnt differences in concentrtion within colonies. IMD exposure ws ssocited with reductions in sperm motility, which lso vried within colonies. Sttisticlly significnt interctions between IMD exposure nd colony were found for ctive mitochondri nd sperm vibility. Our results strongly suggest tht neonicotinoids cn negtively ffect honeybee drone sperm qulity. It is importnt to emphsize tht IMD ctions cn be strongly modulted ccording to the colony. Apis mellifer / imidcloprid / spermtozo / motility / vibility 1. INTRODUCTION During the pst two decdes, neonicotinoids hve been mjor group of highly effective nd widely used insecticides. Imidcloprid (IMD) is one of the most populr nd extensively used neonicotinoid insecticides to dte; in 28, it ws the world s lrgest selling insecticide nd second lrgest selling pesticide (Simon-Delso et l. 215). Neonicotinoid residues in the nectr nd pollen of bee-ttrctive crops such s oilseed rpe constitute the mjor exposure vehicle to pollintors, such s honeybees or bumblebees (Pohoreck et l. 212; Cresswell 214). Neonicotinoids exert n importnt influence on Corresponding uthor: A. Ciereszko,.ciereszko@pn.olsztyn.pl Mnuscript Editor: Dvid Trpy non-trget invertebrtes, including honeybees s the most highly studied species in this respect (Goulson 213). These effects cn be lethl but very often include brod rnge of importnt sublethl impcts chrcterized by non-liner nd non-intuitive ptterns due to the complex interply of receptor binding nd gene reprogrmming effects (Pis et l. 215). Sublethl effects my not directly cuse bee mortlity nd/or colony collpse but rther my become lethl in time nd/or render colonies more sensitive to further stresses ultimtely leding to colony collpse (vn der Sluijs et l. 213; Dively et l. 215). There is growing wreness of the knowledge gp regrding the potentil effects of neonicotinoid insecticides on the reproduction of pollintors (Blcquière et l. 212). Whitehorn et l. (212) hve observed n 85 % reduction in the production of new bumblebee queens

3 212 Ciereszko A. et l. (Bombus terrestris ) fter exposure to fieldrelistic levels of the neonicotinoid imidcloprid. The ltter suggests tht the impct of imidcloprid on the reproduction of wild-bee colonies is likely to be widespred nd significnt. Sndrock et l. (214) hve demonstrted tht chronic neonicotinoid dietry exposure hs severe detrimentl effects on solitry red mson bee (Osmi bicornis ) reproductive output mnifested by 5 % reduction in totl offspring production nd significntly mle-bised offspring sex rtio. Cresswell (211) pointed out tht most vilble dt on IMD effects on honeybees originte from studies of dult worker bees, wheres colony helth depends on the success of ll life stges, including drones. It is well estblished tht poorly inseminted queens is one of the key fctors ffecting honeybee colonies (Severson nd Erickson, 1989). Recently, Willims et l. (215) demonstrted tht the reproductive system of honeybee queens exposed to neonicotinoid pesticides ws severely ffected morphologiclly nd physiologiclly. To our knowledge, the effects of IMD on the mle reproductive system of insects hve not been studied. Such studies re lso justified by recently published results demonstrting impirments of testiculr function in IMD-treted vertebrtes (Bl et l. 212; Crdone 215; Lopez- Anti et l. 215; Lonreetl.215). Testing the sperm qulity of drones is importnt to understnd the mechnism of biology of honeybee mting, including drone fitness, polyndry, nd sperm competition (Ben Abdelkder et l. 214). The usefulness of single tests is very limited for the evlution of sperm qulity in drones (Wegener et l. 212). Therefore, multiple testing pproch is recommended in order to include different spects of drone reproductive performnce. Sperm concentrtion mesurement is importnt to evlute the efficcy nd/or disturbnces to spermtogenesis nd to evlute if sufficient numbers of spermtozo re produced over the period from spermtogenesis to sexul mturity to fertilize queens nd ensure the productivity of colonies (Rhodes et l. 211). Sperm motility reflects both energetic efficcy nd the qulity of the motility pprtus; it is lso importnt for sperm trnsfer to queen s spermthec (Ruttner nd Koeniger 1971; Ber 25). Vibility stining reflects functionl qulity of membrne permebility nd ws found to be especilly useful for the evlution of sperm competition in insects, including honeybees (Hunter nd Birkhed 22; Tofilski et l. 212). Mesurements of mitochondril membrne potentil (MMP) is importnt for evluting the function of mitochondri, which re prticulrly vitl for the production of energy during sperm movement. The use of flow cytometry, rther thn fluorescence microscopy, fcilittes high-throughput nlysis nd enbles the quntifiction of thousnds of sperm cells per smple nd between the implementtion of blind counting nd rndomiztion (Holmn 29; Rzymski et l. 212; Pynter et l. 214). We recently evluted the reltionship between the totl ntioxidnt cpcity of honeybee workers hemolymph nd IMD exposure (Słowińsk et l. 216). The results reveled tht ntioxidnt protection of honeybees is relted to ge nd my be disturbed by exposure to IMD. In the current study, using the sme experimentl design, we evluted whether IMD exposure lso ffected the qulity of drone semen. Although drones do not prticipte in colony tsks, their life is influenced in similr mnner becuse they spend their first dys inside the colony nd re fed with contminted food nd by workers contminted with IMD. The objective of this study ws to evlute the effects of chronicl exposure of honeybee drones to environmentl, fieldrelistic concentrtion (5 ppb) nd nonenvironmentl concentrtion (2 ppb) of IMD on sperm concentrtion, motility, vibility, nd mitochondril membrne potentil mesured in semen obtined from 18 drones originting from 18 colonies. Concentrtions of IMD used in our study were imed to test field-relistic concentrtions (5 ppb) s well s potentilly lethl concentrtions (2 ppb). The ltter concentrtions ws lso selected fter dt of Tppro et l. (211), who reported tht rnge high concentrtions of IMD (up to 34 ppb) cn be found in gutttion drops of corn. The usefulness of bsorbnce mesurements t 6 nm to estimte sperm concentrtions (s estblished for studies of vertebrtes)

4 Sperm qulity fter exposure to IMD 213 (Ciereszko nd Dbrowski 1993) ws lso evluted. 2. MATERIAL AND METHODS 2.1. Animls nd smpling Honeybee colonies The experiment ws crried out during the beekeeping seson (June August 214) nd included drones rered in honeybee (Apis mellifer crnic ) colonies kept in n piry ner Olsztyn, Polnd (lt , long ). We used colonies occupying two supers of Wielkopolski hives with mm frmes of type commonly used in Polnd. Ech colony contined 2-yer-old queen, nturlly mted, pproximtely 4, workers, nd 2 wx combs. Eighteen colonies were rndomly ssigned to three experimentl groups. Colonies from group BE (control group) were given food free from imidcloprid (IMD, Byer Helth Cre AG, Lerverkusen, Germny), while the food dministered to colonies from groups BE-5 nd BE-2 ws contminted with 5 nd 2 ppb of IMD, respectively. The bees were fed with sugr syrup (rtio sugr to wter 5:3) nd pollen pstry mde from fresh pollen lods nd inverted syrup API-Fortune HF 1575 (ICKO, Bollène, Frnce) in the rtio of 2.5:1.4. The colonies were given 5.5 kg of the liquid food nd.3 kg of the pstry in two portions for totl period of 2.5 months. During the experiment, nturl externl food ws bsent. The plnts did not produce nectr. Exposure to IMD resulted in its trnsfer into honeybee bodies s described by Słowińsk et l. (216). We ssumed tht IMD ws trnsferred lso to ll individuls in hive, drones included Honeybee mles nd semen collection Queens in ll colonies were cged on drone comb in 3-comb isoltor 4 weeks fter the strt of feeding. All queens were sisters rered from one mother queen. The queens were relesed fter 24 h, nd the combs contining eggs were isolted to prevent further egg lying by the queen. The brooded cells were seled on the 23rd dy fter egg lying, the combs with broods were tgged, nd then they were plced in isoltors with queen excluder, which were incubted inside the colony. During the next 24 h, the time of drone emergence ws checked t 6-h intervls. The emerged drones were weighed nd mrked with color, nd they were then plced in lower super in the bee colonies where they remined until the end of the study. The supers were isolted from the bottom nd the top with queen excluder, which prevented the drones but not workers from pssing through. The drones styed in the colonies until they were 15 dys old. Three to 4 h before semen collection, the drones were cught nd trnsported to the lbortory together with workers in cges ( mm) supplied with Apifond cndy. The body weight of drones in ll groups ws similr (33.6 ± 21.5, ± 3.3, nd ± 3.7 mg, for, 5, nd 2 ppb of IMD, respectively) nd the differences were not sttisticlly significnt (p =.12). Semen ws collected by provoking the orgn to evert by slightly bending the thorx nd pressing it with fingers, following the method described by Cobey et l. (213). Ech mle provided pproximtely 1 μl of ejculte. For ech tretment, 1 mles from 6 colonies were smpled which resulted in obtining 18 semen smples from 18 drones originting from 18 colonies Anlyticl methods Dilution of semen for nlysis Semen (1 μl) ws diluted to 1:1 rtio in Kiev buffer (Collins 25). Semen suspensions were further smpled (1 μl) for sperm motility nlysis (1 μl), for sperm concentrtion estimtion, nd (8 μl) for flow cytometry nlysis Sperm motility Semen suspensions were further diluted t 1:3 rtio with Kiev buffer contining.5 % bovine serum lbumin (finl dilution of semen ws 1:4). Semen ws incubted t 35 C for 15 min. Sperm motility ws estimted subjectively under the microscope equipped with heted stge (35 C) by single observer in blinded mnner. Live video pictures were tken using n Olympus BX4 microscope (Olympus Opticl, Tokyo, Jpn) with 1 negtive phse objective ndsonyccdblckndwhitecmer(spt- M18CE). Spermtozo were rted s motile if ctive

5 214 Ciereszko A. et l. movement (mostly circulr with sperm hed nd til overlpping) (Wegener et l. 212) ws presented. For ech experimentl tretment, semen ws obtined from 18 mles (three IMD concentrtions for six colonies, 1 mles per dose t ech colony). Ech individul smple ws nlyzed in duplicte (bout 2 spermtozo per single nlysis) Sperm concentrtion Preprtion of stndrd curvesemen obtined from four drones originted for control colonies ws used. All semen smples were crem-colored nd found t the tip of the genitli on bed of white mucus (Rousseu et l. 215). Ejcultes were first diluted 1 times, then 1 μl ws smpled nd further diluted 17, 2, 25, 3, 35, 4, 5, nd 8 times (finl dilutions were 17, 2, 25, 3, 35, 4, 5, nd 8, respectively). From these suspensions, 9 μl ws trnsferred to UVette disposble cuvettes 5 2 μl (Ct. No.: , Eppendorf AG, Hmburg Germny), nd the bsorbnce t 6 nm ws mesured using BioPhotometer (Eppendorf, Hmburg, Germny). Sperm suspensions diluted 4 times were counted using Bürker chmber. For ech drone, counting ws mde in duplicte. Averge number of counted spermtozo per drone ws 91 ± 11. Using these results, the sperm concentrtion ws clculted for other dilutions. The results were plotted nd regression eqution ws clculted. Mesurement of sperm concentrtion For routine sperm concentrtion nlysis, the smples were diluted 4 times, the bsorbnce t 6 nm ws mesured nd the sperm concentrtion expressed s 1 9 spermtozo per microliter ws clculted from the regression eqution Flow cytometry Cell membrne integrity (vibility) The sperm (1 1 6 sperm cells per smple) smples were diluted in 1 μl of Kiev buffer, nd the APC Annexin V poptosis detection kit (Biolegend) ws used ccording to the mnufcturer s instructions to chrcterize the smple. Briefly, the sperm smples were incubted for 15 min in the drk t room temperture (RT) in 5 μl of APC Annexin V nd 5 μl of 7-AAD vibility stining solution. Then,.4 ml of fresh Kiev buffer ws dded Figure 1. Representtive picture for cell membrne integrity (vibility) nlysis 7-AAD is high ffinity DNA dye tht enter nd stin ded cells, but is impermeble to live cells. nd the smples were nlyzed using flow cytometer FACS Ari II (Becton Dickinson, Sn Jose, CA). Results were nlyzed using DIVA softwre (Becton Dickinson). A representtive picture is shown in Figure 1. This kit ws used to evlute cell membrne integrity (vibility) becuse poptosis ws not observed (<.5 %) in the sperm smples. Mitochondril membrne potentil nlysis The sperm smples were diluted to finl concentrtion of sperm cells in 1 μl of Kiev buffer. Then, 5 μg/ml of Rh 123 (Sigm) ws dded nd the sperm suspensions were incubted for 3 min in the drk t RT. Next,.4 ml of Kiev buffer ws dded nd the smples were centrifuged (4 g, 5 min, RT). Next, the stining medium ws removed, the sperm pellets were resuspended in fresh Kiev buffer (1 ml) nd counterstined with 5 μg/ml of 7-AAD (Invitrogen). Immeditely fter the stining, the smples were nlyzed using flow cytometer FACS Ari II (Becton Dickinson, Sn Jose, CA). The results were nlyzed using DIVA softwre (Becton Dickinson). Approximtely 1, spermtozo were nlyzed, nd representtive picture is shown in Figure Sttisticl nlysis All the results re presented s the men ± SD. For sttisticl nlysis, dt percentges were trnsformed by

6 Sperm qulity fter exposure to IMD y = x Figure 2. Representtive picture for mitochondril membrne potentil nlysis Rhodmine 123 selectively ccumultes in mitochondri bsed on the membrne potentil. rcsin squre root trnsformtion. The Gussion distribution of vlues ws confirmed using D Agostino nd Person omnibus normlity tests. The effect of IMD nd colony on semen prmeters ws nlyzed using twowy ANOVA. Where necessry, the mens were seprted with post hoc Tukey s test. Correltion nd regression between sperm concentrtion nd bsorbnce t 6 nm were clculted using Person correltion coefficients. GrphPd Prism (GrphPd Softwre Inc., Sn Diego, CA, USA) softwre ws used for sttisticl clcultions. 3. RESULTS 3.1. Regression between sperm concentrtion nd bsorbnce t 6 nm A significnt regression between sperm concentrtion nd bsorbnce t 6 nm ws recorded (Figure 3), nd correltion coefficient ws found to be.95 (p <.1) Sperm concentrtion in reltion to exposure of honeybees to imidcloprid The verge sperm concentrtion in experimentl groups vried from 5.9 to spermtozo per microliter. A significnt effect of IMD concentrtion on sperm concentrtion ws not observed Sperm concentrtion (x 1 6 / l) 1 5 r 2 =.898, p < A6 nm (UA) Figure 3. The regression between sperm concentrtion nd bsorbnce t 6 nm (n = 32). Semen obtined from four drones. Ejcultes were first diluted 1 times, nd further diluted 17, 2, 25, 3, 35, 4, 5, nd 8 times. Then sperm suspensions were trnsferred to UVette disposble cuvettes nd the bsorbnce t 6 nm ws mesured. Sperm suspensions diluted 4 times were counted using Bürker chmber. The results were plotted nd regression eqution ws clculted (F 2,162 =.19,P =.83)(Figure4). There ws no significnt interction between IMD exposure nd colony effects (F 1,162 = 1.31, P =.23). However, there ws significnt effect of colony on sperm concentrtion (F 5,162 = 3.38, P =.6). This ws due to difference between colony 1 nd 4 (Figure 4b) Sperm motility in reltion to exposure of honeybees to imidcloprid The verge sperm motility in experimentl groups vried from 69 to 79 %. There ws significnt effect of imidcloprid on sperm motility (F 2,162 = 1.13, P <.1) between the control nd 2-μg concentrtionofimd (Figure 5). A significnt difference between colonies ws lso recorded (F 5,162 =3.97,P =.2) between colonies 1 nd 6 (Figure 5b). There ws no significnt interction between IMD exposure nd colony effects (F 1,162 =.65, P =.77) Sperm vibility in reltion to exposure of honeybees to imidcloprid The verge sperm vibility in experimentl groups vried from 95 to 99 %. There ws

7 216 Ciereszko A. et l. A Sperm concentrtion (x 1 6 / l) B Sperm concentrtion (x 1 6 / l) b IMD concentrtion [ppb] b b b Colonies b Figure 4. Sperm concentrtion in reltion to exposure of honeybees to imidcloprid ( ) nd colony (b ). Dt re presented s men ± SD. Mle semen ws collected by provoking the orgn to evert by slightly bending the thorx nd pressing it with fingers. For ech tretment, 1 mles from 6 colonies were smpled which resulted in obtining 18 semen smples from 18 drones originting from 18 colonies. Dt were nlyzed using two-wy ANOVA nd the mens were seprted with post hoc Tukey s test. Sperm concentrtion brs tht shre the sme letter did not differ significntly (P <.5) A Sperm motility (%) B Sperm motility (%) C 5 2 IMD concentrtion [ppb] b b b b b Colonies Figure 5. Sperm motility in reltion to exposure of honeybees to imidcloprid ( ) nd colony (b ). Sperm motility ws estimted subjectively under the microscope equipped with heted stge (35 C). Dt re presented s men ± SD. For ech tretment, 1 mles from 6 colonies were smpled which resulted in obtining 18 semen smples from 18 drones originting from 18 colonies. Dt were nlyzed using twowy ANOVA nd the mens were seprted with post hoc Tukey s test. Sperm motility brs tht shre the sme letter did not differ significntly (P <.5) b significnt effect of imidcloprid on sperm vibility (F 2,162 = 9.6, P =.1). A significnt difference mong colonies ws lso recorded (F 5,162 = 14.33, P <.1). There ws significnt interction between IMD exposure nd colony effects (F 1,162 = 8.18, P <.1). Significnt differences were found within colonies for prticulr IMD concentrtion (Figure 6). The effects of IMD lso vried between colonies; for exmple, no effects were found in colonies 1 nd 6, decrese in vibility ws found for colony 2, nd n increse ws recorded for colonies 3, 4, nd5(figure6b) Sperm mitochondril potentil in reltion to exposure of honeybees to imidcloprid The verge sperm mitochondril potentil in experimentl groups vried from 66 to

8 Sperm qulity fter exposure to IMD 217 A Live sperm (%) b b b b bc b c b bc b c c bc bc Colony IMD concentrtion [ppb] B 1 b b c b b IMD conc. [ppb] Live sperm (%) Colony Figure 6. Percentge of live sperm mesured by flow cytometry in reltion to exposure of honeybees to imidcloprid ( ) nd colony (b ). Dt re presented s men ± SD. For ech tretment, 1 mles from 6 colonies were smpled which resulted in obtining 18 semen smples from 18 drones originting from 18 colonies. Dt were nlyzed using two-wy ANOVA nd the mens were seprted with post hoc Tukey s test. There ws significnt interction between IMD exposure nd colony effects (F 1,162 =8.18,P <.1). Sperm vibility brs tht shre the sme letter within the sme IMD concentrtion ( ) nd colony (b ) did not differ significntly (P <.5) 93 %. There ws significnt effect of imidcloprid on sperm mitochondril potentil (F 2,162 =6.7,P =.3). A significnt difference within colonies ws lso recorded (F 5,162 = 12., P <.1). There ws significnt interction between IMD exposure nd colony effects (F 1,162 = 2.12, P =.3). Significnt differences were found within colonies for prticulr IMD concentrtion (Figure 7). The effects of IMD lso vried between colonies; for exmple, no effects were found for colonies 1 nd 6, decrese in vibility ws found for colony 2, nd n increse ws recorded for colonies 3, 4, nd 5 (Figure 7b). 4. DISCUSSION Most mesurements of sperm concentrtions in honeybee drones re bsed on counting the sperm cells under microscope. This pproch is tedious, time consuming, nd difficult due to highly vrible results tht my result from the clumping of spermtozo nd the poor dispersion of spermtozo in the hemocytometer cells (Collins 25; Koeniger et l. 25). Collins (25) hs tested the spectrophotometric method t 26 nd 55 nm nd found significnt correltion between sperm counts nd bsorbnce (r =.83). In our opinion, however, individul points shown on the regression plot in this pper were quite dispersed, nd therefore the ccurcy of this

9 218 Ciereszko A. et l. A Active mitochondri (%) b b b b b b bc b bc c b b b b b Colony IMD concentrtion [ppb] B 1 b b b b Active mitochondri (%) IMD conc. [ppb] Colony Figure 7. Percentge of ctive mitochondri in reltion to exposure of honeybees to imidcloprid ( ) nd colony (b ). Dt re presented s men ± SD. For ech tretment, 1 mles from 6 colonies were smpled which resulted in obtining 18 semen smples from 18 drones originting from 18 colonies. Dt were nlyzed using two-wy ANOVA nd the mens were seprted with post hoc Tukey s test. There ws significnt interction between IMD exposure nd colony effects (F 1,162 =2.12,P =.3). Brs tht shre the sme letter within the sme IMD concentrtion ( ) nd colony (b ) did not differ significntly (P <.5) regression cn be improved. Using our modifiction, we found the sperm concentrtion estimtion dt less dispersed, which is confirmed by high correltion coefficient vlue (.95). This result suggests tht bsorbnce t 6 nm rther thn t 55 nm provides more ccurte estimtion of sperm concentrtions in bees. Sperm concentrtions stored in spermthec re recognized s mjor component of queen longevity nd consequently productivity of the colony nd low spermtozo numbers results in premture supersedure (Cobey 27). Our results indicted tht sperm concentrtion ws not ffected by IMD tretment nd suggests tht drone spermtogenesis ws not ffected over the period from spermtogenesis to sexul mturity. This is contrry to vertebrtes, where morphologicl nd moleculr dmge in the testis were identified

10 Sperm qulity fter exposure to IMD 219 fter IMD exposure, including chnges in testiculr rchitecture nd rrested spermtogenesis (Crdone 215). The ltter lso indictes n increse in poptotic processes; however, our results did not show significnt numbers of poptotic spermtozo (<.5 %). Perhps this discrepncy reflects significnt difference in spermtogenesis nd sperm production between insects nd mmmls (Bishop 192). The honeybee testes degenerte fter htching, nd sperm is stored in the ccessory testes (den Boer et l. 29). In conclusion, spermtogenesis in honeybees seems to be resistnt to IMD. The mechnism underlying this resistnce deserves further nlysis in order to obtin comprtive dt concerning insect nd vertebrte spermtogenesis. Sperm motility is n importnt prmeter of semen qulity. It is estimted tht only 2.5 % of inseminted spermtozo ctively migrte into long-term storge (Ber 25). Wegener et l. (212) found strong correltion between sperm motility nd indictors of sperm performnce in inseminted queens. In our study, we noticed significnt decline in sperm motility only for drones exposed to n extremely high concentrtion of IMD (2 ppb). This suggests tht sperm motility cn indeed be ffected by neonicotinoids, but such effects seem to be unlikely in environmentl conditions where lower concentrtions of IMD re recorded. However, sublethl chnges in sperm motility cnnot be excluded, for exmple, vi cumultive effects of low IMD concentrtion. Such chnges cnnot be evluted by subjective method for motility estimtions s used in our study, rther more sophisticted methods using computer-ssisted sperm nlysis (Al-Lwti et l. 29) will be required. Recent dt suggest tht despite dequte insemintion, the sperm stored in queens my progressively loose vibility (Trpy nd Olivrez 214). A slight reduction ( 1 %) in sperm vibility occurs nturlly during the second stge of eversion of the endophllus nd during the injection of semen into the lterl oviducts of queens (Gençer et l. 214). A significnt vribility in the vibility of spermtozo stored in queen spermthece (with some queens contining less thn 2 % vible sperm) hs been described in Cnd (Rousseu et l. 215). Trpy et l. (212) recorded 59 to 93 % vribility in sperm vibility in the spermthece of commercil honeybee queens. Trpy nd Olivrez (214) suggested tht one of the mjor resons for lifespn reductions of queens is depletion of stored semen, which results in queen lying unfertilized eggs tht develop into drones. It is tempting to speculte tht spermtozo with decresed vibility cused by exposure to IMD my hve difficulties in coping with the stress nd consequently my die premturely, which would led to decrese in the queen s reproductive potentil. Recent results of Willims et l. (215) seem to support this suggestion. The ltter indicted reduced vibility of spermtozo stored in the spermthece of honeybee queens exposed to IMD. Moreover, recent findings of Sndrock et l. (214b) who reported 6 % queen supersedure in colonies exposed to two neonicotinoids, thimethoxm, nd clothinidin lso suggest long-term effects of neonicotinoids on the qulity of spermtozo inside queen reproductive systems. To our knowledge, mesurements of mitochondril membrne potentil (MMP) ws performed for the first time for honeybee drones. This sperm prmeter is importnt for evluting the function of mitochondri, which re vitl for the production of energy during sperm movement. For this reson, correltion between MMP nd sperm motility hs been reported for vertebrtes (Grhm et l. 199; Espinoz et l. 29; Poli et l. 211). Our results hve demonstrted tht significnt IMD colony interctions occur for MMP (similrly to sperm vibility). This strongly suggests tht IMD toxicity is relted to prticulr colonies, some of which my be more sensitive to IMD thn others. In our study, significnt interctions were found between IMD nd sperm vibility nd MMP. For this reson, only conclusions for prticulr IMD concentrtion colony vrints cn be formulted. Moreover, ll queens were sisters. For this reson, the results of our study cn be explined by pternl effects (or by other unexplined defense mechnisms). Our results suggest tht honeybee spermtozo re susceptible to imidcloprid toxicity nd this susceptibility is strongly ffected by colonies. These results re consistent with the vribility of reported IMD

11 22 Ciereszko A. et l. effects on honeybees. Pis et l. (215) reported tht the honeybee lethl dose (LD 5 ) for imidcloprid vries significntly (by two orders of mgnitude) from 5 to 5 ng, which cn prtilly be ttributed to colonies (but lso to individul vribility relted to mode of contct nd differences in environmentl conditions). Recently, Stürup et l. (213) reported tht ge effects of honeybee drone sperm vibility re relted to individul colony effects. These uthors found negtive correltion between senescence nd sperm vibility, while significnt interction between ge nd colony ws lso observed ( decline in sperm vibility ws observed in three of the five colonies). The potentil mechnism of differences in sperm vibility mong colonies my be relted to differences in seminl fluid protein bundnce (Ber et l. 212). Perhps such differences reflect the mechnism of IMD effects on the reproductive potentil of drones. It is importnt to note tht under field conditions, there might be no observble effect on queen fecundity since queens mte with up to 2 drones nd collect n excess of spermtozo. In such conditions sufficient number of vible spermtozo could be secured. Moreover, under nturl mting conditions, there is n extremely high competition of drones, so it is uncler if IMDexposed drones cn successfully compete with non-exposed drones. All these precutions hve to be tken into ccount in future studies. Recently, Willims et l. (215) demonstrted tht the reproductive system of honeybee queens exposed to neonicotinoid pesticides ws severely ffected morphologiclly nd physiologiclly. Pesticide-exposed queens developed smller ovries (lower number of ovrioles). Moreover, these queens were chrcterized by reduced number nd vibility of spermtozo stored in their spermthece. Our dt significntly complement these results by demonstrting tht mle reproduction cn be disturbed by neonicotinoids s well. Therefore, it cn be suggested tht neonicotinoids ffect the reproduction cpcity of both sexes in honeybees. In conclusion, our results strongly suggest tht neonicotinoids cn negtively ffect the sperm qulity of honeybee drones. Consequently, the exposure of honeybees to neonicotinoids cn potentilly disturb the reproductive potentil of drones nd possibly led to colony filure due poor qulity in mting. Colonies re differentilly ffected by IMD-relted chnges in sperm qulity. Future studies should identify the mechnisms responsible for the vrible susceptibility of colonies to IMD effects, to ttribute how much of observed effects there ws from the colony-level exposure. Knowledge of such mechnisms will be helpful in developing strtegies countercting the negtive effects of IMD on honeybee reproduction. Moreover, due to lrge vrition in IMDresponse mong colony future experiments should focus on feeding individul drones with IMD. ACKNOWLEDGMENTS This work ws funded by the Institute of Animl Reproduction nd Food Reserch nd the University of Wrmi nd Mzury in Olsztyn. We would like to thnk Dr. Gbriel Bodek from the In Vitro Lbortory for performing flow cytometry nlyses. OPEN ACCESS This rticle is distributed under the terms of the Cretive Commons Attribution 4. Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. Prmètres du sperme des mâles d'beille exposés à l'imidclopride Apis mellifer / néonicotinoïde / spermtozoïde / motilité / vibilité Prmeter des Drohnensperms der Honigbiene nch Imidcloprid-Exposition Apis mellifer / Imidcloprid / Spermtozo / Beweglichkeit / Vibilität REFERENCES Al-Lwti, H., Kmp, G., Bienefeld, K. (29) Chrcteristics of the spermthecl contents of old nd young honeybee queens. J. Insect Physiol. 55 (2), Ber, B. (25) Sexul selection in Apis bees. Apidologie 36 (2), 187 2

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