The colony environment modulates sleep in honey bee workers

Transcription

1 215. Pulished y The Compny of Biologists Ltd The Journl of Experimentl Biology (215) 218, doi:1.12/je RESEARCH ARTICLE The colony environment modultes sleep in honey ee workers Ad En-Rothschild* nd Guy Bloch ABSTRACT One of the most importnt nd evolutionrily conserved roles of sleep is the processing nd consolidtion of informtion cquired during wkefulness. In oth insects nd mmmls, environmentl nd socil stimuli cn modify sleep physiology nd ehvior, yet reltively little is known out the specifics of the wke experiences nd their reltive contriution to experience-dependent modultion of sleep. Honey ees provide n excellent model system in this regrd ecuse their ehviorl repertoire is well chrcterized nd the environment they experience during the dy cn e mnipulted while keeping n ecologiclly nd socioiologiclly relevnt context. We exmined whether socil experience modultes sleep in honey ees, nd evluted the reltive contriution of different socil signls. We exposed newly emerged ees to different components of their nturl socil environment nd then monitored their sleep ehvior in individul cges in constnt l environment. We found tht rich wking experience modultes susequent sleep. Bees tht experienced the colony environment for 1 or 2 dys slept more thn sme-ge sister ees tht were cged individully or in smll s in the l. Furthermore, ees plced in mesh-enclosures in the colony, tht prevented direct contct with nestmtes, slept similrly to ees freely moving in the colony. These results suggest tht socil signls tht do not require direct or close distnce interctions etween ees re sufficiently rich to encompss lmost the entire effect of the colony on sleep. Our findings provide remrkle exmple of socil experience-dependent modultion of n essentil iologicl process. KEY WORDS: Apis mellifer, Wke experience, Sleep need, Socil environment INTRODUCTION The uiquity of sleep cross the niml kingdom nd the deteriortion in helth nd performnce following sleep deprivtion suggest tht sleep is restortive nd regenertive iologicl process essentil for survivl. Nevertheless, its dptive vlue remins elusive (Cirelli nd Tononi, ; Mignot, ). Accumulting evidence suggests tht n importnt nd evolutionrily conserved role of sleep is the processing nd consolidtion of informtion cquired during wkefulness (Diekelmnn nd Born, 21). In oth mmmls nd insects, environmentl nd socil stimuli cn modify susequent sleep physiology nd ehvior (e.g. Aou-Ismil et l., 21; Donle et l., 29; Gnguly-Fitzgerld et l., 26; Mquet et l., 2; Miymoto et l., 23), yet only little is known out the specifics of the wke experiences ccounting for experiencedependent modultion of sleep. Deprtment of Ecology, Evolution nd Behvior, The Alexnder Silermn Institute of Life Sciences, The Herew University of Jeruslem, Jeruslem, 9194, Isrel. *Present ddress: Deprtment of Psychitry nd Behviorl Sciences, Stnford University, Stnford, CA 9435, USA. Author for correspondence (guy.loch@mil.huji.c.il) Received 9 July 214; Accepted 4 Decemer 214 Over the pst three decdes, sleep or sleep-like sttes hve een descried for diverse verterte nd inverterte species: fishes (Proer et l., 26; Yokogw et l., 27), insects (Hendricks et l., 2; Kiser nd Steiner-Kiser, 1983; Shw et l., 2; Toler, 1983) nd even nemtode worms (Rizen et l., ), in ddition to mny mmmls. Moleculr nd genetic studies indicte tht the moleculr pthwys ssocited with sleep in vertertes nd invertertes show lrge degree of conservtion. These similrities re consistent with n ncient nd common origin for sleep (Alld nd Siegel, ; Cirelli, 29). In mmmls nd irds, sleep is usully defined y typicl corticl nd musculr ctivity, wheres in other species sleep is defined y ehviorl criteri tht include consolidted periods of inctivity ssocited with reduced muscle tonus, reduced responsiveness to externl stimuli, nd homeosttic regultion (Hendricks et l., 2; Toler, 1983). In oth rodents nd flies, environmentl nd socil enrichment modify sleep durtion nd/or intensity (Gutwein nd Fishein, 198; Mirmirn et l., 1982; Tgney, 1973). In the fruit fly Drosophil melnogster, socil enrichment increses sleep durtion nd lters sleep rchitecture (Bushey et l., 211; Gnguly-Fitzgerld et l., 26). Sleep durtion in socilly enriched flies lso increses proportionlly with the size of the (Gnguly-Fitzgerld et l., 26). Given tht this increse in sleep is not oserved in fly strins with muttions in suset of short- nd long-term memory genes, it hs een suggested tht the socil experience-dependent increse in sleep depends on the ility to form new memories (Donle et l., 29; Gnguly-Fitzgerld et l., 26). In order to etter understnd experience-dependent modultion of sleep it is necessry to define the wke experiences nd environmentl signls tht modify sleep. Honey ees (Apis mellifer Linneus 58) provide n excellent model system with which to study the nture of wking experiences modulting sleep ecuse their ehviorl repertoire is well chrcterized nd the environment they experience during the dy cn e mnipulted while keeping n ecologiclly nd socioiologiclly relevnt context (En-Rothschild nd Bloch, 212). Honey ees re eusocil insects, living in colonies contining single egg-lying queen, tens of thousnds of femle workers nd few hundred mles (Winston, 1987). Among the workers, there is n ge-relted division of lor. During the first ~2 weeks of dult life, the workers perform vrious in-hive ctivities, such s rood cre. Lter, worker ees perform vrious ctivities in the hive periphery, such s honeycom construction nd nectr storing. From ~3 weeks of dult life, honey ee workers typiclly forge for pollen nd nectr outside the hive (Roinson, 1992; Winston, 1987). The sleep ehvior of honey ees hs een studied oth in the nturl context of the colony (Kiser, 1988; Klein nd Seeley, 211; Klein et l., ; Klein et l., 21) nd in detiled lortory studies (En-Rothschild nd Bloch, ; Kiser, 1988; Suer et l., 23). Honey ees exhiit ll three ehviorl chrcteristics of sleep: period of quiescence (En-Rothschild nd Bloch, ; Kiser, 1988; Suer et l., 23; Suer et l., ), n incresed response threshold (En-Rothschild nd Bloch, ; Kiser, 1988; Kiser The Journl of Experimentl Biology 44

2 The Journl of Experimentl Biology (215) doi:1.12/je nd Steiner-Kiser, 1983) nd homeosttic regultion mechnism (Klein et l., 21; Suer et l., ). In honey ee forgers, s in mmmls nd flies, sleep deprivtion impirs lerning nd memory processes (Beyert et l., 212; Hussini et l., 29; Klein et l., 21), nd forger ees induced to lern novel nvigtionl tsks sleep longer thn control ees (Beyert et l., 212). In this study, we exmined the influence of vrious wke experiences on susequent sleep in honey ees. We focused specificlly on wke experiences tht re ssocited with socil interctions in the colony nd found evidence for strong environmentl modultion of sleep in ees. Interestingly, our results suggest tht the colony environment hs strong influence on sleep tht does not require close distnce interctions with other ees or the rood. RESULTS Experiment 1: the influence of the colony environment on susequent sleep Forger ees (typiclly older thn 3 weeks of ge) tht were individully isolted in constnt l environment illuminted y dim red light were predominntly ctive during the sujective dy nd slept during the sujective night (Fig. 1A, Fig. 2B; P<.1 in ll three colonies for the influence of time of dy; for the forgers dt presented in Fig. 2B, repeted mesures one-wy ANOVA, d.f.=3, F colony S73 =23.78; F colony H1 =.98; F colony H14 =37.3). These findings tht re sed on locomotor ctivity dt re consistent with detiled ehviorl oservtions of sleeping honey ee forgers (En-Rothschild nd Bloch, ; Kiser, 1988; Klein et l., ). Young ees tht experienced the colony environment for their first 1 or 2 dys of dult life slept more in the first dy of monitoring compred with sme-ge sister ees tht were isolted individully immeditely fter emergence (Fig. 1B D, Fig. 2A,B). Furthermore, the differences etween the colony-experienced nd individully isolted ees were pprent throughout the dy, nd not restricted to specific circdin phse (Fig. 2B). An increse in oth the numer nd durtion of sleep outs contriuted to the overll increse in totl sleep durtion in young ees experiencing the colony environment compred with individully isolted ees (Fig. 2C,D). These results demonstrte tht single dy of colony experience is sufficient to modulte susequent sleep in young ees. The differences etween forgers nd young ees tht experience the colony environment suggest tht there re dditionl ge- or experience-relted chnges in sleep. Experiment 2: the influence of socil interctions outside the colony on susequent sleep To exmine the influences of socil interctions on sleep, we confined 3 newly emerged ees for 2 dys to wooden cge outside the colony ( L ) nd lter monitored their sleep individully in the lortory. We found tht L ees slept less during the first dy in the monitoring cge (Fig. 1E, Fig. 3A,B) nd hd shorter sleep outs (Fig. 3C) compred with sister ees experiencing the colony environment for the sme period. In two of the trils (with ees from colonies H11 nd H12), the L ees ppered to sleep more thn individully isolted ees, ut these differences were sttisticlly significnt only in the tril with colony H12 (Fig. 3A). In complementry two-wy ANOVA tht included ees from ll three trils, the differences in sleep durtion etween ll three s were sttisticlly significnt (Tle 1). Tken together, these results suggest tht socil interctions with 3 peers outside the colony cn modify susequent sleep, ut not to the sme extent s the entire colony. Experiment 3: the influence of direct nd indirect interctions with other ees in the colony on susequent sleep To uncouple the influence of different components of the colony environment on sleep, we cged 3 newly emerged ees inside field colony in either single-mesh () or doule-mesh () enclosures (enling or preventing close contct with nestmtes outside the enclosure, respectively; see Mterils nd methods). Bees tht during their first 2 dys of dult life were llowed to move freely per hour A E B F C G Dys in the monitoring cge Fig. 1. Representtive sleep plots. Ech plot depicts the proportion of sleep per hour during the entire monitoring session for n individully isolted honey ee. The experimentl chmer ws illuminted with constnt dim red light. (A) A forger of unknown ge (forgers re typiclly >3 weeks of ge). (B,C) A young ee tht spent her first h ( h, B) or inside field colony (, C). (D) A young ee tht spent her first individully isolted in the l ( L individully ). (E) A young ee tht spent her first inside cge contining 3 dditionl sme-ge ees in the l ( L ). (F,G) A young ee tht spent her first confined with 3 dditionl sme-ge ees to single-mesh () enclosure ( colony, F) or doule-mesh () enclosure ( colony, G) inside colony. Sleep ws defined s lck of movement for five consecutive minutes. Differences in the x-xis stem from vrition in monitoring durtion cross experiments. D The Journl of Experimentl Biology 45

3 The Journl of Experimentl Biology (215) doi:1.12/je per dy No. of sleep outs per dy Men sleep out durtion (min) A C S73, P<.1 H1, P=.5 H14, P<.1 6,, 4, 2 D S73, P< S73, P=.1 1 forgers B S forgers : 6: h h 6: 12: h h 12: 18: h 18 L individully 18: : h L individully H1, P<.1 forgers 18 H1, P<.1 18 H14, P<.1 forgers h L individully H1 : 6: h forgers h 6: 12: h h 12: 18: h L individully 18: : h L individully forgers H143, P=.2, H14 : 6: h forgers h 6: 12: h h 12: 18: h, L individully 18: : h L individully Fig. 2. Exposure to the colony environment influences susequent sleep in young ees. (A) Proportion of sleep on the first dy of isoltion in monitoring cge in constnt l environment. (B) during the first dy divided to 6 h ins. (C) Numer of sleep outs during the first dy of monitoring. (D) Men sleep out durtion during the first dy of monitoring. Numers inside rs depict smple size. Ech column depicts repetition with ees from different source colony. Dt re mens ± s.e.m. The P-vlues were otined from one-wy ANOVA nlyses (old indictes significnce). Groups with different lowercse letters re significntly different in Tukey s post hoc test. in the colony or were confined to or enclosure showed similr totl mount of sleep, numer of sleep outs nd men sleep out durtion (Fig. 1F,G, Fig. 4). However, only in the tril with ees from colony S85 did L ees show reduced totl sleep durtion nd out numer (Fig. 4A,C) compred with sme-ge sister ees exposed to the colony. The mount of sleep for the L ees from colonies H2 nd HS76 ws comprle to tht of colony ees nd higher thn in other experiments with L ees (see Fig. 3). Nevertheless, the men sleep out durtion of L ees from colonies H2 nd HS76 (Fig. 4D) tended to e shorter compred with their sister ees experiencing the colony environment. These findings suggest tht socil interctions in smll s cn hve strong influence on sleep in some genotypes. The similrity etween the ees freely moving in the colony, in enclosures nd in enclosures suggests tht direct contct with other ees in the colony, the rood or the queen is not necessry for the influence of the colony on sleep. The influence of time in isoltion on susequent sleep durtion To etter understnd the influence of experiencing rich environment on susequent sleep, we compred sleep durtion s function of time isolted in monitoring cge in the l. For this nlysis we pooled dt cross experiments, nlyzing together ll the ees tht experienced the sme environment efore eing isolted in the l. This nlysis shows tht the influence of previous socil experience ws not limited to the first dy of monitoring ut insted lsted for severl dys (Fig. 5). Nevertheless, we found highly significnt decrese in the mount of sleep with time in isoltion for ll s of ees tht experienced complex socil environment prior to monitoring (Fig. 5; P<.1 for ll these s). Only the ees tht were individully isolted shortly fter emergence showed no significnt chnges in the mount of sleep with time in the monitoring cge (Fig. 5, P=.7). DISCUSSION Our findings show tht socil wke experience hs profound influences on susequent sleep in honey ees. Bees tht experienced rich colony environment slept significntly more compred with sme-ge sister ees tht were individully isolted in poor l environment. The influence of rich socil experience on sleep persisted for severl dys fter the ees were trnsferred to individul cges in constnt l conditions. These findings for the honey ee support nd extend previous evidence for strong socil influences on sleep in Drosophil nd mmmls (Bushey et l., 211; Gnguly-Fitzgerld et l., 26; Gutwein nd Fishein, 198; The Journl of Experimentl Biology 46

4 The Journl of Experimentl Biology (215) doi:1.12/je per dy A H11, P<.1 2 L L individully H12, P<.1 23 L c 23 L individully H6, P< L, 26 L individully Fig. 3. Socil interctions with smege ees outside the colony led to n increse in sleep, ut not to the sme extent s exposure to the entire colony environment. For detils of A D, see Fig. 2. L, young ees tht were cged in s of 3 in the l during the first fter emergence. B L L individully 1 H11 H12 H6 No. of sleep outs per dy Men sleep out durtion (min) H11, P=.5 H12, P=.5 H6, P= H11, P=.3 H12, P<.1 H6, P< C D : 6: h 6: 12: h 2 12: 18: h 2 L L individully 18: : h : 6: h : 12: h L 12: 18: h L individully 18: : h : 6: h 6: 12: h L 12: 18: h L individully 18: : h Mirmirn et l., 1982; Tgney, 1973). Tking dvntge of the honey ee socioiology, we further suggest tht the colony influence on sleep is potent even without direct or close interctions with other ees in the colony. Together, our findings suggest tht the socil signls tht influence sleep include voltile pheromones, sounds, com virtions or other environmentl vriles tht re ssocited with the ctivity or ehvior of other ees in the colony. Our findings confirm nd extend previous evidence for n gerelted decrese in honey ee sleep (En-Rothschild nd Bloch, Tle 1. Two-wy ANOVA with multiple comprisons for the dt presented in Fig. 3 d.f. MS F P Tretment < Tretment colony vs L <.1 vs L individully <.1 L vs L individully.5 ; Klein et l., ; En-Rothschild et l., 212). Young ees slept more thn their older sister forgers when the two s of ees were trnsferred from the sme colony to individul cges in the sme l environment. A similr decrese in sleep durtion with ge hs lso een reported for flies (Shw et l., 2) nd mmmls (Dijk et l., 1999; Shw et l., 2). The higher sleep requirement in young nimls is commonly ttriuted to the drmtic elortion of the centrl nervous system during erly life (Kyser et l., 214). The similrity in ge-relted chnges in sleep is consistent with the premise tht mny sleep functions re conserved cross diverse niml tx (Alld nd Siegel, ). Given the importnce of sleep t n erly ge (Kyser et l., 214), it is interesting to note tht our study further shows tht the sleep of young honey ees is very sensitive to the socil environment they experience during the first post-pupl emergence. Young ees tht experienced the colony environment slept ~5 h dy 1 more nd hd longer nd overll more sleep outs when monitored individully compred with sme-ge sister ees tht were individully isolted shortly fter emergence. Wht in the colony environment modultes sleep? Bees recurrently touch nd The Journl of Experimentl Biology 47

5 The Journl of Experimentl Biology (215) doi:1.12/je per dy No. of sleep outs per dy A B C S85, P<.1 H2, P=. HS76, P= S85, P< S85 H2 HS76 : 6: h 6: 12: h 12: 18: h L L 18: : h : 6: h 6: 12: h H2, P=.14 12: 18: h L 18: : h 27 3 : 6: h 6: 12: h HS76, P=.9 12: 18: h L : : h Fig. 4. Direct contct with other ees in the colony is not needed for colony influence on sleep. For detils of A D, see Fig. 2., 3 newly emerged ees tht were confined to enclosure inside colony during their first fter emergence;, sme s ut the enclosure hd two meshes. Men sleep out durtion (min) D S85, P= L H2, P=.5,, 27 3 L HS76, P=.2,, L ntennte other ees nd these socil interctions my ffect susequent sleep. Indeed, in Drosophil, sleep durtion incresed proportionlly with the numer of flies with which the individul ws cged (Gnguly-Fitzgerld et l., 26; Donle nd Shw, 29). Consistent with these findings for the fly, we found tht interctions with other individuls lso incresed susequent sleep durtion in honey ees. Young ees tht were housed for 2 dys in poor l environment together with 3 peers tended to sleep more thn sme-ge ees tht were individully isolted in similr environment. But the nturl socil environment of honey ees is much more complex thn intercting with 3 peers. The honey ee colony is n extremely rich socil environment composed of thousnds of dult nd young individuls relesing numerous chemicl, tctile, uditory nd virtion signls into the smll hive cvity (Winston, 1987). Our findings indeed suggest tht experiencing the complexity of the colony led to n even greter increse in susequent sleep durtion (Figs 3, 4). The specifics nd chrcteristics of the socil signls tht influence sleep re currently unknown. Using genetic pproch, it ws found tht muttions in genes involved in vision nd olfction, ut not in uditory communiction, locked the increse in sleep following socil enrichment in the fruit fly (Gnguly-Fitzgerld et l., 26). These findings suggest tht visul nd olfctory socil signls re importnt for socil modultion of sleep in Drosophil. To strt proing the nture of the signls in the colony tht influence sleep in honey ees, we used different pproch. We cged focl ees in or enclosures inside the colony. Both mnipultions prevented the ees from moving freely in the colony nd performing ctivities such s rood cre or cell clening, which re the typicl tsks of ees t the tested ges ( 2 dys of ge). The doule mesh lso prevented close interctions with other ees in the colony. It is thus remrkle tht in three out of three trils, sleep durtion nd rchitecture (out numer nd durtion) did not differ etween ees moving freely in the colony nd those confined to or enclosures. In this experiment, in one of the trils, sleep durtion ws significntly shorter in the L ees compred with the ees tht experienced the colony, while in the two other trils, only the men sleep out durtion ws shorter. The lck of difference in totl sleep durtion in two of the trils differs from the results presented in Fig. 3, nd perhps stems from the high sensitivity of these genotypes to socil interctions such tht even contct outside of the colony context could modulte their sleep. The finding tht the doule mesh did not compromise the influence of the colony environment on sleep is intriguing ecuse it suggests tht tctile signls, contct pheromones or trophllxis (i.e. the trnsfer of food or other fluids mong conspecifics), which re ll importnt mens The Journl of Experimentl Biology 48

6 The Journl of Experimentl Biology (215) doi:1.12/je per dy.8 forgers P< L P<.1 P<.1 P<.1 h P<.1 P<.1 L individully P=.7 Fig. 5. The experience-medited influence on sleep durtion decreses with time of isoltion in constnt l environment. The plots show the proportion of sleep (mens ± s.e.m.) for ech dy of isoltion in constnt l environment. Ech plot summrizes pooled dt for ll individuls experiencing the specified tretment cross experiments. Smple sizes: forgers, N=84;, N=213; h, N=65; L individully, N=113; L, N=152;, N=83;, N=81. P-vlues were otined from repeted mesures ANOVA..2. Dys in the monitoring cge of communiction in honey ees, re not necessry for the socil experience-dependent modultion of susequent sleep. These remrkle influences of the colony environment on sleep contrst with recent study suggesting tht in honey ee forgers (which were older thn the young ees studied in the current report), sleep durtion ws not ffected y the distnce flown etween the hive nd food source during the preceding dy (Beyert et l., 212). In the sme study, forgers tht were forced to lern new nvigtion tsks indeed slept longer during the following night, ut the differences were round 1 h nd there were no differences etween ees forced to lern long nd short routes (Beyert et l., 212). Although the ees nd techniques vry significntly etween the findings of Beyert et l. (Beyert et l., 212) nd our work, the comprison of the two studies suggests the intriguing possiility tht socil signls cn hve stronger influence on honey ee sleep thn cognitively demnding tsk such s lerning new route. This interesting hypothesis deserves dditionl reserch. Another importnt question is why do individuls exposed to rich socil environment sleep more thn those exposed to poorer environment? Perhps ees in the densely populted hive re sleep deprived ecuse they lmost continuously need to respond to numerous socil signls nd frequently interct with their nestmtes, nd when removed from the colony they compenste y sleeping longer (sleep reound). This hypothesis is consistent with the finding tht sleep durtion decresed with time in isoltion for ll the ees experiencing socil interctions ut not for the ees individully isolted in poor l environment (Fig. 5). However, severl studies hve suggested tht honey ees do not compenste for sleep loss y incresing sleep durtion; rther, they suggest tht ees recover from sleep deprivtion y intensifying their lter sleep (Beyert et l., 212; Hussini et l., 29; Klein et l., 21; Suer et l., ). Moreover, even if young honey ees were compensting for sleep loss y incresing sleep durtion, one would predict tht 2 dys of sleep deprivtion will result in greter sleep reound thn single dy of sleep deprivtion, which is not consistent with our findings tht sleep durtion ws similr for ees experiencing the colony environment for 1 or 2 dys (Fig. 2). An lterntive explntion for our findings is tht ees experiencing the rich colony environment sleep longer thn their individully isolted peers ecuse they need more sleep for processing the informtion they cquired while wke. Severl different hypotheses hve een rised to explin the ssocition etween sleep nd cognitive performnce, mong which is the synptic homeostsis hypothesis, which predicts strong ssocition etween wke experience nd sleep durtion (Tononi nd Cirelli, 214). According to this hypothesis, new synpses require oth spce nd energy, nd therefore their numer must e limited nd mny re downscled, mostly during sleep. The synptic homeostsis hypothesis further predicts tht the richer the environment nimls re exposed to, the longer is their susequent sleep. This prediction is supported y studies showing incresed synptic re following rich wke experience, nd susequent reduction during sleep (Bushey et l., 211; Mret et l., 211). Thus, lthough the underlying neuronl mechnisms re still elusive, it is possile tht the richness of the colony environment entils demnding informtion processing tht leds to n increse in sleep need. The findings presented ove dd to the ody of evidence on the pervsive influence of the socil environment on the ehvior nd physiology of honey ees (En-Rothschild nd Bloch, 212; Ichikw nd Sski, 23; Mleszk et l., 29). The socil mnipultions reported here lso influenced the development of circdin rhythms in the sme young honey ees (En-Rothschild et l., 212). It is thus interesting to note tht the influence of the two processes differed to some extent. First, ees experiencing 1 or 2 dys in the colony showed similr increse in sleep ut the development of circdin rhythms required 2 dys in the colony nd single dy ws not sufficient for expressing consistent rhythmicity (En-Rothschild et l., 212). Second, confinement with 3 peers in cge outside the colony led to significnt increse in sleep durtion compred with isoltion in n individul cge, ut did not seem to ffect the development of circdin rhythms (En- Rothschild et l., 212). These pprent differences suggest tht the influences of the socil environment on sleep nd on the development of circdin rhythmicity re medited, t lest prtilly, y different mechnisms. Thus, the sme socil environment my led to multiple physiologicl nd ehviorl modifictions (En- Rothschild nd Bloch, 212). Our findings show tht rich wking experience ffects susequent sleep in young honey ees. The nture of the wking experience is importnt nd socil signls, specificlly, pper to e pivotl. Additionl studies re needed for corroorting our findings suggesting tht socil informtion trnsmitted without direct or close contct encompsses lmost the entire influence of the colony environment on sleep nd for identifying the most potent socil signls tht modulte sleep in honey ees. The Journl of Experimentl Biology 49

7 The Journl of Experimentl Biology (215) doi:1.12/je MATERIALS AND METHODS Generl procedure Honey ees were derived from mixture of Europen rces of A. mellifer typicl to Isrel. We kept the colonies ccording to stndrd eekeeping techniques in the Bee Reserch Fcility t the Edmond J. Sfr cmpus of the Herew University of Jeruslem, Givt-Rm, Jeruslem, Isrel. To otin newly emerged ees, we removed honeycoms contining pupe from source colonies nd immeditely trnsferred them to light-proof continer. We plced the continer inside n environmentl chmer [31±1 C, reltive humidity (RH)=55±5%], which ws illuminted y constnt dim red light. We mrked newly emerged ees, under dim red light, with pint dot on their thorx within 3 min of emergence, nd ssigned them rndomly to one of the test socil environments ( tretments, see elow). After or, during which the ees experienced the experimentl socil environments, they were collected nd plced individully in monitoring cge, mde from modified Petri dish (dimeter 9 mm, height 15 mm) provisioned with d liitum sugr syrup. On the sme dy, we lso collected sister forgers from the entrnce to the source colony ( forgers ). We identified forgers y pollen lods on their hindlegs, nd only collected those with undmged wings (suggesting reltively little flight experience). In ech tril, the ees of ll tretment s originted from the sme source colony ( genotype ). We repeted ech experiment three times, ech with ees from different, unrelted colony, in order to estlish tht our findings were generl nd not limited to certin specific genotype (i.e. source colony). The queens of two of the source colonies (S73 nd S85) were instrumentlly inseminted with semen from single (different) drone, wheres the other nine colonies were heded y nturlly mted queen ( honey ee queen typiclly mtes with 1 2 drones). Becuse of the hplodiploid sex-determintion system of ees, femles from colony in which the mother queen mted with single mle re closely relted ( full-sisters ) with coefficient of reltionship of R=.75. Sleep monitoring We plced the monitoring cges with the focl ees in n environmentl chmer (29±1 C, RH=45±5%), which ws illuminted y constnt dim red light. We monitored locomotor ctivity with the ClockL dt cquisition system (Actimetrics Co., Wilmette, IL, USA), four light-sensitive lck nd white Pnsonic WV-BP334.8 lx CCD cmers nd high-qulity monochrome imge cquisition ord (IMAQ 149, Ntionl Instruments Co., Austin, TX, USA). Ech cmer monitored 3 cges simultneously. Empty cges served s control for ckground noise. Dt were collected continuously for 4 dys, t frequency of 1 Hz (Shemesh et l., 27; Shemesh et l., 21). The findings on the influence of environment on the development of circdin rhythms for these ees were reported previously (En-Rothschild et l., 212). We nlyzed sleep ehvior using customwritten softwre (Mtl), sed on the dt collected y the ClockL dt cquisition system. Previous studies indicted tht lck of movement for five consecutive minutes served s relile proxy for sleep in honey ees (Beyert et l., 212; En-Rothschild nd Bloch, ). Accordingly, we defined sleep s continuous period lsting 5 min or more during which the monitored ee did not move. A similr index for sleep is routinely used in studies with flies (e.g. Huer et l., ; Shw et l., 2). For ech ee, we clculted the percentge of time sleep, the numer of sleep outs, nd the men sleep out durtion during ech dy of the experiment. Sttisticl nlyses For nlyzing the influence of the environment on the vrious sleep prmeters, we used the Prism 6. softwre pckge (GrphPd Softwre). We used one-wy nlysis of vrince (ANOVA) tests followed y Tukey post hoc test to compre the proportion of time sleep, men out durtion nd the numer of sleep outs per dy. For the dt from experiment 2, we performed complementry two-wy ANOVA nlysis, ecuse the differences etween the three tretments showed similr trend in two of the colonies, yet the post hoc nlysis only reched sttisticl significnce in one of them. For the nlysis of sleep durtion s function of time in the monitoring cges, we performed repeted mesures ANOVA. Experiment 1: the influence of the colony environment on susequent sleep We ssigned newly emerged ees to one of the following tretments: (i) h in field colony ( h), (ii) in field colony ( ) nd (iii) in n individul cge in the l ( L individully ). The individul cges were similr to the monitoring cges (see ove) ut contined pollen, nd were kept in drk environmentl chmer (31±1 C, RH=55±5%). We used sister forgers from the sme source colony s positive control ( forgers; see ove). To compre ees tht spent nd in the colony, we mrked newly emerged ees during two consecutive dys. Between these 2 dys the com with the pupe ws plced in host field colony. Becuse the ees tht were introduced to field colonies were exposed to dylight twice (during the time of introduction nd collection from the colony), we lso exposed the ees isolted outside the colony to similr dylight experience. The exposure to light ws for less thn minute during the introduction, nd for period lsting 5 1 min t the time of collection. We trnsferred the ees tht were isolted individully to new monitoring cges just efore the eginning of the monitoring session. This procedure ws followed in order to expose them to similr hndling stress to tht experienced y the ees from the other s tht were trnsferred from the colony to the l. We repeted this experiment three times, ech with ees from different source colony. Experiment 2: the influence of socil interctions outside the colony on susequent sleep We ssigned newly emerged ees to the following tretments: (i) in field colony ( ), (ii) in wooden cge ( cm) with 3 sme-ge sister ees in the l ( L ) nd (iii) in n individul cge in the l ( L individully ). We plced the individul nd cges in drk environmentl chmer for (see ove). The ees in ll tretments experienced similr exposure to dylight (sme s ove). We repeted this experiment three times, ech with ees from different source colony. Experiment 3: the influence of direct nd indirect interctions with other ees in the colony on susequent sleep We ssigned newly emerged ees to one of the following tretments: (i) in field colony ( ), (ii) in enclosure together with 3 sme-ge sister ees inside field colony ( ), (iii) in enclosure together with 3 sme-ge sister ees inside field colony ( ) nd (iv) in wooden cge ( cm) with 3 sme-ge sister ees in the l ( L ). The ees in ll four tretments experienced similr exposure to dylight (sme s ove). For the tretment, we used enclosure ( cm) mde of n 8-holes per inch mesh tht ws emedded on n empty com. For the tretment, we surrounded enclosure with lrger 8-holes per inch mesh cge ( cm), plced 1.5 cm wy from the first mesh. Both the nd enclosures prevented the cged ees from intercting with the rood, ut llowed exposure to the colony microenvironment (e.g. temperture, humidity nd gses such s CO 2 ), com virtions nd the colony odors. The enclosure, ut not the, lso prevented direct contct with ees outside the enclosure. We plced the frme with the enclosures contining the focl ees in the center of the colony, such tht the cged ees were flnked y rood-contining honeycoms. We provisioned the nd cges with d liitum sugr syrup nd pollen, similr to the ees cged outside the colony (see ove). The density of ees inside the nd enclosures ws similr to tht in wooden cges in the l. Prior to the experiment (efore introducing the focl ees), we plced the empty honeycom frmes with the nd enclosures inside the source colonies for 2 3 dys. This ws done in order to llow the honeycom to sor colony odors nd minimize confounding fctors ssocited with exposing ees from the different tretment s to different honeycoms. During this period, nd in ll the trils of this experiment, there were no eggs lid on the honeycoms into which we emedded the mesh enclosures. We repeted this experiment three times, ech with ees from different source colony. 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