2841 The Journl of Experimentl Biology 215, 2841-2848 212. Published by The Compny of Biologists Ltd doi:1.1242/jeb.7252 RESEARCH ARTICLE Interlocking of chele is key fctor for dominnce hierrchy formtion in cryfish Ryusuke Ueno 1 nd Toshiki Ngym 2, * 1 Division of Biology, Grdute School of Science nd Engineering, Ymgt University, Ymgt 99-856, Jpn nd 2 Deprtment of Biology, Fculty of Science, Ymgt University, Ymgt 99-856, Jpn *Author for correspondence (ngym@sci.kj.ymgt-u.c.jp) SUMMARY We chrcterized the role of chele during gonistic encounters of the cryfish Procmbrus clrkii. Physicl symmetries in body length, body mss nd chele size were directly relted to dominnce hierrchy formtion. More thn 8% of winning cryfish hd longer body nd chele lengths, nd winners were usully hevier in body mss, even if their differences were less thn 3% compred with losing opponents. In mismtched pirings, lrge cryfish with short chele were beten by smll cryfish with lrge chele. Three physicl elements of body length, body mss nd chele length eqully ffected the outcome of gonistic bouts. Chele restriction, in which chele were tightly closed using rubber bnds, ffected the outcome of gonistic bouts between lrge nd smll cryfish. Before chele restriction, lrge cryfish won in ll pirings. Following chele restriction for t lest 3 dys from the first encounters, the winning rte of lrge cryfish tht were previous winners decresed significntly in the second encounters ginst the sme opponents tht were previous losers. The hndicp of chele restriction significntly prolonged the time to formtion of the winner loser reltionship. Individul fights esclted during gonistic bouts between lrge cryfish with one chel nd smll cryfish with intct chele, wheres the number of fights incresed but the durtion of individul fights did not increse in lrge cryfish with both chele. Furthermore, when the chele of both lrge nd smll cryfish were disbled, the dominnce order ws frequently not formed during 3 min of gonistic bouts. Preventing chele from interlocking prevented escltion of gonistic bouts. We show tht interlocking of chele cted s key fctor for the formtion of dominnce hierrchy. Key words: cryfish, dominnce hierrchy, gonistic behviour, physicl body size, chele. Received 12 Mrch 212; Accepted 23 April 212 INTRODUCTION Sttus-dependent hierrchy formtion is essentil for territoril nimls to mintin socil stbility. Dominnt nimls hve incresed opportunity to ccess good shelter, food nd mting prtner, wheres subordinte nimls cn be rescued from severe injury or deth. Dominnce hierrchies re formed in vrious species of rthropods including crickets (Alexnder, 1961), spiders (Aspey, 1977), cockroch (Ewing, 1974), lobsters (Fiedler, 1965), hermit crbs (Hzlett, 1968) nd cryfish (Bovbjerg, 1953). Decpod crustcens, in prticulr, re good models with which to chrcterize dominnce order both physiologiclly nd behviourlly (Fujimoto et l., 211; Sto nd Ngym, 212). Physicl symmetries hve been shown to be dequte predictors of dominnt subordinte reltionships. For exmple, crpce length (Pvey nd Fielder, 1996), body length (Bovbjerg, 1956; Lowe, 1956; Rubenstein nd Hzlett, 1974), chele size (Grvey nd Stein, 1993; Rutherfold et l., 1995; Brki et l., 1997; Sneddon et l., 1997; Gbbnini et l., 26), body mss (Pvey nd Fielder, 1996) nd sex (Stein, 1976; Figler et l., 1995) hve ll been implicted s determining fctors of the outcome of gonistic encounters. Lrger nimls usully win in gonistic encounters. Furthermore, extrinsic nd intrinsic fctors, such s hbitt nd socil experience, lso ffect the outcome of gonistic bouts. For exmple, cryfish gonistic interctions re longer nd more intense when the gonistic bout took plce ner shelter thn on or ner food-resource hbitts (Bergmn nd Moore, 23), nd residence owners re more likely to win thn intruders (Rnt nd Lindstrom, 1992; Peeke et l., 1995). The winner nd loser effect is lso importnt, with previous winners winning significntly more encounters thn previous losers during their second fight (Goessmnn et l., 2; Dws et l., 22; Seebcher nd Wilson, 27; Zulndt et l., 28). In mny decpods, especilly Astcide, Anomur nd Brchyur, the front pir of wlking legs hve enlrged pincers, the chele. The chele re unique, being verstile ppendges for both offence nd defence. The chele re used s wepons during gonistic encounters, nd disply ptterns of chele, e.g. extension, grsping, lifting, scissoring, striking, thrust, embrce, nip nd push, hve n importnt role in gonistic nd ggressive interctions (Mrippn et l., 2). During growth, the body proportions re similr, with the cephlothorx nd bdomen being pproximtely equl; however, the chele show positive llometry (Bovbjerg, 1956; Aiken nd Wddy, 1992). There is positive correltion between the pinching force nd body length, between the pinching force nd body mss, nd lso between chele size nd chele force (Clussen et l., 28; Wilson et l., 29). Rutherfold et l. (Rutherfold et l., 1995) showed tht chel length ws n importnt indictor of fighting bility in the cryfish Orconectes rusticus, s there ws no significnt difference in cephlothorx length but winners hd significntly longer chele thn losers. Furthermore, lrger chele clerly result in greter dominnce, but the pinching force of the chel hd no bering on dominnce in slender cryfish Cherx dispr (Wilson et l., 27). In the crb Potmon fluvitile, chel force is
2842 The Journl of Experimentl Biology 215 (16) the min fctor influencing the outcome of gonistic bouts only in femles, but chel size, more thn chel force, is used to settle gonistic bouts in mles (Gbbnini et l., 26). There is, however, lck of clrity in the role of chele interlocking during gonistic bouts of the cryfish Procmbrus clrkii. In this study, we hve chrcterized the reltionship between body length, body mss nd chele size during gonistic encounters, nd found tht interlocking of the chele ws key fctor in forming dominnce hierrchies. MATERIALS AND METHODS Animls Adult mle cryfish Procmbrus clrkii (Girrd 1852) of 6 9 cm in length, from rostrum to telson, were purchsed from commercil supplier nd mintined individully in seprte opque continers of 19 33 15 cm (width length height) filled with wter to depth of 1 cm for t lest 3 dys. Ech cryfish ws fed equl mounts of smll food pellets once week nd ws lst fed t lest 5 dys before pirings. Cryfish were mintined under 12 h:12 h light:drk cycle. Experimentl trils were crried out in dimly lit lbortory t room temperture of pproximtely 23 C. Piring Two cryfish with length difference between 1 nd 15% were selected nd pired in new opque continer (26 38 24 cm width length height) filled with wter to depth of pproximtely 12 cm. The dy before piring, body length (from rostrum to telson), wet body mss nd verge length of the right nd left chele were mesured. Prior to ech tril, n opque plstic brrier ws plced in the centre of the tnk seprting it into two res. A cryfish ws plced on ech side of this brrier nd llowed to cclimte for t lest 1 min before the divider ws removed. The gonistic bouts of the cryfish were recorded using video cmer (Victor GZ-MG33-S, Okoym, Jpn) mounted on tripod bove the continer for 3 min. The behviour of ech cryfish ws nlyzed using single frme mesurement to construct n ethogrm of ech second. Behviourl cts tht occurred during gonistic bouts were ctegorized s one of seven types: cpture, fight, contct, pproch, retret, tilflip nd neutrl (Sto nd Ngym, 212). Cpture, fight, contct nd pproch were ggressive behviourl cts, wheres retret nd tilflip were submissive cts. Cpture ws defined s the ct in which one cryfish held n opponent using its chele. Fight ws defined s the ct in which both cryfish fought using their chele. Contct ws defined s the ct in which both cryfish mde physicl contct without fighting, nd pproch ws defined s the ct in which one cryfish moved forwrd towrds the other. Retret ws defined s the ct in which one cryfish wlked wy from n pproching or ttcking opponent, nd tilflip ws defined s the ct in which one cryfish escped from n pproching or ttcking opponent using rpid tilflipping. Neutrl ws defined s other behviourl cts including puse, wlking with no correltion to n opponent nd no response. The winner loser reltionship ws determined with severl fights. Before determintion of dominnce sttus, the cryfish tht initited the pproch ws frequently beten in the following bouts by their opponents. After estblishment of dominnce order, subordinte cryfish lmost lwys showed retret or tilflip following the dominnt s pproch without fight. We determined the time of estblishment of the winner loser reltionship when the subordinte cryfish showed retret or tilflip following the dominnt s pproch t lest three times in succession (Sto nd Ngym, 212). Mismtched piring Juvenile cryfish tht were htched in our lbortory from commercilly obtined femles were collected t the third stge (fter the second moult). They were isolted nd rered to over 6 cm in length. At specific stges during growth, both chele were removed to regenerte new, smll chele t the next moult. In some experiments, these nimls with short chele were pired with smll cryfish (body size) with big chele. Chele restriction In some experiments, dominnt nd subordinte cryfish were seprted from ech other fter their first encounter. To negte the effect of the result of first encounter, nimls were re-isolted in seprte tnks for more thn 3 dys. Then, they were pired gin in new continer nd tested once more. Two dys before the second encounter, the right or left chel of the dominnt nimls, both chele of dominnt nimls, or both chele of both the dominnt nd subordinte nimls were closed tightly using rubber bnds round ech chel. Cryfish were then pired gin nd, fter the dominnt subordinte reltionship ws determined, they were reisolted for t lest 3 dys. After the second re-isoltion, the chele restriction ws relesed by removl of the rubber bnds. The sme nimls were pired gin for third encounter with the exception of pirs in which either one or both nimls moulted during isoltion. Sttisticl nlyses were crried out using SigmPlot v11 (Systt Softwre, Chicgo, IL, USA) nd R 2.14.1 (R Foundtion for Sttisticl Computing, Vienn, Austri). Dt re presented s mens ± s.e.m. unless otherwise indicted. RESULTS Body size nd gonistic encounters of cryfish After piring of two cryfish in the fighting ren, they rised both chele nd ttcked ech other. The period of ech fight ws vrible from severl seconds to some minutes. The probbility of fighting declined grdully over time nd the reltionship between dominnt nd subordinte ws usully determined within 2 min fter piring. The subordintes were then usully observed to move wy rpidly from the pproch of dominnts using minly retret. In 43 pirs (Fig. 1), cryfish with longer body length, from rostrum to telson (1.3 15.1% longer thn smll opponents), won in 37 pirings. The smller cryfish (2.9 4.3% shorter thn lrge opponents) won in three pirings, wheres in the remining three pirings (length difference rnged between 2.4 nd 8.1%) no winner found in 3 min period. Hevier cryfish (1. 28.8% hevier thn smll opponents) won in 4 out of 43 pirings wheres in the remining three pirings (mss difference between 14.5 nd 19.8%) no winner ws found in 3 min period. The cryfish tht hd longer chele Outcome of gonistic bout (%) 1 75 5 25 Length Mss Chele Lose Drw Win Fig. 1. Winning rte of lrger cryfish during gonistic bouts ginst smller cryfish bsed on 43 pirings. The percentges of outcomes of gonistic bouts of lrger cryfish re plotted s wins (blck), drws (light grey) nd losses (drk grey). The left br represents the results of nimls of greter body length, the middle br represents tht of nimls of greter wet mss nd the right br represents tht of nimls of greter chele length.
Interlocking of chele during cryfish gonistic bouts 2843 (.7 28.8% longer thn smll opponents) won in 34 pirings nd lost in six pirings (2.5 16.2% longer chele). In the remining three pirings in which the chele length difference ws between 4.1 nd 9.%, no winner ws found in 3 min period. The winning percentge of lrge cryfish (in terms of body length) ws 86.%, tht of hevy cryfish ws 93.% nd tht of cryfish with long chele ws 79.1%. Cryfish lrger in terms of body length, body mss nd chele length were thus likely to win (binomil test, P<.1). Furthermore, the probbility of lrge nimls winning ws not sttisticlly different mong these three groups (Fisher s exct test, P.166). Mismtched pirings Body length nd mss re highly correlted (Wilson et l., 27), nd the chele re lrge structures tht mke up 35 5% of cryfish totl mss (Stein, 1976). Furthermore, s shown in Fig. 2, length nd mss of chele were highly correlted (r 2.925, P<.1). Becuse cryfish routinely use their chele for both intimidtion nd fighting (Wilson et l., 27), lrge cryfish with short chele were pired with smll cryfish (1 9% difference in body length nd 3 12% difference in wet mss) tht hd long chele (7 25% difference). As control, norml cryfish with different body mss (3 12% difference) were pired (N 17). In ll norml pirings (tringles in Fig. 3), hevy cryfish won irrespective of whether they hd long or short chele (from 16 to 9% difference). Sixteen out of 17 winners hd longer body length thn losing opponents. In contrst, hevy nd lrge cryfish won in one out of 1 pirings in mismtched pirings (circles in Fig. 3). In the remining nine pirings, cryfish with long chele becme dominnts despite their lighter body msses nd shorter body lengths. The possibility of winning of hevy cryfish ws significntly different between control nd mismtched pirings (Fisher s exct test, P<.1), suggesting tht the length of chele reltive to body size ws importnt in predicting the outcome of gonistic bouts. In control pirings, dominnt subordinte reltionships were formed within 75 to 13 s (725.8±9.4 s). The time to form hierrchy in mismtched pirings ws within 4 to 11 s (696.8±78.9 s), similr to control pirings (Mnn Whitney rnk sum test, P.829; Fig. 4A). The totl number of fights during gonistic encounters of 3 min ws 7.3±1.2 in control pirings nd 6.8±.8 in mismtched pirings (Fig. 4B). Furthermore, the totl fight durtion ws 192.6±37.6 s in control pirings nd 291.6±81.8 s in mismtched pirings (Fig. 4C). The number nd durtion of fights between control nd mismtched pirings were not sttisticlly different (Mnn Whitney rnk sum test, P 1. nd P.353, respectively;). As shown in Fig. 4D, the number of pproches of hevy nimls in the control pirings ws 26.7±2.5, wheres tht Chel mss (g) 6 4 2 3 4 5 6 7 8 9 1 Chel length (cm) Fig. 2. The reltionship between chel length nd chel mss. There ws significnt reltionship (y 4.221+1.3x, r 2.925, P<.1), bsed on 32 chel from mle cryfish between 6.5 nd 8.4 cm in body length. These cryfish were not used for piring experiments throughout this study. Reltive chele length difference (%) +3 +2 +1 1 2 3 12 6 +6 Reltive body mss difference (%) Norml pirings Mismtched pirings +12 Fig. 3. The reltionship between reltive differences in body mss nd chele length for outcomes of gonistic bouts in norml pirings (tringles) nd mismtched pirings (circles). Open symbols represent winners nd filled symbols represent losers of the bouts. of hevy nimls in the mismtched pirings significntly decresed to 9.8±2.9 (Mnn Whitney rnk sum test, P<.1). In contrst, the number of submissive behviourl cts, i.e. retrets nd tilflips, of hevy nimls in the mismtched pirings significntly incresed to 5.1±7.9 compred with tht of hevy nimls in the control pirings (3.8±.5) (Mnn Whitney rnk sum test, P<.1; Fig. 4E). Chele restriction In ddition, we exmined the effect of chele interlocking during gonistic bouts using chele restriction pirings. Before the restriction of chele, lrge (from 1 to 12% difference) nd hevy (from 1 to 29% difference) cryfish with long chele (from 1 to 27% difference) were pired with smll nd light opponents with short chele length s first encounter. Lrge cryfish won in ll pirings (N 36). After the dominnt subordinte order ws determined, cryfish were re-isolted for more thn 3 dys. Then they were pired gin with the sme opponents following three different tretments of chele restriction. In the first tretment, one chel ws in lrge, previously dominnt cryfish. Either the right or the left chel of the dominnt cryfish ws closed tightly using rubber bnd before second piring with the previous loser. In 11 pirings, lrge cryfish won in seven pirings. Smll, previously subordinte cryfish won in one piring nd in the remining three pirings no winner ws found in 3 min period (Fig. 5). The winning probbility of lrge cryfish with one chel restriction ws not significntly different before nd fter chel restriction (McNemr s test, P.125), but they were not likely to win (binomil test, P.549). In the second tretment, both chele were in lrge, previously dominnt cryfish. Both the right nd left chele of lrge cryfish were closed tightly using rubber bnds before the second pirings with previous losers. In 15 pirings, lrge cryfish won on six occsions. Smll, previously subordinte cryfish with intct chele won in four pirings wheres in the remining five pirings no winner ws found in 3 min period (Fig. 5). Lrge cryfish with both chele were not likely to win (binomil test, P.67) nd their winning probbility fter chele restriction significntly decresed from the first encounter (McNemr s test, P.4). In four new pirings, the bse of both chele of the lrge cryfish ws surrounded by rubber bnds s dummy tretment. In this cse, lrge cryfish could use chele
2844 The Journl of Experimentl Biology 215 (16) Time (s) Fight number Fight durtion (s) Approch number Retret number 1 5 1 5 4 2 3 15 6 3 A B C D E Control Mismtched Fig. 4. Comprison of gonistic behviourl cts of hevier cryfish in both norml nd mismtched pirings. (A) The time in which the dominnt subordinte reltionship ws determined. (B) The number of fights of pirings for 3 min gonistic bouts. (C) The totl durtion of fights of pirings for 3 min gonistic bouts. (D) The number of pproches of hevier nimls for 3 min gonistic bouts. (E) The number of retrets nd tilflips of hevier nimls for 3 min gonistic bouts. Brs represent mens ± s.e.m. from 15 pirs of cryfish in norml pirings s controls nd 11 pirs of cryfish in mismtched pirings. Asterisks indicte tht the number of responses differed significntly between norml nd mismtched pirings (Mnn Whitney rnk sum test, P<.1). to interlock with opponents during gonistic bouts. These lrge cryfish lwys won ginst smll opponents. In the third tretment, the chele of both the lrge nd smll cryfish were. Both the right nd left chele of the previous losing smll cryfish s well s the previous winning lrge cryfish were closed tightly using rubber bnds before second encounters. In 1 pirings, lrge cryfish won in three pirings. No smll cryfish won, but in the remining seven pirings no winner ws found in 3 min period (Fig. 5). Lrge, previously dominnt cryfish with both chele were not likely to win (binomil test, P.344) nd their winning probbility ws significntly different between the first nd second encounters (McNemr s test, P.16). After the second Outcome of gonistic bout (%) 1 75 5 25 First piring before restriction One chel Second piring Both chele Both nimls Third piring fter relese Lose Drw Win Fig. 5. Winning rte of lrger cryfish during gonistic bouts ginst smller cryfish before, during nd fter chele restriction. The percentges of outcomes of gonistic bouts of lrger cryfish re plotted s wins (blck), drws (light grey) nd losses (drk grey). The left br represents the result of lrge nimls in the first pirings before chele restriction (N 36). Middle brs represent the results of lrge nimls in the second pirings during chele tretments (left: either the left or right chel ws in the lrge nimls, N 11; centre: both left nd right chele were in the lrger nimls, N 15; nd right: both chele of both lrge nd smll nimls were, N 1). Right br represents the result of lrge nimls in the third pirings fter relese from chele restriction (N 16). pirings with chele restriction, nimls were re-isolted for more thn 3 dys following relese from chele restriction by removing the rubber bnds. In 16 pirings, the lrge nimls were pired gin with the sme smll opponents for third encounter. The lrge nimls won in ll pirings regrdless of the results of the second encounters (Fig. 5). Fig. 6 shows the time in which the dominnt subordinte reltionship ws determined in ech piring. In the first pirings before chele restriction, dominnce hierrchy ws formed within 143 to 168 s (97.9±69.7 s). The time to dominnce formtion in the second pirings with one chel in lrge nimls (N 8 out of 11 pirings) ws within 626 to 1658 s (1314.5±137.3 s), tht in the second pirings with both chele in lrge nimls (N 1 out of 15 pirings) ws within 53 to 1772 s (1214.±113.4 s), nd tht in the second piring with chele restriction of both the lrge nd smll nimls (N 3 out of 1 pirings) ws within 1129 to 152 s (1364.±118.1 s). In the remining second pirings of ech tretment (N 1, 5 or 7, respectively), dominnce hierrchy ws not formed in 3 min period. The time in which the dominnt subordinte reltionship ws determined in the third encounters fter relese of chele restriction (N 16) ws within 143 to 1127 s (752.9±72.2 s). There ws no sttisticl difference (t-test, P.174) in the time between the first nd the third encounters. Compred with the first nd third encounters, the time for formtion of dominnce hierrchy in the second pirings with tretment of chele restriction ws significntly longer (t-test, P<.5 for the first versus second pirings with one chel or with both chele, P<.1 for the third versus second pirings with ll three tretments), lthough there ws no difference sttisticlly mong the second pirings of three different tretments of chele restriction (t-test, P.577 for one chel versus both chele, P.842 for one chel versus both cryfish, nd P.512 for both chele versus both cryfish ). The totl number of pproches during gonistic encounters of 3 min of the lrge cryfish nd smll opponents is plotted in Fig. 7A. In the first encounters before chele restriction, the number of
Interlocking of chele during cryfish gonistic bouts 2845 Time (s) 16 12 8 4,b First piring before restriction c One chel pproches of the lrge cryfish ws 23.±2.6 wheres tht of smll opponents ws significntly lower (6.8±.8; Mnn Whitney rnk sum test, P<.1). In the second pirings, the number of pproches in the lrge cryfish with one chel ws 11.8±1.8 wheres tht of the opponents ws 6.±1.2. The number of pproches between them ws sttisticlly different (Mnn Whitney rnk sum test, P<.5). The number of pproches in the lrge nimls with both chele ws 2.2±4. wheres tht of smll opponents with intct chele ws 19.9±2.6. There ws no sttisticl difference (Mnn Whitney rnk sum test, P.944). The number of pproches in the lrge nimls with both chele ws 28.1±4.4 wheres tht of smll opponents with both chele ws 12.±2. tht ws significntly lower (Mnn Whitney rnk sum test, P<.1). In the third encounters fter the relese of chele restriction, the number of pproches in the lrge nimls ws 26.3±2.4 wheres tht of smll opponents significntly decresed (Mnn Whitney rnk sum test, P<.1) to 7.7±1.2. The totl number of retrets nd tilflips during gonistic encounters of 3 min of the lrge cryfish nd smll opponents is plotted in Fig. 7B. In the first encounter before chele restriction, the number of retrets/tilflips of the lrge cryfish ws 1.9±.4 wheres tht of smll nimls ws 23.1±2.9. In the second pirings, the number of retrets/tilflips in the lrge cryfish with one chel ws 1.2±.3 wheres tht of smll opponents ws 9.4±1.6. The number of retrets/tilflips of the lrge nimls with both chele ws 8.9±2.4 wheres tht of smll opponents with intct chele ws 14.9±4.6. The number of retrets/tilflips in the lrge nimls with chele ws 4.5±1.3 wheres tht of smll opponents with chele ws 18.9±4.1. In the third encounter fter the relese of chele restriction, the number of retrets/tilflips of the lrge cryfish ws 2.1±.4 wheres tht of smll opponents ws 31.3±4.5. There were significnt differences in the number of retrets/tilflips between lrge nd smll cryfish (Mnn Whitney rnk sum test, P<.1 for the first nd third pirings, P<.1 for the second pirings with one chel restriction, P<.5 for the second pirings with chele restriction of both nimls), with the exception of the second pirings between lrge cryfish with chele nd smll opponents tht hd intct chele (Mnn Whitney rnk sum test, P.56). c Both chele Second piring b,c Both nimls Third piring fter relese Fig. 6. The time in which the dominnt subordinte reltionship ws determined before, during nd fter tretment of chele restriction. Brs represent mens ± s.e.m. from 36 pirs of the cryfish in the first pirings, 11 pirs in the second pirings with one chel in lrge cryfish, 15 pirs in the second pirings with both chele in lrge cryfish, 1 pirs in the second pirings with chele restriction of both cryfish, nd 16 pirs in the third pirings fter relese from chele restriction. Letters bove plots show sttisticl differences (t-test). Approch number Retret/tilflip number 4 2 4 2 A B First piring before restriction Lrge Smll * ** One chel Both chele Second piring The totl number of fights during gonistic encounters of 3 min ws 6.6±.7 in the first piring before chele restriction, 6.±.9 in the second pirings with one chel, 15.1±2.5 in the second pirings with both chele, 11.3±1.7 in the second pirings with chele in both nimls, nd 6.7±.9 in the third pirings fter relese from chele restriction (Fig. 8A). In tretments with both chele, the number of fights incresed significntly. The totl durtion of fights ws 321.6±47.3 s in the first piring before chele restriction, 66.±121.3 s in the second pirings with one chel, 575.8±68.4 s in the second pirings with both chele, 48.5±53.5 s in the second pirings with chele restriction of both nimls, nd 197.4±35.9 s in the third pirings fter relese from chele restriction (Fig. 8B). Tretments of chele restriction in the lrge cryfish incresed the fight durtion significntly. The men durtion of individul flight ws 59.8±1.4 s in the first piring, 115.5±25.3 s in the second pirings with one chel, 46.9±6.9 s in the second pirings with both chele, 47.8±11. s in the second pirings with chele restriction of both nimls, nd 27.9±3.7 s in the third pirings fter relese from chele restriction. Individul fight durtion between lrge nimls with one chel nd smll opponents with intct chele ws considerbly longer thn tht of other pirings of the first, second nd third pirings (Mnn Whitney rnk sum test, P<.5). ** * Both nimls Third piring fter relese Fig. 7. Comprison of ggressive nd submissive behviourl cts in both lrge nd smll cryfish before, during nd fter tretment of chele restriction. (A) The number of pproches of lrge (filled brs) nd smll (open brs) nimls for 3 min gonistic bouts. (B) The number of retrets nd tilflips of lrge nd smll nimls for 3 min gonistic bouts. Brs represent mens ± s.e.m. from 36 pirs of the cryfish in the first pirings, 11 pirs in the second pirings with one chel in lrge cryfish, 15 pirs in the second pirings with both chele in lrge cryfish, 1 pirs in the second pirings with chele restriction in both cryfish, nd 16 pirs in the third pirings fter relese from chele restriction. Asterisks indicte tht the number of responses differed significntly between lrge nd smll nimls (t-test or Mnn Whitney rnk sum test, *P<.5, **P<.1, P<.1).
2846 The Journl of Experimentl Biology 215 (16) Fight durtion (s) Number of fights 2 1 A B 8 4,b First piring before restriction c,d One chel Fight dynmics lso chnged between the first nd second pirings. Before chele restriction, cryfish used chele to strike nd to grsp their opponent s chel or wlking leg. Chele were lso observed to cut opponent s ntenne. Cryfish ttempted to upset their opponent by lifting nd twisting interlocking chele. After chele restriction, cryfish used chele to strike nd push their opponent. They ttempted to press the opponent from the upper side. DISCUSSION Reltive size is sufficient to predict the outcome of gonistic bouts Mny previous studies hve indicted tht physicl symmetries re dequte predictors of the dominnt subordinte reltionship in cryfish (Bovbjerg, 1956; Lowe, 1956; Rubenstein nd Hzlett, 1974; Grvey nd Stein, 1993; Rutherfold et l., 1995; Pvey nd Fielder, 1996). We hve confirmed this in present study, which showed tht the reltive size of cryfish (in terms of body length, body mss s well s chele size) ws directly relted to dominnce hierrchy formtion. More thn 8% of winning cryfish hd longer bodies nd/or chele length nd winners were usully hevier in mss, even if their differences were less thn 3% of losing opponents. In shore crbs, winning crbs hve hevier chele with greter surfce re thn losing opponents. Sneddon et l. (Sneddon et l., 1997) showed tht wepon size is more relible predictor of the outcome of pirwise fights thn body size. Brki et l. (Brki et l., 1997) lso showed tht cheliped size is used by mle prwns in the ssessment of fighting bility. Reltively smll symmetries in b c,d Both chele Second piring b b,c Both nimls Third piring fter relese Fig. 8. Comprison of gonistic fights of pirings before, during nd fter tretment of chele restriction. (A) The number of fights for 3 min gonistic bouts. (B) The totl durtion of fights for 3 min gonistic bouts. Brs represent mens ± s.e.m. from 36 pirs of cryfish in the first pirings, 11 pirs in the second pirings with one chel in lrge cryfish, 15 pirs in the second pirings with both chele in lrge cryfish, 1 pirs in the second pirings with chele restriction of both cryfish, nd 16 pirs in the third pirings fter relese from chele restriction. Letters bove plots show sttisticl differences (t-test or Mnn Whitney rnk sum test). chele size overrode lrge contrdictory symmetries in body size, nd the lrge symmetry in body length did not bis the probbility of winning the contest towrd the lrger opponent, even when the opponents were symmetric in chele size. Furthermore, Rutherfold et l. (Rutherfold et l., 1995) showed tht chel length is relible predictor of reltive fighting bility in the cryfish Orconectes rusticus. In the present study, however, six cryfish out of 43 pirings who hd longer chele (even more thn 1% difference) were beten by opponents with smller chele. In these cses, winning opponents were usully lrger in terms of body length nd body mss. The cryfish with lrge body lengths with hevy body msses nd with long chele lengths lwys won in this study. These three physicl elements were eqully importnt in predicting the outcome of gonistic bouts of cryfish. Mismtched piring experiments supported this ide. Long nd hevy cryfish usully won in norml pirings in which the chele length difference with opponents ws less thn 1%. Longer nd hevier cryfish were, however, beten when they hd very smll chele ginst opponents with short body lengths nd light msses but with long chele of more thn 1% in difference. The effect of chele restriction As well s gonistic interctions, chele of decpods hve mjor role in defensive postures, copultion, prey mnipultion, cquisition of shelters nd courtship displys (Hzlett, 1962; Hzlett, 1972; Slmon et l., 1978; O Neill nd Cobb, 1979; Ameyw-Akumfi, 1981; Ngym et l., 1986; Mrippn et l., 2). As wepon, chele length, more thn pinching force, is thought to be the min fctor influencing fighting bility in cryfish (Rutherfold et l., 1995; Wilson et l., 27; Wlter et l., 211), though Seebcher nd Wilson (Seebcher nd Wilson, 27) hve indicted tht greter chele closing force significntly increses the chnces of winning. Our chele restriction experiments showed tht preventing chele from opening nd/or closing or decresing their interlocking bility considerbly ffected the outcomes of gonistic bouts. Restriction of the right or left chel in the lrge nimls who won in previous encounters decresed the winning probbility in the second encounters with the sme smll opponents who hd intct chele. Restriction of both chele in the lrge nimls significntly decresed the winning probbility in the second encounters with the sme previous losing opponents who hd intct chele. Furthermore, when right nd left chele of both the lrge nd smll cryfish were, their dominnt subordinte reltionship frequently ws not formed. These results suggest tht chele opening nd closing strongly influenced outcome of fight. Becuse the length nd mss of chele were highly correlted, which suggested tht lrge chele hd greter mss of muscles, the pinching force of chele ffected the outcome of the gonistic encounter. Seebcher nd Wilson (Seebcher nd Wilson, 27) performed similr chele restriction experiments with the chele of winner cryfish disbled by ttching the dctylus to the propodus with crylmide glue. Previous winners with their chele disbled fter the first pirings won significntly more encounters thn previous losers with intct chele during their second fight 3 min or 24 h fter their initil pirings. When they were mtched ginst unknown opponents with similr body size, there were no significnt differences in the number of victories between previous winners with chele restriction nd new opponents with intct chele. This difference seemed to be explined by winner effect, i.e. previous winners won significntly more encounters thn previous losers during their second fight (Goessmnn et l., 2; Dws et l., 22). Winner effects re known to lst for 1 2 weeks (Hemsworth et l., 27); therefore, in
Interlocking of chele during cryfish gonistic bouts 2847 the present study there ws no effect of previous experience in our experiments when the sme lrge nd smll cryfish were pired gin fter t lest 3 dys from the previous encounters. A hndicp tht prevented chele interlocking thus decresed the winning probbility of cryfish tht hd physicl dvntge of lrge body size. The finding tht the dominnt cryfish drop to low rnk when one chel ws removed fter only 5 dys with group (Lowe, 1956) supports our conclusion. The role of interlocking chele When two cryfish encountered ech other, gonistic bouts esclted from chele disply to interlocking (Bruski nd Dunhm, 1987). As cryfish were fcing ech other, the chele extended lterlly nd upwrds. One of them stroked its opponent with chel, nd the chele of both cryfish opened nd interlocked with those of the opponent. With the chele interlocked, the wlking legs were fully extended to forcefully push the nimls towrds ech other. They took hold of ech other s chele nd ttempted to unblnce ech other. The winner loser reltionship ws suddenly determined when subordintes moved wy from opponents nd subsequently subordintes voided pproching dominnts with either retret or tilflip. The time in which the winner loser reltionship ws determined ws significntly prolonged in the second encounters with chele restriction even if only one chel ws. Furthermore, the number of outcomes in which the winner loser reltionship ws not clerly determined incresed significntly when the chele of both lrge nd smll nimls were. The number of pproches nd retrets would reflect result of outcomes of gonistic bouts, s subordintes voided the pproch of dominnts by mens of retret. The number of pproches nd retrets between lrge nd smll cryfish in the first encounters before chele restriction, s well s in the third encounters fter the relese from chele restriction, ws sttisticlly different. Furthermore, there ws significnt difference between lrge nd smll nimls in the second pirings of the tretments with one chel in lrge nimls nd with chele restriction of both nimls. The occurrence probbilities of pproches were not sttisticlly different between lrge nd smll cryfish in the second pirings of the tretment with both chele in lrge cryfish. The number of retrets between lrge nd smll cryfish in the second encounters ws lso sttisticlly indifferent. In sense, dominnce order might depend upon the rection of subordintes. When one contestnt gve up nd disengged, the dominnt subordinte reltionship ws determined. No ction of interlocking from lrge opponents might motivte fighting of smll cryfish. The number of fights in the second piring between lrge cryfish with one chel nd smll cryfish with intct chele ws not sttisticlly different from tht of their first piring, but the durtion of the fights of their second encounters ws significntly longer thn tht of the first piring. These results indicted tht individul fights between the lrge cryfish with one chel nd the smll cryfish with intct chele esclted owing to the hndicp of one chel restriction of the lrge cryfish. However, the number of the fights in the second pirings of the remining two tretments of both chele restriction of the lrge cryfish nd chele restriction of both lrge nd smll cryfish significntly incresed, wheres the durtion of individul fights ws not sttisticlly different from tht of their first encounters. Disturbnce of chele interlocking behviour prevented escltion of gonistic bouts, which resulted in lck of formtion of the dominnce order. Thus, interlocking of chele functioned s key fctor for dominnce hierrchy formtion of the cryfish. At present, it is still uncler whether pinching force of interlocking chele is n essentil element for dominnt hierrchy formtion. Further experiments mnipulting chele force or fixing chele in n open position would be needed to clrify this point. Further interesting observtions would be the rections of smll cryfish in the second encounters with lrge opponents with both chele. Smll cryfish with intct chele seemed to hve certin dvntge over opponents s only smll cryfish performed chele interlocking. However, smll cryfish with intct chele were beten frequently nd disengged. By contrst, no dominnce hierrchy ws determined in gonistic bouts ginst lrge opponents with chele restriction when chele of smll cryfish were lso. At present, it is still uncler how subordintes decide to give up fighting. Further behviourl, physiologicl nd phrmcologicl nlyses would be indispensble to clrify the intrinsic stte underlying the ggressive motivtion of subordinte nimls. ACKNOWLEDGEMENTS We thnk Dr K. Shirishi for help with sttisticl nlyses. FUNDING This work ws supported by Grnts-in-Aid from the Ministry of Eduction, Science, Sport nd Culture to T.N. REFERENCES Aiken, D. E. nd Wddy, S. L. (1992). The growth process in cryfish. Rev. Aqut. 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