Production of chemical alarm cues in convict cichlids: the effects of diet, body condition and ontogeny

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1 Ann. Zool. Fennici 41: ISSN 3-455X Helsinki 22 June 24 Finnish Zoologicl nd Botnicl Pulishing Bord 24 Production of chemicl lrm cues in convict cichlids: the effects of diet, ody condition nd ontogeny Grnt E. Brown 1 *, Ptrici E. Fom 1, Hilry E. Cowell 1, Plestin Guevr Fiore 1 & Dougls P. Chivers 2 1) Deprtment of Biology, Concordi University, 7141 Sherrooke St. W., Montrél, Queéc, H4B 1R6, Cnd (*corresponding uthor s e-mil: grown@lcor.concordi.c) 2) Deprtment of Biology, University of Ssktchewn, 112 Science Pl., Ssktoon, SK, S7N SE2 Cnd Received 5 Fe. 24, revised version received 12 Mr. 24, ccepted 4 Mr. 24 Brown, G. E., Fom, P. E., Cowell, H. E., Guevr Fiore, P. & Chivers, D. P. 24: Production of chemicl lrm cues in convict cichlids: the effects of diet, ody condition nd ontogeny. Ann. Zool. Fennici 41: While much is known regrding the role of chemicl lrm cues in the medition of predtor prey dynmics within qutic ecosystems, little is known regrding the production of these criticlly importnt informtion sources. In series of lortory experiments, we tested the possile effects of diet, ody condition nd ontogeny on the production of chemicl lrm cues in juvenile convict cichlids (Archocentrus nigrofscitus, Cichlide, Acnthopterygii). Juvenile cichlids were fed one of two diets, tuifex worms (Tuifex spp.) or rine shrimp (Artemi spp.). Shrimp fed cichlids grew longer nd hevier nd were in etter condition thn were tuifex fed cichlids. In Experiment 1, cichlids exhiited stronger ntipredtor response to conspecific skin extrcts from donors fed shrimp versus tuifex, regrdless of test cichlid diet. In Experiment 2, juvenile cichlids were exposed to the skin extrcts of high versus low condition donors fed either tuifex or shrimp diets. Cichlids exhiited significntly stronger ntipredtor response to skin extrcts of high condition donors, regrdless of donor diet. Finlly, in Experiment 3, juvenile cichlids were exposed to skin extrcts of similr sized juvenile conspecifics, dult conspecifics, swordtil (Xiphophorus helleri) or distilled wter control. We found no evidence of n ontogenetic chnge in the production of lrm cues s cichlids exhiited similr intensity ntipredtor responses when exposed to juvenile nd dult conspecific lrm cues. Tken together, these dt suggest tht individul diet my influence ody condition with the consequence of influencing chemicl lrm cue production in juvenile cichlids. Introduction A wide vriety of freshwter fishes rely on dmge-relesed chemicl lrm cues to detect nd void potentil predtors (Chivers & Smith 1998, Brown 23). These lrm cues re typiclly stored in the epidermis, nd re relesed following mechnicl dmge to the skin, s would occur during predtion events (Chivers & Smith 1998, Smith 1999). When detected y nery conspecifics nd some symptric heterospecifics, these chemicl lrm cues cn elicit

2 488 Brown et l. ANN. ZOOL. FENNICI Vol. 41 drmtic, short-term increses in species-typicl ntipredtor ehviour (Chivers & Smith 1998, Smith 1999, Brown 23). In ddition, chemicl lrm cues cn elicit suite of longterm responses, including cquired recognition of novel predtors nd induced morphologicl nd life history chnges (Chivers & Smith 1998, Smith 1999). Responding to chemicl lrm cues, either directly or s recognizle component of predtor s dietry odour, hs een shown to led to incresed survivl during encounters with potentil predtors (Mthis & Smith 1993, Mirz & Chivers 2, 23, 23, Chivers et l. 22). In ddition, senders of n lrm cue my enefit through incresed escpe proilities s result of the recruitment of secondry predtors (Mthis et l. 1995, Chivers et l. 1996) or through kin selected indirect fitness gins (Brown & Smith 1994, Wisenden & Smith 1998, G. E. Brown & R. S. Mirz unpul. dt). While much is known regrding the function of chemicl lrm cues to oth the senders nd receivers, reltively little is known regrding the production mechnisms nd ssocited energetic costs of chemicl lrm cues. Fishes of the Superorder Ostriophysi possess specilized epiderml clu cells, which produce nd store the chemicl lrm cue (Pfeiffer 1977, Smith 1992). Wisenden nd Smith (1997, 1998) exmined the effects of diet on epiderml clu cell production in fthed minnows (Pimephles promels). Their results demonstrte tht minnows rered on higher food rtion were le to produce greter numer of clu cells. These results suggest tht there is n energetic cost ssocited with the production of chemicl lrm cues nd lso suggest tht diet (or diet qulity) my directly influence the production of chemicl lrm cues in freshwter prey fishes. Recently, Brown et l. (21) rgued tht the Ostriophysn lrm cue is produced from metolic y-products ssocited with the degrdtion of proteins. The use of metolic y-products is thought to e n energeticlly efficient method of cue production (Brown et l. 21). A similr production mechnism my lso e selected in non-ostriophysn fishes. Ontogenetic stge my lso hve significnt impct on the production of chemicl lrm cues. Mirz nd Chivers (22) exposed smll (~4. cm) nd lrge (~9.5 cm) rook chrr (Slvelinus fontinlis) to the skin extrcts of smll versus lrge conspecifics. They report tht for oth size clsses, individuls exhiited significntly stronger ntipredtor response to likesized donors. Hrvey nd Brown (24) likewise exposed juvenile nd dult yellow perch (Perc flvescens) to the skin extrcts of juvenile versus dult donors nd found no ontogenetic effect on the production of chemicl lrm cues. Thus, the role of ontogeny in the production of chemicl lrm cues remins equivocl. Convict cichlids (Archocentrus nigrofscitus, Cichlide, Acnthopterygii) possess dmge-relesed chemicl lrm cue, nlogous to tht seen in the well-studied Ostriophysn fishes (Wisenden & Srgent 1997, Alemdi & Wisenden 22). However, unlike Ostriophysn fishes, cichlids do not possess the specilized epiderml clu cells known to store the lrm cue (Pfeffier 1977). As such, it remins unknown if fctors such s diet qulity, individul condition nd/or ontogenetic stge hve ny influence on the production of dmge-relesed chemicl lrm cues in non-ostriophysn species. The gol of this study is to exmine the potentil influence of diet type, ody condition nd ontogenetic stge on the production of chemicl lrm cues y juvenile convict cichlids. In Experiment 1, we fed juvenile convict cichlids either high or low qulity diet to test for the possile effects of diet type on oth the production of chemicl lrm cues nd the ehviourl response of signl receivers. Differences in response of lrm cue receivers to the cues of donors fed high versus low qulity diets could result from differences in donor ody condition or difference in diet qulity, per se. In Experiment 2, we specificlly test for the possile effects of stimulus donor condition, s seprte from diet type. Finlly, in Experiment 3, we test for the potentil effects of ontogenetic stge on the production of lrm cues. Mteril nd methods For ll three experiments, test nd stimulus donor cichlids originted from our lortory stock

3 ANN. ZOOL. FENNICI Vol. 41 Production of lrm cues in juvenile convict cichlids 489 popultion. Prior to testing, stock popultions were held in 11-l glss quri, filled with continuously filtered, dechlorinted tp wter (27 C, ph 7.2), contining grvel sustrte, nd were fed d liitum, twice dily with commercil flke food. All stock tnks were held under 12:12 light:drk cycle. Swordtils (Xiphophorus helleri) used in Experiment 3 (see elow), were otined from commercil supplier nd held in 37-l glss qurium under identicl conditions s descried ove for convict cichlids. As oth Experiment 1 nd 2 required test fish nd/or stimulus donors fed different diets, we plced 3 juvenile cichlids into ech of eight 37-l holding tnks, under identicl conditions s descried ove. Four of these tnks were fed high qulity diet (previously frozen dult rine shrimp, Artemi spp.) nd four were fed low qulity diet (re-hydrted tuifex worms, Tuifex spp.). According to the mnufcturer s informtion, rine shrimp hve similr crude protein levels nd higher crude ft levels thn tuifex worms. As such, we predicted tht the rine shrimp diet ws of higher qulity thn the tuifex diet. Cichlids were fed dily for four weeks during this growth phse of the study. Ech tnk ws fed the sme mss of either shrimp or tuifex (1.5 ±.4 g, dry weight per dy). At the initition nd completion of the growth phse, individul cichlids were mesured (length to the nerest.5 mm nd weight to the nerest.1 g). In ddition, we clculted n llometric index of ody condition (weight g length mm 1 1) for ech fish. We compred the weight nd length differences etween fish fed different diets for strting nd finl mesurements using unpired t-tests with =.13 to control for incresing Type 1 error rtes (Sokl & Rohlf 1981). At the onset of the growth phse, there ws no significnt difference in men length (t 6 =.26, P =.8; Fig. 1A), or weight (t 6 = 1.25, P =.26; Fig. 1B). However, cichlids fed the shrimp diet were longer (t 6 = 12.25, P <.1; Fig. 1A), nd hevier (t 6 = 12.74, P <.1; Fig. 1B) thn those fed tuifex worms t the completion of the growth phse. To test for significnt effect of the diet type on the condition index, we conducted n nlysis of covrince using weight s the dependent vrile, diet type s the independent vrile nd length s the covrite (Grcí-Berthou 21). Stndrd length (mm) Mss (g) Condition index A B C Week 1 Week 4 Fig. 1. Men (± S.E.) (A) stndrd length, (B) mss, nd (C) condition index (mss length 1 1) for juvenile cichlids fed shrimp (circles) nd tuifiex (dimonds) diets. Week 1 = initition of growth phse, Week 4 = completion of growth phse.

4 49 Brown et l. ANN. ZOOL. FENNICI Vol. 41 We found significnt interction etween the diet type nd length (F 1,12 = 14.61, P =.2; Fig. 1C), indicting tht cichlids fed shrimp were in etter condition (hevier for length) thn those fed tuifex worms. Experiment 1: Influence of diet qulity Experimentl stimuli Skin extrcts were collected from eight donor cichlids from ech of the two diet tretments (men ± S.E. stndrd length = 3.11 ±.48 nd 2.52 ±.42 cm for shrimp nd tuifex diets respectively). Donor fish were killed with low to the hed (in ccordnce with Concordi University Animl Cre Protocol #AC-22- BROW). We collected skin fillets from either side of donors nd immeditely plced them into 5 ml of chilled, glss-distilled wter. We then homogenized the smples, filtered them through polyester floss (to remove ny prticulte mtter) nd djusted the finl volume with the ddition of distilled wter. We collected totl of 25.5 cm 2 (in 29 ml) nd (in 292 ml) of skin for shrimp- nd tuifex-fed donors, respectively. Skin extrcts were frozen in 15 ml liquots until needed. As control, we lso froze 15 ml liquots of distilled wter. Experimentl protocol All oservtions were conducted in series of 37-l test tnks, equipped with single irstone nd n dditionl length of tuing to llow for the injection of control nd experimentl stimuli from distnce of t lest 2 m. The tnks contined grvel sustrte nd were filled with dechlorinted tp wter, ut were not filtered. Temperture nd lighting were identicl to these in the holding tnks. We positioned the test tnks ehind lck plstic viewing lind. All oservtions were videotped for lter ehviourl nlysis. For ech tril, we ritrrily selected two cichlids from one of the holding tnks (tuifexfed or shrimp-fed) nd plced them into test tnk 24 h prior to testing. All test (cue receiver) fish, regrdless of diet tretment, were fed d liitum with commercil flke food prior to testing, in order to reduce potentil confounds of forging-ntipredtor ehviour trde-off (Smith 1981, Brown & Smith 1996, Brown & Cown 2). Trils consisted of pired control nd experimentl oservtions. For oth control nd experimentl oservtions, we conducted 1-min pre-stimulus nd 1-min post-stimulus injection oservtion period. Control nd experimentl oservtions were conducted on sequentil dys, with 24 h etween. Order of presenttion (control versus experimentl stimuli) ws rndomized. Men (± S.E.) stndrd length t testing ws 2.89 ±.43 nd 2.71 ±.31 cm (shrimp nd tuifex diet respectively). Prior to the pre-stimulus oservtions (for oth control nd experimentl), we withdrew nd discrded 6 ml of tnk wter through the stimulus injection tue (to remove ny residul cues from the tue). We then withdrew nd retined n dditionl 6 ml of wter. Following the 1-min pre-stimulus oservtion period, we injected either 1 ml of distilled wter (control trils) or 1 ml of either tuifex-fed or shrimpfed cichlid skin extrct (experimentl trils) nd slowly flushed it into the tnk using the retined 6 ml of tnk wter. Ech pir of cichlids ws used only once. During oth pre- nd post-stimulus oservtion periods, we recorded suite of four ehviourl mesures typicl of n ntipredtor response for juvenile cichlids (Wisenden & Srgent 1997). Verticl re use ws recorded (every 15 s) s the position of ech cichlid within the tnk. Are use scores rnged from 2 (oth cichlids ner the sustrte) to 8 (oth cichlids ner the wter surfce). Time spent moving ws recorded s the totl time ech cichlid ws swimming (expressed s per cpit vlue). Distnce etween individuls ws recorded every 15 s s mesure of shol cohesion. Finlly, we recorded the totl occurrence of ggressive interctions (chsing nd iting). Decresed re use, time spent moving, distnce to neighour nd ggressive interctions re indictive of n ntipredtor response in juvenile convict cichlids (Wisenden & Srgent 1997).

5 ANN. ZOOL. FENNICI Vol. 41 Production of lrm cues in juvenile convict cichlids 491 Sttisticl nlysis For ech ehviourl mesure, we clculted the difference etween pre- nd post-stimulus oservtion periods (post pre), nd used these difference scores s dependent vriles for ll susequent nlyses. We ssessed the effects of stimulus donor diet nd receiver diet using twowy repeted mesures ANOVAs, with distilled wter control versus skin extrct experimentl tretments s the repeted mesure. We tested totl of 1 pirs of cichlids per tretment comintion. Individul receivers were used only once. Experiment 2: Influence of ody condition Test fish Test fish were fed, d liitum, twice dily with commercil flke food prior to the experiment. Men (± S.E.) length t testing ws 2.84 ±.12 cm. Unlike Experiment 1, ll test fish for Experiment 2 were fed the sme diet (flke food). Stimulus preprtion We collected skin extrct from tuifex nd shrimp fed cichlids s descried ove. However, for ech diet, we collected skin from high condition index nd low condition index fish. Thus, we hd totl of four stimulus types: (1) shrimp diet, high condition, (2) shrimp diet, low condition, (3) tuifex diet, high condition, nd (4) tuifex diet, low condition. Donor cichlids were chosen such tht the men condition index ws similr for oth diet tretments for high nd low condition tretments (Tle 1). The finl concentrtion of ech of the four skin extrcts ws the sme s tht used in Experiment 1 (Tle 1). Experimentl protocol Pirs of cichlids were tested s descried ove for Experiment 1 with one exception. We did not use the distilled wter control, s the results of Experiment 1 demonstrted tht juvenile cichlids do not respond to the introduction of distilled wter. Sttisticl nlysis As in Experiment 1, we clculted the difference etween pre- nd post-stimulus oservtion periods nd used these difference scores s dependent mesures in susequent nlyses. We tested the potentil influence of donor diet nd donor condition index using two-wy ANOVAs, with donor diet (shrimp vs. tuifex) nd donor condition (high versus low) s independent vriles. We tested 1 pirs of cichlids for ech tretment comintion. Experiment 3: effects of ontogeny on lrm cue production Test fish Test fish were of the sme popultion, nd were held under identicl conditions s descried in Experiment 1. Cichilds were likewise fed s descried ove. Men (± S.E.) length t testing ws 2.86 ±.9 cm. Tle 1. Men (± S.E. in prentheses) length (cm), weight (g), nd condition index for skin extrct donors nd totl re of skin collected nd finl djusted volumes of stimuli used in Experiment 2. Length (cm) Weight (g) Condition Totl skin Finl volume index re (cm 2 ) (ml) Shrimp high condition 4.1 (.9) 2.18 (.11).54 (.3) Shrimp low condition 3.7 (.15).7 (.8).23 (.2) Tuifex high condition 3.62 (.12) 1.9 (.7).53 (.2) Tuifex low condition 2.63 (.14).56 (.5).21 (.2)

6 492 Brown et l. ANN. ZOOL. FENNICI Vol. 41 Pirs of juvenile convict cichlids were exposed to skin extrcts collected from similr sized conspecifics nd from lrger dult cichlids. In ddition, we tested two control stimuli, distilled wter nd swordtil skin extrct. While poecilids possess n nlogous chemicl lrm cue system (Grci et l. 1992, Brown & Godin 1999, Mirz et l. 21), it is not recognized y juvenile cichlids, nd therefore serves s control for the odour of ny injured prey fish. Stimulus preprtion We collected skin extrcts from 11 juvenile (men ± S.E. length = 2.99 ±.11 cm) nd three dult (8.19 ±.3 cm) cichlids nd from seven swordtils (5.38 ±.44 cm) s descried ove. We selected donors tht hd similr weight to length rtio (i.e. condition index, see ove). Men condition index ws.35 (rnge:.32.38) for the dult donors nd.33 ±.4 for juvenile donors. We collected totl of cm 2 (in 221 ml) of juvenile cichlid skin, cm 2 (in 312 ml) of su-dult cichlid skin nd cm 2 (in 379 ml) of swordtil skin. The finl concentrtions of ll three stimuli were the sme s used in Experiments 1 nd 2. Prior to the preprtion of skin extrcts, dult nd juvenile cichlid nd swordtil donors were fed rine shrimp nd commercil flke food d liitum, twice dily. Skin extrcts were frozen in 2 ml liquots t 2 C until required. As control, we lso froze 2 ml liquots of distilled wter. Experimentl protocol Trils were conducted, s descried ove for Experiment 2. Pirs of cichlids were exposed to 1 ml of one of the four stimuli. Antipredtor ehviour ws recorded s ove. We compred the chnge in ehviour in response to the four stimuli using one-wy ANOVAs. We conducted totl of 1 trils for ech of the four stimuli. Post-hoc multiple comprisons were mde using Fisher s Protected Lest Squred Differences. Results Experiment 1: Influence of diet qulity For ech of the four ehviourl vriles, we found significnt repeted mesures effect (Tle 2 nd Fig. 2). In ddition, for time spent Tle 2. Results of repeted mesures ANOVAs for ech response vrile tested in Experiment 1. Repeted mesure denotes control (distilled wter) versus experimentl (skin extrct) trils, test diet denotes diet of test (cue receiver) cichlids nd stimulus diet denotes diet of skin extrct donors. N = 1 per tretment comintion. F df P Time moving Repeted mesure ,36 <.1 Repeted mesure test diet.19 1,36 =.66 Repeted mesure stimulus diet ,36 <.5 Repeted mesure test diet stimulus diet.7 1,36 =.93 Are use Repeted mesure ,36 <.1 Repeted mesure test diet.1 1,36 =.97 Repeted mesure stimulus diet ,36 <.5 Repeted mesure test diet stimulus diet.89 1,36 =.35 Distnce to neighour Repeted mesure ,36.1 Repeted mesure test diet ,36 =.19 Repeted mesure stimulus diet ,36 <.1 Repeted mesure test diet stimulus diet ,36 =.19 Aggressive interctions Repeted mesure ,36 <.1 Repeted mesure test diet.39 1,36 =.53 Repeted mesure stimulus diet.13 1,36 =.72 Repeted mesure test diet stimulus diet.1 1,36 =.76

7 ANN. ZOOL. FENNICI Vol. 41 Production of lrm cues in juvenile convict cichlids A.5 B Time moving (s) 25 Are use C 4 D 2 2 Distnce to neighour (cm) 2 4 Aggressive interctions Tuifex donor Shrimp donor Tuifex receiver Tuifex donor Shrimp donor Shrimp receiver 8 Tuifex Shrimp donor donor Tuifex receiver Tuifex donor Shrimp donor Shrimp receiver Fig. 2. Men (± S.E.) chnge (post pre) in (A) time moving (seconds), (B) re use, (C) distnce to neighour (cm), nd (D) occurrence of ggressive interctions for juvenile cichlids fed either tuifex or shrimp diets (cue receivers) nd exposed to distilled wter control (open rs) or tuifex or shrimp fed skin extrct (cue donors, solid rs). N = 1 per tretment comintion. moving, re use nd distnce etween individuls, we found significnt interction etween stimulus donor diet nd the repeted mesures effect (Tle 2 nd Fig. 2). There ws no significnt interction etween the repeted mesures effect nd test fish diet for frequency of ggressive interctions (Tle 2 nd Fig. 2). Regrdless of their own diet, juvenile cichlids exposed to shrimp diet skin extrct exhiited significntly greter reductions in time moving, re use nd distnce to neighour thn those exposed to tuifex diet skin extrcts. Test fish diet did not ffect the response ptterns. There ws no significnt interction etween tretment (repeted mesures effect) nd either skin extrct or test fish diet for the frequency of ggressive interctions (Tle 2 nd Fig. 2). The results of Experiment 1 suggest tht cichlids fed high qulity diet produce chemicl lrm cue tht elicits stronger ntipredtor response thn tht of cichlids fed lower qulity diet. A potentil confound resulting from the growth phse is tht the shrimp fed cichlids were in generlly etter condition thn were cichlids fed tuifex. As result, it is possile tht the oserved response ptterns found in Experiment 1 my e due to overll ody condition versus diet per se. We, therefore, conducted Experiment 2 to exmine the effects of growth (ody condition) on the production of chemicl lrm cues.

8 494 Brown et l. ANN. ZOOL. FENNICI Vol A.5 B Time moving (sec) Are use C 5 D Distnce to neighour (cm) Aggressive interctions Tuifex Shrimp 15 Tuifex Shrimp Fig. 3. Men (± S.E.) chnge in (A) time moving (seconds), (B) re use, (C) distnce to neighour (cm), nd (D) occurrence of ggressive interctions for juvenile cichlids exposed to skin extrct from low condition index (open rs) versus high condition index (solid rs) donors fed either tuifex or shrimp diets. N = 1 per tretment comintion. Tle 3. Results of two-wy ANOVAs for ech response vrile recorded in Experiment 2. Diet denotes diet tretment (shrimp versus tuifex) of stimulus donors, Condition denotes condition index (high versus low) of stimulus donors. N = 1 per tretment. F df P Time moving Diet.15 1,36 =.7 Condition ,36 <.5 Diet condition.2 1,36 =.88 Are use Diet.1 1,36 =.75 Condition ,36 <.5 Diet condition.1 1,36 =.91 Distnce to neighour Diet.56 1,36 =.46 Condition ,36 <.5 Diet condition.8 1,36 =.78 Aggressive interctions Diet.1 1,36 =.92 Condition ,36 =.29 Diet condition.22 1,36 =.64 Experiment 2: Influence of ody condition For time spent moving, re use nd distnce to neighour, we found significnt effects of stimulus donor condition index, ut no significnt effects of stimulus donor diet (Tle 3 nd Fig. 3). As in Experiment 1, there ws no significnt difference in the frequency of ggressive interctions (Tle 3 nd Fig. 3). We found no significnt interctions etween stimulus donor diet nd condition index for ny of the response vriles (Tle 3). Juvenile cichlids exposed to the skin extrct of high condition index donors, regrdless of donor diet, exhiited significntly more intense ntipredtor ehviours thn did those exposed to the skin extrcts of low condition index donors. The results of Experiment 2 demonstrte tht the skin extrct of fish of higher condition elicits stronger ntipredtor response thn does the skin extrct of lower condition donors, regrdless of the diet type. However, the growth phse

9 ANN. ZOOL. FENNICI Vol. 41 Production of lrm cues in juvenile convict cichlids A.5 B Time moving (s) Are use C 12 D 1 8 Distnce to neighour (cm) 1 2 Aggressive interctions Lrge Smll SWT DW Lrge Smll SWT DW Stimulus Stimulus Fig. 4. Men (± S.E.) chnge in (A) time moving (seconds), (B) re use, (C) distnce to neighour (cm), nd (D) occurrence of ggressive interctions for juvenile cichlids exposed to su-dult conspecific (Lrge), juvenile conspecific (Smll), or swordtil (SWT) skin extrcts or distilled wter (DW) control. N = 1 per tretment comintion. Different letters denote significnt differences (P <.5) sed on Fisher s Protected Lest Squred Differences. of our study suggests tht cichlids fed higher qulity diet (shrimp) hd higher growth rte thn those fed lower qulity diet (tuifex). As such, differences in growth rte (ontogeny) rther thn condition per se my ccount for the oserved results. We, therefore, conducted Experiment 3 to ddress the question of the role of ontogeny in chemicl lrm cue production. Experiment 3: Effects of ontogeny For ech of the four ehviourl mesures, we found significnt effect of stimulus type. Cichlids exposed to the sme size nd lrger cichlid skin extrct significntly decresed time spent moving (F 3,36 = 3.93, P =.16), re use (F 3,36 = 3.32, P =.31), distnce to neighour (F 3,36 = 3.11, P =.39), nd the frequency of ggressive interctions (F 3,36 = 3.8, P =.39) when compred with either distilled wter or swordtil skin extrct (Fig. 4). Moreover, there ws no significnt difference in the response to sme size versus lrger conspecific skin extrcts (Fig. 4), suggesting tht ontogeny does not ccount for the response ptterns reported for Experiment 1 nd 2. Discussion Tken together, the results of these experiments strongly support the hypothesis tht individul

10 496 Brown et l. ANN. ZOOL. FENNICI Vol. 41 diet significntly influences chemicl lrm cue production in juvenile convict cichlids. Experiment 1 demonstrted tht cichlids fed higher qulity diet (rine shrimp) produced n lrm cue, which elicited significntly stronger ntipredtor response in conspecifics. Experiment 2 demonstrted tht the mechnism responsile for the oserved difference in response intensity ws overll condition. Cichilds of higher ody condition (i.e. hevier for given length) were le to produce either more lrm cue per cm 2 of skin or produced chemicl, which ws more recognizle. Experiment 3 further supports this hypothesis y demonstrting tht when ody condition is held constnt, the ge of skin donors does not result in significnt differences in chemicl lrm cue production. Two possile non-mutully exclusive mechnisms my ccount for the oserved diet/ody condition effects. Initilly, higher qulity diets, leding to etter overll condition, my result in the production of more chemicl lrm cue per re of skin. As result, the skin extrct of high condition donors would result in higher functionl concentrtion of chemicl lrm cue. Such mechnism ssumes tht the response to conspecfic chemicl lrm cues is grded (Brown 24). There is, however, contrdictory evidence regrding grded versus non-grded responses. Brown et l. (21) exposed shols of fthed minnows to hypoxnthine-3-n-oxide (H3NO; the puttive Ostriophysn lrm pheromone ; Brown et l. 2, 21, 23) t concentrtions rnging etween 6.7 to.1 nm. Minnows exhiited consistent ntipredtor ehviour responses when exposed to H3NO t concentrtions of.4 nm nd ove. At concentrtions elow this point, there ws no mesurle chnge in overt ntipredtor ehviour (Brown et l. 21). In ddition, they found tht if chemicl lrm cues ove this threshold re detected, individuls respond in n ll-or-nothing fshion. Similr results hve een shown for juvenile rinow trout (Oncorhynchus mykiss; Mirz & Chivers 23) nd pumpkinseed sunfish (Lepomis giosus; Mrcus & Brown 23). Conversely, Zho nd Chivers (24), found evidence in support of grded response in juvenile goldfish (Crssius urtus). Juvenile goldfish exhiited ntipredtor responses tht decresed in intensity proportionl to the concentrtion of lrm cue, suggesting true grded response pttern. Alterntively, higher qulity diet my llow individuls to llocte more resources to lrm cue production. There is some indiction in the literture tht proteins my ply significnt role s either recognizle lrm cues or s crrier compounds (Ksumyn & Ponomrev 1987). If individuls cn ccumulte higher proportion of proteins in their diet from selectively forging on higher qulity food items, this my then result in n lrm cue tht is more redily detected y signl receivers or is trnsmitted through the wter column more redily. Brown et l. (21, 23) rgued tht Ostriophysn lrm cues re produced from the metolic yproducts of protein degrdtion. Such mechnism would e energeticlly inexpensive, s the precursors to the lrm cue re redily ville. If such mechnism is operting in the cichlid lrm cue system, then incresing the overll qulity or quntity of the diet (leding to n overll increse in ody condition) my serve s proximte mechnism ccounting for our oserved response ptterns. Previous studies hve demonstrted significnt trde-offs etween hunger level nd response to conspecific chemicl lrm cues. When fooddeprived for reltively short time periods (~24 hours), Iow drters (Etheostom exile) exhiited significnt reductions in their response to conspecific lrm cues (Smith 1981). Likewise, fthed minnows (Brown & Smith 1996), finescle dce (Phoxinus neogeus; Brown & Cown 2), nd reticulte sculpins (Cottus perplexus; Chivers et l. 2) fil to respond to conspecific lrm cues when food is deprived for periods of 24 to 48 hours. Thus, it could e rgued tht since the tuifex diet resulted in n overll poorer qulity diet, test fish fed tuifex might e expected to e energeticlly stressed nd hence, show weker response to lrm cues. This, however, is not the cse in the current study, s we found no significnt effect of test fish diet. If the tuifex fed test fish were energeticlly stressed (reltive to shrimp fed test fish), we would expect to see significnt effect of test fish diet. However, cichlids fed tuifex or shrimp responded with similr intensities to the sme donor-diet tretments. Likewise, Vilhunen nd Hirvonen (23) filed to find n effect of hunger level on the response

11 ANN. ZOOL. FENNICI Vol. 41 Production of lrm cues in juvenile convict cichlids 497 of juvenile Arctic chrr (Slvelinus lpinus) to conspecific lrm cues. Alemdi nd Wisenden (22) exposed juvenile convict cichlids within nd just eyond the size rnge t which individuls would e defended y prents. They report tht there ws no difference in the response y smller versus lrger juveniles to the lrm cue of either size clss tested. This suggests tht even t n erly developmentl stge, juvenile cichlids re producing recognizle lrm cue. Our results expnd on this finding, demonstrting tht there ws no significnt difference etween juvenile nd dult cichlids in the production of the lrm cue. Similr results hve lso een demonstrted for yellow perch (Hrvey & Brown 24). It remins unknown if the cichlid lrm cue consists of some specific molecule or group of molecules (s in the Ostriophysn lrm pheromone system; Brown et l. 2, 21, 23) or is some generlized cue. Cichlids lck the specilized epiderml clu cells found in Ostriophysn fishes (Pfeiffer 1977), which might suggest more generlized nture. However, we found no response to swordtil skin extrct. Likewise, Wisenden nd Srgent (1997) reported no response of juvenile convict cichlids to the skin extrct of gmusi (Gmusi ffinis) nd Brown et l. (23) found no response to hypoxnthine-3-n-oxide, the puttive Ostriophysn lrm pheromone. Comined, these results suggest tht convict cichlids do not respond to the generlized cue of ny injured prey fish, supporting the existence of specilized cichlid lrm cue. To dte, insufficient work hs een conducted to ddress this question. Responding to chemicl lrm cues cn significntly increse n individul s proility of surviving n encounter with predtor (Mthis & Smith 1993, Chivers et l. 22, Mirz & Chivers 23, G. E. Brown & R. S. Mirz unpul. dt). G. E. Brown nd R. S. Mirz (unpul. dt) showed tht individuls exhiiting stronger ntipredtor response gin proportionlly greter survivl enefits. As such, potentil enefit ssocited with sholing ner conspecifics in good condition might e incresed potentil of detecting nd responding to these criticlly importnt cues. Experiments re ongoing to directly test this hypothesis. Size specific sholing is well documented mong prey fishes (Wrd & Kruse 21), especilly under conditions of incresed perceived predtion risk (Hore et l. 24). Preferentilly sholing with similr sized nd/or morphologiclly similr individuls hs een rgued to reduce conspicuousness nd hence predtion risk (Lndeu & Terorgh 1986, Theodorkis 1989, ut see Mthis & Chivers 23). In ddition, differences in competitive ility would result in smller individuls eing out-competed y lrger sholmtes (Peuhkuri 1997, Sepp et l. 1999, Kim et l. 24). Individuls sholing with similr sized or conditioned conspecifics versus smller (Wrd & Kruse 21) or those in lower ody condition (Brer et l. 1998, Poulin et l. 1999) my gin dditionl enefit ssocited with stronger response intensities upon detection of conspecific lrm cues. Given the demonstrted survivl enefits ssocited with responding to dmge relesed chemicl lrm cues (see ove), selection should fvour such size ssorttive sholing under conditions of high predtion risk (Hore et l. 24). Thus, in ddition to the well documented direct enefits ssocited with size ssorttive sholing, our current results suggest there my exist indirect enefits s well. Acknowledgements We thnk Antoine Leduc, Mrk Hrvey, Justin Golu, Jmes Grnt nd Iselle Désormeux for helpful comments on erlier versions of the mnuscript. Chrystl Hely nd Jennifer Cotter provided ssistnce in the lortory. Finncil support ws provided y n NSERC of Cnd Discovery Grnt nd Concordi University to G.E.B. All work reported herein ws conducted in ccordnce with Concordi University Animl Cre Protocol AC-22-BROW. References Alemdi, S. D. & Wisenden, B. D. 22: Antipredtor response to injury-relesed chemicl lrm cues y convict cichlid young efore nd fter independence from prentl protection. Behviour 139: Brer, I., Downey, L. C. & Brithwite, V. A. 1998: Prsitism, oddity nd the mechnism of shol choice. J. Fish Biol. 52: Brown, G. E. 23: Lerning out dnger: chemicl lrm cues nd locl risk ssessment in prey fishes. Fish Fish. 4:

12 498 Brown et l. ANN. ZOOL. FENNICI Vol. 41 Brown, G. E. 24: Locl predtion risk ssessment sed on low concentrtion chemicl lrm cues in prey fishes: evidence for thret-sensitivity. In: Mson, R. T., LeMster, M. & Müller-Schwrze, D. (eds.), Chemicl signls in vertertes, vol. 1. Plenum Press, New York. [In press]. Brown, G. E. & Cown, J. 2: Forging trde-offs nd predtor inspection in n Ostriophysn fish: switching from chemicl to visul cues. Behviour 137: Brown, G. E. & Godin, J.-G. J Chemicl lrm signls in Triniddin guppies: lortory nd field evidence. Cn. J. Zool. 77: Brown, G. E. & Smith, R. J. F Fthed minnows use chemicl cues to discriminte sholmtes from unfmilir conspecifics. J. Chem. Ecol. 2: Brown, G. E. & Smith, R. J. F. 1996: Forging trde-offs in fthed minnows (Pimephles promels): cquired predtor recognition in the sence of n lrm response. Ethology 12: Brown, G. E., Adrin, J. C. Jr., Ptton, T. & Chivers, D. P. 21: Fthed minnows lern to recognize predtor odour when exposed to concentrtions of rtificil lrm pheromone elow their ehviourl-response threshold. Cn. J. Zool. 79: Brown, G. E., Adrin, J. C. Jr., Kufmn, I. H., Erickson, J. L. & Gershneck, D. L. 21: Responses to nitrogen-oxides in chriciform fishes suggest evolutionry conservtion in Ostriophysn lrm pheromones. In: Mrchlewsk-Koj, A., Lepri, J. J. & Müller-Schwrze, D. (eds.), Chemicl signls in vertertes, vol. 9: Plenum Press, New York. Brown, G. E., Adrin, J. C. Jr., Nderi, N. T., Hrvey, M. C. & Kelly, J. M. 23: Nitrogen oxides elicit ntipredtor responses in juvenile chnnel ctfish, ut not in convict cichlids or rinow trout: conservtion of the Ostriophysn lrm pheromone. J. Chem. Ecol. 29: Brown, G. E, Adrin, J. C. Jr., Smyth, E., Leet, H. & Brennn, S. 2: Ostriophysn lrm pheromones: lortory nd field tests of the functionl significnce of nitrogen-oxides. J. Chem. Ecol. 26: Chivers, D. P. & Smith, R. J. F. 1998: Chemicl lrm signlling in qutic predtor prey systems: review nd prospectus. Écoscience 5: Chivers, D. P., Brown, G. E. & Smith, R. J. F The evolution of chemicl lrm signls: ttrcting predtors enefits lrm signl senders. Am. Nt. 148: Chivers, D. P., Mirz, R. S. & Johnston, J. 22: Lerned recognition of heterospecific lrm cues enhnces survivl during encounters with predtors. Behviour 139: Chivers, D. P., Puttlitz, M. H. & Blustein, A. R. 2: Chemicl lrm signling y reticulte sculpins, Cottus perplexus. Environ. Biol. Fish. 57: Grcí, C., Rolán-Alvrez, E. & Sánchez, L Alrm rection nd lert stte in Gmusi ffinis (Pisces, Poeciliide) in response to chemicl stimuli from injured conspecifics. J. Ethol. 1: Grcí-Berthou, E. 21: On the misuse of residuls in ecology: testing regression residuls vs. the nlysis of covrince. J. Anim. Ecol. 7: Hrvey, M. C. & Brown, G. E. 24: Dine or dsh?: Ontogenetic shifts in the response of yellow perch to conspecific lrm cues. Environ. Biol. Fish. [In press]. Hore, D. J., Couzin, I. D., Godin, J.-G. J. & Kruse, J. 24: Context-dependent group size choice in fish. Anim. Behv. 67: Ksumyn, A. O. & Ponomrev, V. Yu. 1987: Biochemicl fetures of lrm pheromone in fish of the order Cypriniformes. J. Evol. Biochem. Physiol. 23: Kim, J.-W., Brown, G. E. & Grnt, J. W. A. 24: Interctions etween ptch size nd predtion risk ffect competitive ggression nd size vrition in juvenile convict cichlids. Anim. Behv. [In press]. Lndeu, L. & Terorgh, J. 1986: Oddity nd the confusion effect in predtion. Anim. Behv. 34: Mrcus, J. P. & Brown, G. E. 23: Response of pumpkinseed sunfish to conspecific chemicl lrm cues: n interction etween ontogeny nd stimulus concentrtion. Cn. J. Zool. 81: Mthis, A. & Chivers, D. P. 23: Overriding the oddity effect in mixed-species ggregtions: group choice y rmored nd nonrmored prey. Behv. Ecol. 14: Mthis, A. & Smith, R. J. F. 1993: Chemicl lrm signls increse the survivl time of fthed minnow (Pimephles promels) during encounters with northern pike (Esox lucius). Behv. Ecol. 4: Mthis, A., Chivers, D. P. & Smith, R. J. F Chemicl lrm signls: predtor deterrents or predtor ttrctnts? Am. Nt. 145: Mirz, R. S. & Chivers, D. P. 2: Predtor-recognition trining enhnces survivl of rook trout: evidence from lortory nd field-enclosure studies. Cn. J. Zool. 78: Mirz, R. S. & Chivers, D. P. 22: Brook chr (Slvelinus fontinlis) cn differentite chemicl lrm cues produced y different ge/size clsses of conspecifics. J. Chem. Ecol. 28: Mirz, R. S. & Chivers, D. P. 23: Response of juvenile rinow trout to vrying concentrtions of chemicl lrm cue: response thresholds nd survivl during encounters with predtors. Cn. J. Zool. 81: Mirz, R. S. & Chivers, D. P. 23: Predtor diet cues nd the ssessment of predtion risk y juvenile rook chrr: do diet cues enhnce survivl? Cn. J. Zool. 81: Mirz, R. S., Scott, J. J. & Chivers, D. P. 21: Differentil responses of mle nd femle red swordtils to chemicl lrm cues. J. Fish Biol. 59: Peuhkuri, N. Rnt, E. & Sepp, P. 1997: Size-ssorttive schooling in free-rnging sticklecks. Ethology 13: Pfeiffer, W. 1977: The distriution of fright rection nd lrm sustnce cells in fishes. Copei 1977: Poulin, R., Mrcogliese, D. J. & McLughlin, J. D. 1999: Skin-penetrting prsites nd the relese of lrm sustnces in juvenile rinow trout. J. Fish Biol. 55:

13 ANN. ZOOL. FENNICI Vol. 41 Production of lrm cues in juvenile convict cichlids Sepp, T., Peuhkuri, N., Hirvonen, H., Luril, A., Piironen, J. & Rnt, E. 1999: Nrrow size regime mong individuls fvours rpid growth in Arctic chr (Slvelinus lpinus) juveniles. Cn. J. Fish. Aqut. Sci. 56: Smith, R. J. F. 1981: Effect of food deprivtion on the rection of Iow drters (Etheostom exile) to skin extrct. Cn. J. Zool. 59: Smith, R. J. F. 1992: Alrm signls in fishes. Rev. Fish Biol. Fish. 2: Smith, R. J. F. 1999: Wht good is smelly stuff in the skin? Cross function nd cross tx effects in fish lrm sustnces. In: Johnston, R. E., Müller-Schwrze, D. & Sorensen, P. W. (eds.), Advnces in chemicl signls in vertertes: Kluwer Acdemic Press, New York. Sokl, R. R. & Rohlf, F. J. 1981: Biometry, 2nd ed. W.H. Freemn, New York. Theodorkis, C. 1989: Size segregtion nd the effects of oddity on predtion risk in minnow schools. Anim. Behv. 38: Vilhunen, S. & Hirvonen, H. 23. Innte ntipredtor response of Arctic chrr (Slvelinus lpinus) depend on predtor species nd their diet. Behv. Ecol. Socioiol. 55: 1 1. Wrd, A. H. W. & Kruse, J. 21: Body length ssorttive sholing in the Europen minnow, Phoxinus phoxinus. Anim. Behv. 62: Wisenden, B. D. & Srgent, R. C. 1997: Antipredtor ehviour nd suppressed ggression y convict cichlids in response to injury-relesed chemicl cues of conspecifics ut not to those of n lloptric heterospecific. Ethology 13: Wisenden, B. D. & Smith, R. J. F. 1997: The effect of physicl condition nd sholmte fmilirity on prolifertion of lrm sustnce cells in the epidermis of fthed minnows. J. Fish Biol. 5: Wisenden, B. D. & Smith, R. J. F. 1998: A re-evlution of the effect of sholmte fmilirity on the prolifertion of lrm sustnce cells in fthed minnows. J. Fish Biol. 53: Zho, X. & Chivers, D. P. 24: Response of juvenile goldfish (Crssius urtus) to chemicl lrm cues: reltionship etween response intensity nd the level of predtion risk. In: Mson, R. T., LeMster, M. & Müller-Schwrze, D. (eds.), Chemicl signls in vertertes, vol. 1. Plenum Press, New York. [In press]. This rticle is lso ville in pdf formt t