转基因鱼Aggregation Behavior in Wildtype and Transgenic Zebrafish(第4组) (1)

Aggregation Behavior in Wildtype and Transgenic Zebrafish Jennifer L.Snekser*,Scott P.McRobert*,Caleb E.Murphy &Ethan D.Clotfelter*Department of Biology,Saint Joseph’s University,Philadelphia,PA,USADepartment of Biology,Amherst College,Amherst,MA,USAIntroductionZebrafish(Danio rerio)have been genetically engin-eered to express greenfluorescent protein(GFP)and redfluorescent protein(RFP)derived from the cnid-arians Aequorea victoria and Discosoma sp.,respectively (Wan et al.2002;Gong et al.2003).To create these transgenicfish,DNA constructs were made via inser-tion of a fast-muscle specific promoter gene into red and GFP genes,which were then microinjected into one-or two-cell zebrafish embryos.Fluorescent pro-teins are expressed in the skeletal muscles and are visible under normal light conditions.Designed for use in developmental and toxicological studies,these fish have been marketed by the aquarium industry under the trademark GloFish TM.To date,only the RFP zebrafish are commercially available.They are thefirst transgenicfish sold to the general public (currently restricted to the U.S.).While zebrafish have been an important biomedi-cal model system for years(Haffter et al.1996;Lele &Krone1996;Heindel2000),our understanding of their social behavior is relatively recent(Kitevski& Pyron2003;Mann et al.2003;Pyron2003;Turnell et al.2003;Peichel2004).However,it is known that zebrafish,like many teleostfishes,form social groups termed‘shoals’(Pitcher1986).Shoaling behavior confers many advantages upon individualfish,inclu-ding reduction of predation risk through a phenom-enon known as the‘confusion effect’(Krause& Ruxton2002).According to the confusion effect model,a predator faced with a group of similarfish has difficulty targeting an individual to attack(Land-eau&Terborgh1986).This phenomenon is directly related to the‘oddity effect,’which predicts that physically distinct(or‘odd’)individuals within a shoal are more likely to be targeted by predators. Therefore,an individual’s ability to recognize and associate with similarfish should provide a selective advantage.Previous studies have shown thatfish prefer to shoal with phenotypically similar individu-als(reviewed in Krause et al.2000).CorrespondenceScott P.McRobert,Department of Biology, Saint Joseph’s University,Philadelphia,PA 19131,USA.E-mail:smcrober@Received:March19,2005Initial acceptance:March29,2005Final acceptance:June2,2005(S.Forbes)AbstractRecent advances in the development and availability of genetically modified animals enable researchers to examine the effects of pheno-typic characters on social behavior.Infish,shoaling behavior is known to be influenced by characteristics such as body coloration,striping pat-tern,body shape,and size.GloFish TM are genetically engineered zebra-fish(Danio rerio)that express redfluorescent protein(RFP),resulting in on overall red coloration under the dark longitudinal stripes.The Glo-Fish pattern is distinct from the light body coloration underlying the dark longitudinal stripes seen in wildtype zebrafish.We presented wild-type and transgenic RFP zebrafish with same-sex shoals of both strains offish in dichotomous choice tests.No preference for either of the shoals was shown,however,both strains showed significant preferences for swimming near shoals vs.swimming near an empty tank compart-ment.When presented with opposite-sex individuals of both strains,no preference was shown by either sex of either strain.Thus,the red body coloration of transgenic zebrafish does not appear to affect choice of social partner,in either a shoaling or a potentially reproductive context.EthologyEthology112(2006)181–187ª2006The AuthorsJournal compilationª2006Blackwell Verlag,Berlin181Shoaling behavior can also provide increased opportunity for mating(Krause&Ruxton2002).By choosing shoal-mates of similar phenotype,indivi-duals naturally form conspecific social groups.This brings males and females together and provides them with an opportunity to preview future mating partners.In zebrafish,a variety of phenotypic cues have been examined to determine the influence on social associations such as body size(Pyron2003),fin length(Kitevski&Pyron2003),group size(Ruhl& McRobert2005),and stripe pattern(McCann et al. 1971;Turnell et al.2003).The effect of overall body coloration has yet to be established.Many other cyp-rinidfishes show a preference for a reddish or ‘brassy’nuptial coloration(Winfield&Nelson1991; Braithwaite&Barber2000),like that of the RFP transgenic zebrafish.These observations lead us to the formulation of our two hypotheses.First,when given the choice between a shoal of wildtype zebrafish and a shoal of RFP transgenic zebrafish that are of the same gender, both strains would choose shoals of their own strain, as predicted by the‘oddity effect.’Second,when placed in a potentially reproductive context,wild-typefish would prefer the redness of the transgenic RFPfish.MethodsAnimal SubjectsWildtype and transgenic zebrafish were obtained from commercial suppliers(World Wide Aquarium, Philadelphia,PA and5-D Tropical in Florida).Fish used for stimulus shoals were housed separately from testfish,in40-or80-l holding tanks.Males and females were housed separately.All tanks were covered on three sides with opaque white paper in order to visually isolate the holding tanks.All tanks were maintained underfluorescent lighting on a 12L:12D cycle,at23–25°C.Fish were fedflake food to satiation daily.Test TanksFor shoaling experiments(Shoaling I–III),test tanks were constructed by dividing aquaria (74·31·31cm)into three compartments(see McRobert&Bradner1998).Each end compartment (18·31·31cm)was separated from the central compartment(38·31·31cm)by clear glass parti-tions sealed with silicone caulk.The central compart-ment was further divided into left and right sides by opaque Plexiglas partitions(25·18cm).These par-titions allowed testfish to swim to either side of the central compartment but prevented a testfish on one side from seeing the stimulusfish on the oppos-ite side.Each of the end compartments contained a heater and boxfilter.Mate choice experiments,in which a testfish was given the opportunity to choose between wildtype and transgenic individuals of the opposite sex were performed in two stages.Mate choice I tests were performed in a7-l polycarbonate tank.Stimulusfish were held in1-l polycarbonate containers placed at each end of the test tank.A line was drawn on the glass to separate the tank into halves.Mate choice II tests,which were more conservative,were per-formed in a modified aquarium(50·25·31cm) divided into three compartments by the addition of glass panels,secured with silicon caulk.Each end compartment(which held the stimulusfish)meas-ured14·25·31cm.Lines drawn on central com-partment glass were used to identify two‘choice zones,’ 6.5cm(approximately two body lengths) from each end compartment.Behavioral ObservationsAll observations were performed between09:00and 17:00hours from Jan.2004to Feb.2005.Prior to each test,a single testfish was placed into the central compartment and allowed1h to acclimate.Testfish were chosen at random from a holding tank and no fish was used more than once for a given experi-ment.Stimulus shoals were also placed into the end compartments of the test tanks prior to testing.Dur-ing the1h of acclimation to the test tank,allfish were able to swim freely in their chambers.For all three shoaling experiments,an equal number of tests were run withfish in the left and right end compart-ments to eliminate the risk of side biases.Each test-ing session consisted of a600-s observation period during which the time(in seconds)that the testfish spent on each side of the central compartment(for the three shoaling experiments and mate choice experiment I)or within the two-body-length‘choice’zones in mate choice experiment II was recorded.In shoaling experiment I,three wildtype zebrafish were placed into one of the end compartments, while the other end compartment was empty.In shoaling experiment II,three transgenic zebrafish were placed into one of the end compartments, while the other was empty.In shoaling experiment III,three wildtype zebrafish were placed into oneAggregation Behavior in Wildtype and Transgenic Zebrafish J.L.Snekser,S.P.McRobert,C.E.Murphy&E.D.ClotfelterEthology112(2006)181–187ª2006The Authors 182Journal compilationª2006Blackwell Verlag,Berlinend compartment and three transgenic zebrafish were placed into the other end compartment.Both wildtype and transgenicfish were used as testfish (in the central compartment)in each experiment. The end compartments always containedfish of the same gender as the focal testfish.For the mate choice experiments,testfish were given the choice between a single wildtype zebrafish and a single transgenic zebrafish of the opposite sex. Mate choice experiment I was performed with wild-type testfish only,mate choice II examined the pref-erences of both wildtype and transgenic testfish. Statistical AnalysisFor each shoaling experiment one-sample t-tests, compared with a null expectation(0.5total time), were used to compare time spent on each side of the tank.For the mate choice experiments,paired sam-ple t-tests were used to compare time spent in each part of the test tank.ResultsShoaling ExperimentsIn shoaling experiments I and II,in which testfish were given the choice between a shoal of three ze-brafish(either transgenic or wildtype)and an empty compartment,the testfish always showed a signifi-cant preference for the compartment containing the shoal offish(responses to a wildtype shoal and a transgenic shoal,respectively:n=20,wildtype female testfish:t=5.45,p<0.001and t=8.39, p<0.001;wildtype male testfish:t=3.56,p<0.01 and t=3.26,p<0.01;transgenic female testfish: t=4.31,p<0.001and t=3.62,p<0.01;transgenic male testfish:t=6.28,p<0.001and t=5.08, p<0.001).However,no significant preferences were shown in shoaling experiment III,when testfish were given a choice between a shoal of three wild-typefish and a shoal of three transgenicfish (n=20,wildtype female testfish:t=0.21,p=0.84; wildtype male testfish:t=0.48,p=0.64;transgenic female testfish:t=0.18,p=0.86;transgenic male testfish:t=0.042,p=0.97,Fig.1).Mate Choice ExperimentsIn mate choice experiment I,wildtype male zebra-fish spent significantly more time swimming near a transgenic female than near a wildtype female (n=35,t= 2.54,p<0.05).Similarly,wildtype females spent significantly more time swimming near a transgenic male,than a wildtype male (n=35,t=2.40,p<0.05;Fig.2).However,in the more conservative mate choice experiment II,wildtype males and females showed no significant preference for either wildtype or trans-genicfish of the opposite sex(n=20,wildtype female testfish:t=0.65,p=0.52;wildtype male testfish:t=2.02,p=0.058).Similarly,transgenic male and female testfish showed no significant pref-erence for either wildtype or transgenicfish of the opposite sex(n=20,transgenic female testfish: t=0.68,p=0.51;transgenic male testfish:t=1.47, p=0.16,Fig.3).DiscussionThe development of a red transgenicfish creates an opportunity for studying the importance of body col-oration in both social and reproductive contexts. This question is important asfish are known to dis-criminate between potential shoal-mates on the basis of phenotype,including body coloration.In mollies (Poecilia latipinna),for example,black and white color morphs choose shoal-mates of similar color-ation(McRobert&Bradner1998).Interestingly,the results presented here show that the red coloration found in transgenic GloFish TM does not influence choice of same-sex shoal-mates.Yet,in some cir-cumstances,body coloration does influence prefer-ence for individual,opposite-sexfish.By shoaling withfish with a similar phenotype,it is thought thatfish reduce the likelihood that they will be preyed upon(Landeau&Terborgh1986). Thus,choosing shoal-mates wisely is evolutionarily beneficial.Many studies have shown thatfish will tend to shoal withfish that are alike in appearance, based not only on body coloration(McRobert& Bradner1998),but also on body shape(Hauser et al. 1998),body size(Pritchard et al.2001;Pyron2003), parasite load(Krause&Godin1996),and body pattern(McCann et al.1971;Turnell et al.2003; Engeszer et al.2004).Through the use of transgenic zebrafish,this study attempts to hold other pheno-typic characters constant and focus solely on the role of coloration.In shoaling experiment III,in which testfish were given the choice between a shoal of similarly coloredfish and dissimilarly coloredfish, no preference was shown for either shoal.The lack of preference for,or avoidance of,the RFP trans-genic zebrafish may indicate that overall body color-ation may not play an important role in shoal-mate preference in this species.J.L.Snekser,S.P.McRobert,C.E.Murphy&E.D.Clotfelter Aggregation Behavior in Wildtype and Transgenic Zebrafish Ethology112(2006)181–187ª2006The AuthorsJournal compilationª2006Blackwell Verlag,Berlin183Despite the fact that the red body coloration ofGloFish TM does not influence shoaling choices,itseems apparent that zebrafish can detect the colorred.Zebrafish are tetrachromatic,with four types of cones,in addition to rods,that absorb light at red,green,blue,and UV wavelengths (Robinson etal.Fig.1:Mean association time (±SE)of wild-type females and males (a)and transgenicfemales and males (b)when given a choice inthree shoaling preference experiments:(1)three RFP transgenic zebrafish (gray)and anempty chamber (white);(2)three wildtypezebrafish (black)and an empty chamber(white);and (3)between three RFP transgeniczebrafish (gray)and three wildtype fish (black).**p <0.01;***p <0.001Fig.2:Mean association time (±SE)of wild-type and RFP transgenic females and maleswhen given a choice in an experiment thatcontained one RFP transgenic (gray)or onewildtype fish (black)of the opposite sex in adichotomous mate choice test.*p <0.05Aggregation Behavior in Wildtype and Transgenic Zebrafish J.L.Snekser,S.P.McRobert,C.E.Murphy &E.D.ClotfelterEthology 112(2006)181–187ª2006The Authors184Journal compilation ª2006Blackwell Verlag,Berlin1993).In fact,zebrafish actually have two distinctred opsin photopigments:one maximally sensitive at558nm and the other at 548nm (Takechi &Kawa-mura 2005).In behavioral screens,zebrafish haveexhibited the optokinetic response,displayingsmooth pursuit and saccade eye movements inresponse to illuminated rotating stripes under bothwhite and red lights (Brockerhoff et al.1997).TheRFP of the transgenic zebrafish has an emission spec-trum of 550–675nm,with emission peaks of 558and 583nm (Matz et al.1999).It is thereforeassumed that the zebrafish can detect the red color-ation of the transgenic fish.To further demonstrate that the zebrafish inthese studies were able to see red,wildtype zebra-fish females were presented with a choice betweenthree female wildtype zebrafish and an emptychamber,with red light (600–620nm)as the onlylight source.In these assays the test fish spent sig-nificantly more time near the shoal of fish(n =10,t =4.333,p <0.01).If the zebrafish wereunable to see in this limited red light condition,then no preference would have been expected.The strong preference for the shoal of fish showsthat wildtype zebrafish are capable of perceivingthe color red.To determine whether ultraviolet cues affectshoaling decisions,wildtype test fish were given thechoice between a shoal of three wildtype fish andthree transgenic fish under ultraviolet lights.Inthese tests,as in tests under fluorescent lighting,nosignificant choice was made by the test fish (n =20,transgenic female test fish:t =0.950,p =0.354).These results indicate that ultraviolet cues have noeffect on shoaling preferences of zebrafish.In a dichotomous choice shoaling study done with modified photographs of zebrafish,wildtype fish showed a preference for striped fish over fish lacking stripes,even if the stripe pattern was altered so that the normal light and dark stripes were reversed (McCann et al.1971).Turnell et al.(2003)recently used video stimuli to demonstrate that the orienta-tion of zebrafish stripe patterns significantly affected associative preferences.These results,along with the results from the current study,indicate that it may be the presence of stripes and not general body col-oration that is the important cue in shoal-mate pref-erence in zebrafish.With respect to mate choice,female zebrafish,as in many vertebrates,are the ‘choosier’sex (Pyron 2003).In some species of fish,such as two-spotted gobies (Gobiusculus flavescens ),three-spined stickle-backs (Gasterosteus aculeatus ),and brook sticklebacks (Culaea inconstans ),males also show a preference for females,usually in correlation with the large size of the female,and also for the female’s coloration (McLennan 1995;Amundsen &Forsgreen 2001).The larger size and noticeable ‘nuptial’coloration of the females in these species are indicators of sexual receptiveness,which would lead to a more successful mating attempt by the soliciting males.In zebrafish,however,females do not gain a distinct coloration associated with sexual behavior.The level of sexual dimorphism in zebrafish is low,with females being slightly more robust and males developing a slightly more yellowish coloration.The red coloration of the transgenic zebrafish only seemed to affect mate choice within certain experi-mental parameters.In the first mate choice experi-ment,wildtype fish showed a preferencefor Fig.3:Mean association time (±SE)of wild-type and RFP transgenic females and maleswhen given a choice in an experiment thatcontained one RFP transgenic (gray)or onewildtype fish (black)of the opposite sex in adichotomous mate choice testJ.L.Snekser,S.P.McRobert,C.E.Murphy &E.D.Clotfelter Aggregation Behavior in Wildtype and Transgenic Zebrafish Ethology 112(2006)181–187ª2006The AuthorsJournal compilation ª2006Blackwell Verlag,Berlin 185transgenicfish of the opposite sex over wildtypefish of the opposite sex.However,in these tests,choice was determined by the testfish swimming in the general vicinity of the stimulusfish.In the more conservative mate choice experiment II,in which choice was determined by the testfish swimming within two body lengths of the stimulusfish for a significant fraction of the observation period,no sig-nificant preference was shown for the transgenic fish.Similarly,the transgenicfish showed no prefer-ence for either strain when tested in the mate choice experiment II.These results appear to be supported by Seah,W.K.,Li, D.,Chen,M.,&Gong, Z.(unpublished data),who reported a lack of advan-tage for transgenic zebrafish in a dichotomous choice test(see Gong et al.2003).As red coloration is not usually apparent in zebra-fish,it will be interesting to examine patterns of mate choice using a stimulus that may invoke a more natural bias,such as the GFP transgenic zebra-fish that will soon be available to the general public. 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