Received 22April 2003 Accepted 25 June 2003 Publishedonline 5September 2003 Antimicrobialegg cleaningby the fringed darter (Perciformes: Percidae: Etheostomacrossopterum ): implicationsof anovel component of parentalcare in fishes Jason H.Knouft 1* ,Lawrence M.Page 1 and Michael J.Plewa 2 1Centerfor Biodiversity, Illinois NaturalHistory Survey, 607East Peabody Drive, Champaign, IL 61820,USA 2Departmentof Crop Sciences, University ofIllinois atUrbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801, USA Broad-spectrumantimicrobial compoundshave recentlybeen identified in theepidermal mucusof fishes andprobably serveas a first line ofdefenceagainst microbial pathogens.Because of the ubiquitous nature offungi andbacteria in aquatic systems,defence against thesepathogens should be required throughout thelifespan offishes, including theegg stage.We conducted experiments on Etheostomacrossopterum (Percidae: Catonotus),thefringed darter, todetermine if thepresence of a guarding male inhibits microbial colonization ofeggs. Based on results from acombination ofin-stream experiments, in vitro microbial assays,and morphological characteristics andbehaviour ofbreeding males,we proposethat antimicrobial egg cleaning by theguarding male is aneffective component of parental care in thesefish. Although innate antimicrobial compoundshave beenidentified in avariety oforganisms ranging from insectsto vertebrates, integration ofthese compounds into aspecies’s reproductive life history has beenidentified only in asmall numberof insect species. The resultsfrom this studynot only indicate that E.crossopterum males provide anovel form ofvertebrate parental care,but also have implications regarding theevolution ofparental care in fishesand transitional evolutionary stagesfrom noparental care tomale parental care. Keywords: nestguarding; parental care; Etheostomacrossopterum ;egg cleaning; antimicrobial compounds 1. INTRODUCTION Although aconsiderableamount of effort has been directedtowards understanding reproductive behaviour in Fishesdisplay aremarkable variety ofparental care,with fishes(Breder &Rosen1966; Perrone& Zaret 1979; formsof parental investmentquite different from other Baylis 1981; Gross& Sargent 1985), almost noattention vertebrate groups (Breder &Rosen1966; Perrone& Zaret has beengiven tothe relationship betweenparental care, 1979; Gross& Sargent 1985). Nestguarding is themost microbial infectionand egg viability. This is surprising commonparental behaviour exhibited among families of consideringthat all organic surfacesin aquatic systemsare fishes,with males more likely than femalesto be the coveredby biofilms that are composedprimarily ofhetero- guarding sex(Blumer 1979). This bias may bedue to the trophic bacteria andfungi (Lock et al. 1984). Localized greater netfitness advantage tomales resulting from growth inhibition ofthese microbes shouldbe essential to guarding (Gross& Sargent 1985). In any case,this par- egg survival. ental presencepresumably inhibits predation and Recentstudies have identifiedthe presence of anti- decreasesthe amount of debrison the eggs, provides oxy- microbial compoundsin theepidermal mucusof avariety genand assists in theremoval ofwaste (Moyle & Cech offishes (e.g. Oncorhynchusmykiss (Austin& McIntosh 1996). 1988); Cyprinus carpio (Cole et al. 1997); Pleuronectes Male guarding behaviour andthe evolution ofparental americanus (LeMaitre et al. 1997); Moronesaxatilis ´ M. care in fishesare well studied(Balshine-Earn &Earn chrysops hybrid (Silphaduang &Noga 2001)). These 1998; Goodwin et al. 1998; Lindstrom2000; StMary et speciesare members ofdifferent orders (Salmoniformes, al. 2001). Male guarding in externally fertilizing species Cypriniformes, Pleuronectiformesand Perciformes, has beenproposed to be the result of an evolutionary tran- respectively), suggesting that thepresence of antimicrobial sitionfrom noparental care tomale parental care compoundsin fish mucusis notphylogenetically con- (Gross& Sargent 1985). However,this transition doesnot strainedand such compounds may beubiquitous among automatically ensureparental care tothe offspring. In fishes.These compounds have beenproposed to serve as somecases, an intermediate stage is evidentin which the afirst line ofdefence against pathogens(Boman 1995). male gains somebenefit from remaining at thenest while Becausenest-guarding fishes are in contactor closeprox- theoffspring realize nobenefit from themale’ s presence imity totheir eggs,antimicrobial compoundsin fish (Gross& Sargent 1985; Clutton-Brock1991). mucusmay servean adaptive purposeby preventing microbial colonization ofeggs. Etheostoma (Perciformes:Percidae), containing approxi- *Authorand address for correspondence: Department of Biology, CampusBox 1137,Washington University, StLouis, MO 63130-4899, mately 140 species,is themost diverse genus of freshwater USA([email protected]). fishesin North America (Page 2000). Catonotus, Proc.R. Soc.Lond. B (2003) 270, 2405–2411 2405 Ó 2003 TheRoyal Society DOI10.1098/ rspb.2003.2501 2406J. H.Knouft andothers Antimicrobial egg cleaning bythe fringed darter containing 18 species,is asubgenusof Etheostoma in opaque and/orfungus-covered eggs. Wealso noted whether or which all speciesexhibit egg-clustering behaviour charac- not the malewas stillguarding the eggs. No eggs hatched during terizedby male guarding (Page 1983, 1985; Porterfield et the experiment.A comparisonof the percentageof infectedeggs al. 1999). Within Catonotus,adultsare sexually dimorphic versusuninfected eggs among treatments was madeusing data during thebreeding season.Males are larger andmore collectedon day 4(paired t-test, a = 0.05).Percentage data were boldly patterned.During thereproductive period arcsinetransformed before statistical analyses to achievenor- (typically March –May),a male establishesa territory mality.Based on dailyegg counts,a minimalnumber of eggs underthe cavity ofa flat stone.Multiple femaleswill wereconsumed by guarding males(less than 5%)and ouranaly- sequentially attach eggs totheunderside of the stone that sesof the percentageof infectedeggs assumes that maleswere are simultaneouslyfertilized by themale. This process not selectiveregarding which eggs they consumed.Males and resultsin asingle layer ofup to 1500 eggs ( ca. 2 mm in nest stones werereturned to the streamafter terminationof diameter) depositedon thenest stone, i.e. on the ‘ceiling’ the experiment. ofthe nest cavity directly above themale. The male then Weperformeda colony-formingunit (CFU) assay to demon- remains at thenest until theeggs hatch.Time tohatching strate that opaque eggs containeda greater numberof bacteria is dependenton water temperature andcan range from 5 than cleareggs. Fiveclear and fiveopaque eggs wereselected to10 daysamong nestsacross the spawning season (Page froma nest and placedin separate 1.5ml microcentrifugetubes 1983), while time tohatching within aspecificnest is simi- and 250 m l of Luria–Bonnerbroth (LB)was addedto both lar for all eggs. tubes. Eggs werepulverized with asteriletoothpick and vortexed Although several studieshave investigated facetsof for 1min;100 m lquantities of serialdilutions (10 2 1 to 102 6) Catonotus reproductive behaviour (Knapp &Sargent fromeach egg type werethen incubatedon LB plates for 24h 1989; Page &Bart 1989; Lindstrom& Sargent 1997; at 37 °C.After incubation, CFUs were counted and averaged Page &Knouft2000; Porter et al. 2002), whether Caton- across serialdilutions to determinethe numberof CFUsper five otus males actually provide parental care is unclear. eggs. This experimentwas replicatedthree times. Becausefemales prefer todeposit eggs in nestsalready containing eggs (Knapp& Sargent 1989), males may (b) Collection ofepidermal mucus remain at thenest simply toobtain spawningswith To obtain samplesof epidermalmucus, we collected5 –12 females.The benefitof male guarding toeggs is evenmore E.crossopterum breedingmales from a stream.Epidermal mucus uncertainconsidering that Lindstrom& Sargent (1997) was acquiredby gently drawing asterilespatula across the pre- foundhigh levels offilial cannibalism by guarding males. dorsalnape of eachmale. We then combinedmucus from all The objectiveof this studywas to determineexperimen- malesfrom a particularstream and collectiondate ina 1.5ml tally whethermucus from male E.crossopterum confers an microcentrifugetube containing300 m lof 0.1M potassium antimicrobial benefitto guarded eggs. Observations indi- phosphate buffer (PPB)pH 7.4.Pooling of samplesfrom mul- catethat nestsabandoned by theguarding male oftencon- tiplemales was necessarybecause of the smallamount ofmucus tain non-viable eggs with fungal ( Saprolegnia spp.) and that canbe obtained fromeach male. Samples were either used bacterial infection.We predict that becauseof the behav- immediatelyor frozen at 280 °Cuntilrequired. iour andproximity tothe eggs ofthe breeding male, anti- microbial compoundscontained in themale ’s mucus will (c) Microbial assays inhibit microbial growth oneggs, increase egg viability, Previousstudies of vertebrate antimicrobialcompounds indi- andthus support the hypothesis that speciesof Catonotus catethat these substances arenot specific,with inhibitoryeffects provide male parental care. demonstratedon abroad range of microbes(Zasloff 1987,2002; Boman1995; Cole et al. 1997;LeMaitre et al. 1997;Silphadu- ang &Noga 2001).Because a limitedamount of mucuscould 2. MATERIALAND METHODS be acquiredfrom each E.crossopterum male,and relativelysmall (a) In-stream study population sizesin eachstream limited