食物种类对萼花臂尾轮虫种群增长率的影响

食物种类对萼花臂尾轮虫种群增长率的影响 食物种类对萼花臂尾轮虫种群增长率的影 响 动物52(1):70—78,2006 ActaZoologicaSinica Influenceoffood.typeonthepopulationgrowthrateoftherotifer Brachionuscalyciflorusinshort-chronicassays M.L0RLING",W.BEEKMAN DepartmentofEnvironmentalSciences,AquaticEcologyandWaterQualityManagementGroup,WageningenUniversi ty,P.0.Box8080,6700DDWageningen,TheNetherlands AbstractThetypeoffoodgivenduringshortchronicassayswiththerotiferBrachionuscalyct,ZOrUSmightbeoneofthe sourcesofvariationinthereproductiverateoftherotifers.Tengreenalgalspeciesweresuppliedasmonospecificdietsto examinevariabilityinrotifergrowthrate.Inaddition,rotiferswerefedcyanobacteriaasmonospecificfoodandinmix— tureswiththegreenalgaSeenedesmusobliquustotestthehypothesisthatcyanobacteriaareavaluablesupplementincorn— binationwithfooditemssuchasScenedesmus.ThepopulationgrowthrateofB.calycifloruswasinfluencedsignificantly bythedifferentgreenalgalfoodspecies.S.obliquuspromotedthehighestgrowthinB.catyci~orus(1.6perday), whilegrowthonDesmodesmusabundanswasthelowe.st(O.3perday).Aspercentageofmaximalgrowthon S.obliquus,thesequenceofgreenalgalfoodspeciesis:Desmodesmussubspicatus(88%),lorellavulgaris (83%),Monoraphidiumminutum(77%),D.quadricauda(74%),S.知 lcatus(71%),S.acuminatus (69%),S.pectinatus(64%),Chlamydomonasreinhardtii(57%)andD.abundans(19%).Thegrowthdiffer— enceswerenotexplainedfromsizedifferencesofthealgae.ThecyanobacteriaMicrocystisaeruginosaandSynechococcus elongatusdepressedgrowthofB.calyct,Zorusnotonlyasmonospecificfoodbutalsoinmixeddietswiththegoodfood sourceS.obliquus.Thedetrimentaleffectappearednotrelatedtomicrocystins.Theresultsdonotsupportthehypothe— sisthatnontoxiccyanobacteriaareavaluablesupplementincombinationwithothergreenalgalfoodspecies.Thevariation ingrowthratesobservedincurrentstudyunderlinestheinfluencefoodspeciesmighthaveonthegrowthofB.calyclflorus andpotentiallyontheoutcomeoftoxicitytestslActaZoologicaSinica52(1):70—78,2006J. KeywordsRotifer,Brachionuscalyciflorus,Food,Populationgrowth 食物种类对萼花臂尾轮虫种群增长率的影响 M.LURLINGW.BEEKMAN * DepartmentofEnvironmentalSciences,AquaticEcologyandWaterQualityManagementGroup,WageningenUniversi ty,P.O.Box8080,6700DDWageningen,TheNetherlands 摘要在短期慢性观测过程中,食物类型可能是造成萼花臂尾轮虫 (Brachionuscalyciflor"s)种群繁殖率变化 的一种原因.共观测了分别单独投喂1O种不同绿藻对轮虫种群增长率的影响.为 验证藻青菌是藻类饵料(如绿 藻Scenedesmus)有价值的佐剂这一假说,还用蓝细菌单独投喂或与斜生栅藻 (Scenedesmusobliquus)混合投喂 轮虫进行实验观测.结果发现食物种类对萼花臂尾轮虫种群增长率影响显着.斜生 栅藻组获得最大种群增长 (1.6/d),而Desmodesmus组增长率最低(0.3/d).以占斜生栅藻组最大增长的百分率来表示,其它几种绿藻组 种群增长由高到低依次为:Desmodesmussubspicatus88%,小球藻 (Chlorellnvulgaris)83%,单壳缝藻(Mono— raphidiumminutum)77%,D.quadricauda74%,S.falcatus71%,S.acuminatus69%,S.pecti natus64%, 莱茵衣藻(Chlamydomonasreinhardtii)57%,D.abundans19%.轮虫增长率的差异不能用藻类饵料的大小差异 来解释.蓝细菌(Microcystisaeruginosa)和(Synechococcuselongates)不论是单独投喂还是与优良藻类饵料(斜 生栅藻)混合投喂都对萼花臂尾轮虫种群增长有抑制作用.这种副作用似乎与微囊藻素无关.该结果不支持无 毒蓝细菌可作为与其他绿藻饵料配合使用的优良佐剂这一假说.本研究所观察到的生长变化显示了饵料种类对 ReceivedJuly11,2005;acceptedNov.16,2005 *ThisresearchwasfundedbyafellowshipfromtheRoyalNetherlandsAcademyofArtsandS ciences **Correspondingauthor.E—mail:mique1.1urling@wur.nl ◎2006动物ActaZoologicaSinica M.LORL1NGeta1.:AlgaeandBrachionusgrowth71 萼花臂尾轮虫种群增长的影响,也预示了对毒性实验结果的影响[动物52(1):70—78,2006]. 关键词萼花臂尾轮虫食物种群增长 Planktonicrotifersareknowntoplayanlmpor— tantroleintheenergyflowinlakesandponds,and numerousstudieshaveindicatedtheimportanceofro— tifersasprimaryconsumers(e.g.,Dumont,1977; Pilarska,1977;Pourriot,1977;Walz,1995). Becauseoftheirimportanceinzooplanktoncommuni— tiesandaquaticfoodchainsrotifershavebeenusedas modelorganismsinfeedingandfoodchainstudies,in aquacultureresearchandecotoxicologicalresearch (e.g.,Bogdaneta1.,1980;Schlfiteretal 1987;Walz,1995;ChialandPersoone,2003). Inecotoxicologicalresearch,two—daylifecycle testswiththerotiferBrachionuscalyciflorusin whichmultiplebroodsareproducedandtheFxgener— ationalsoreproducesareusedextensively(e.g., Snelleta1.,1991;SnellandMoffal,1992;Radix eta1.,1999;ChialandPersoone,2003;Margdlek andB16ha,2004).Incontrasttoacuteassayswith mortalityasendpoint,thetwo—daylifecycletesthas growthorreproductionasendpoint,whichrequires feedingofthetestorganisms.However,consider— ablevariabilityintestresultsbetweenstudiesmight occurduetouseofdifferentfoodspeciesandgrowth conditions(ChialandPersoone,2003).Ingeneral, greenalgalspeciessuchasmembersofthecommon generaChlamydomonas,Clorella,Desmodesmus andScenedesmusarefedtoB.calyciflorus,but thereisstillnoconsensusonthenutritionaladequacy ofthefoodspecies.Theresultsofsomestudies,for example,suggestChlorellasp.tobeabetterfood thanScenedesmusspecies(e.g.,Xieta1.,2001; 2002;Flores—Burgoseta1.,2003),whereasinoth— ersB.calyciflorusgrewbetteronScenedesmusthan onChlorella(e.g.,Rothhaupt,1990a).Compar— isonoftheresultsfromthesestudies,however,is difficultasdifferentmethodsforlong—termassays wereused. Inseveralstudies,B.calycii一. orusnthe2d growthassayswereofferedamonospecificdietofonly livegreenalgae(e.g.,CecchineandSnell,1999; Radixeta1.,2000,2002).However,inother studies.therotiferswerefedacombinedfoodoflive greenalgaeanddriedcyanobacteria,Spirulina (ChialandPersoone,2003,2005).Cyanobacterial speciesmightbeavaluablesupplementincombination withothergreenalgalfoodspeciesas,forexample, astimulatoryeffecthasbeenreportedfor B.calyciflorusfeda3:1mixtureofthegreenalga MonoraphidiumandthecyanobacteriumPlank. tothrix(WeithofandWalz,1995).Incontrast, mixedfeedsofthecyanobacteriumAnabaenaandthe cryptophyteCryptomonas B.calyciflorus(Gilbert, hamperedgrowthof 1994,1996).These studieswereperformedwithfilamentouscyanobacte— ria.buttoourknowledgeeffectsofmixeddietsof greenalgaewithnon—filamentouscyanobacteriaare virtuallyabsentinliterature. Theaimofthecurrentstudywastoexaminethe effectoftypeoffoodgivenduringshortchronicas— sayswiththerotiferBrachionuscalyci. orusonthe reproductiverateoftherotifers.AccordingtoSnell andMoffat(1992)growthratesshouldnotbeless thanthe0.65acceptabilitythresholdforshortchron— ictestsinordernottoinfluencetheoutcomeofthe toxicityassays.Consequently,feedorganisms shouldbeofgoodnutritionalvaluetoavoidunsatisfac— toryreproduction.Inasmuchasthetypeofgreenal— galfoodmightseriouslyaffectthegrowthof B.calyciflorus,inthecurrentstudy,10greenalgal speciesasavariableinthegrowthrateoftherotifers werecomparedsimultaneouslyina2-dgrowthassay. Inadditiontotheeffectofthetypeofgreenalgalfood alsotheeffectofthenon—filamentouscyanobacteria MicrocystisaeruginosaandSynechococcuselongatus asmonospecificfoodandinmixtureswiththegreen algaScenedesmusobliquusisstudied.Thiswasdone totestthehypothesisthatcyanobacteriaareavalu— ablesupplementincombinationwithfooditemssuch asScenedesmus. 1Materialsandmethods 1.1Organisms Thegreenalgaeandcyanobacteriausedinthe currentstudyarelistedinTable1.Semi—continuous stockculturesweremaintainedin300mlcellulose plugclosedErlenmeyerflaskscontaining150mlof modifiedWC(WoodsHoleChu)一medium(Larling andBeckman,1999)withvitaminsadded(Biotin B1andB12at50ng/L,ThiamnineHCLat 100ng/L).Flaskswithgreenalgaewerekeptincli— matecontrolledroomat20?incontinuouslightof 60一ttmolquanta/m/sandeverythreeweeksforat leastthreemonthspriortotheexperiments,aliquots weretransferredseparatelyintofreshsterilemedium. Flaskswithcyanobacteriawerekeptat20?andin 45ttmolquantam/m2/slightina16:8hlight:dark rhythm. TherotiferBrachionuscalyci疗. orusPallaswas obtainedfromMicrobiotestsInc.(Nazareth,Bel— glum)andwassuppliedascysts.Cystswerekept refrigerateduntiluse. 72动物52卷 FoodtypesusedinExp11: 100%cyanobacterium一0%S.obliquus;75%一25%;50%一50%;25%一75%; 100%;75%;50%;25%;0%S.obliquus 1:NorwegianInstituteforWaterResearch 1.2Exp.I:effectofgreenalgae Thefirstexperimentevaluatedtheeffectof10 differentgreenalgalspecies(Table1)onrotifer growthrate.Toobtainexponentiallygrowingalgae withgoodnutritionalvalue,aliquotsofgreenalgae fromthesemi..continuousstockculturesweretrans.. ferredpriortotheexperimentinto100一mlErlenmey— erflasks:containing50mlWCmediumwithvita— minsaddedandthatwereclosedwithacellulose plug.Algaldensitiesandparticlesizedistributions weredeterminedinthesizerange2.5—35btmequiv— alentsphericaldiameterusingaCoulteMultisizer II(capillary100p.morificewidth,CoulterElec— tronicsLimited,Luton,England).Eachflaskre— ceivedidenticalinitialconcentrationofalgae.i.e.5 ×100um/m1.Theseflaskswereplacedonarotat— ingshakingtable(60r/m)at24?incontinuous lightof100一t~molquanta/m/s.Afterthreedaysthe algaeweretransferredagainatidenticalconcentra— tionsof5×10.um/mlinto100mlErlenmeyer'sthat contained50一mlWC—medium.Afterthreedaysthe algaewereusedintheexperiment.MeanwhileB. calycifloruscystswerehatchedin100一mlsuspensions ofeachofthecorrespondinggreenalgaeintheabove mentionedWC-medium.Thehatchingflaskswere placedonarotatingshakingdeviceat24?incontin— UOUSlightof100一ttmolquanta/m2/s.Thecystswere hatchedinthedesiredfoodinasmuchasashort— prefeedingperiod(2hafterhatching)hasbeen foundtohaveapronouncedpositiveeffectongrowth rates(ChialandPersoone,2005). Theexperimentwasrunin6一welledcoveredcul— tureplatespositionedfortwodaysonarotatingshak— ingtable(60r/m)indarknessinaclimate-con— trolledroomat24?.Eachwellwasfilledwith10一 mlgreenalgalfoodsuspensionsatidenticalconcentra— tionsof10m/ml(?5mgC/L).F0reachfood type(Table1)sixwellswereusedofwhichthree receivedsixfreshlyhatched(2—3hold)BTa— chionus,whilethreeotherreplicatesreceivednoro— tifers.Thenewbornrotiferswerecollectedinadish with15mlfreshWC—mediumandwerecountedand transferredfromthedishtothecultureplatesundera dissectingmicroscopeat20×magnificationusinga preparedglassfinetippipette.Theexperimentwas runfor48hoursafterwhichchlorophyl1.aconcentra. tionsweredeterminedin1.5一mlsamplesfromeach wellusingaPHYTo—PAMphytoplanktonanalyzer (HeinzWalzGmbH,Effeltrich,Germany).Thisis anon—destructivemethodinwhichchlorophyllcon— centrationsareusedtoestimatealgaldisappearance ratesandtoestimaterotiferclearancerates(Lfirling andVerschoor,2003).C1earancerates(,ml/ ind./h)werecalculatedusingtheequation: l期M.LORLINGeta1.:AlgaeandBrachionusgrowth73 CR:f\ln(At1)一In(Atife)At)× whereAc0ntrolisthealgalconcentration(aschloro— phyll—a(ChlFn)int~g/LI1)after2daysincontrols, Ar0tiferisthealgalconcentrationintherotifertreat— ments,?,isthetime(h),Vistheculturevolume (m1)andNisthenumberofanimals.ForNmean valueswereestimatedusingthepopulationgrowth rateandtheexponentialgrowthmode1.Algaldisap— pearancerates(Dr,perday)wereestimatedfrom: [In(Chla?盯fe)一In(Chlacontro1)]. r=——————————————?————————————一,where Dristhealgaldisappearancerate(perday),Chla. cantrolisthechlorophyll—aconcentration(int~g/L)af— ter2daysincontrolsandChlarotiferisthechlorophyll— aconcentration(int~g/L)after2daysinpresence ofrotifers. Thenadding0.5mlofLugol'sfixativetoeach wellterminatedtheexperiment.Thenumbersofro— tiferswerecountedunderadissectingmicroscopeand populationgrowthrateswerecalculatedfrom: : ,where.theristher——————————————一, populationgrowthrate(perday),N2isthenumber ofanimalsafter2daysandN1theinitialnumberof rotifers(=6). 1.3Exp.1I:effectofcyanobacteria Asecondexperimentwasruntostudytheeffect onrotifergrowthrateofmixturesofcyanobacteria withS.obliquusthatincluded0%,25%,50%, 75%and100%cyanobacteria(Table1).The cyanobacteriawereharvestedfromone..weekoldcu1.. turesmaintainedatidenticalconditionsasthe cyanobacterialstockcultures(20?andin45/tmol quanta/m/slightina16:8hlight:darkrhythm), S.obliquusfromacultureasoutlinedabove.Each food—typewasrunintriplicateat10/tm'/m1.The experimentwasruninsix—welledcultureplatesin darknessinaclimate—controlledroomat20?1?. After2days,thenumbersofrotiferswerecounted underadissectingmicroscope.Growthrateswere calculatedasoutlinedabove(Exp.I). 1.4Statistics Populationgrowthratesandalgaldisappearance ratesinExp.1wereanalyzedbyone—wayANoVA inthestatisticaltoolpackSPSS10.1.0(SPSS. 2000).InExp.1I,meanpopulationgrowthrates werecomparedrunningtwo—wayANOVAwith cyanobacterialspeciesandfoodcompositionasthe fixedfactors.Forcomparisonsamongtreatmentsand speciesalsoone—wayANoVAwererun.Differences betweenmeansweredistinguishedbyTukey'spost hoccomparisontest(P<0.05).InExp.I,rela— tionshipsbetweengreenalgalparticlevolume,algal disappearancerates,rotiferclearanceratesandpopu— lationgrowthwereestimatedbynon—linearregression usingasigmoidmodel(four-parameterGompertfunc— tion)inthetool-packSigmaPlot2000,version6.O0. 2Results 2.1Exp.I:effectofgreenalgae The10differentgreenalgalfoodspeciesexerted distincteffectsonthepopulationgrowthrateofB. calyciflorus(Fig.1).Theone—wayANoVAindi— catedthatgrowthratesweresignificantlydifferent betweenfoodtypes(F9.2o=155.3,P<0.001). Tukey'spost—hoccomparisonrevealed8homogenous groupsthatweresignificantlydifferent(Fig.1). WiththeexceptionofD.abundansallgreenalgal speciessupportedgoodtoexcellentgrowthin B.calyciflorus.Thehighestgrowthrateswereob— servedwhenS.obliquuswasofferedasfood. Growthasapercentageofthemaximum(on S.obliquus)growthrateforothergreenalgaevaried between19%(D.abundansasfood)and88% (D.subspicatusasfood).Formostspeciesthevari— ancecoefficientswerelow(<7%).however,for D.abundansitwas47%(Fig.1).Greenalgaldis— appearancerateswerealsosignificantlydifferent (F9.2o:41.6,P<0.001)betweenfood.types (Fig.2).Tukey'spost—hoccomparisonrevealed6 homogenousgroupswiththehighestdisappearance rateforS.obliquusandthelowestforD.abundans. C.reinhardtiiandS.pectinatus(Fig.2).Esti— matedclearanceratesdifferedsignificantly(F9.2o 3.05,P=0.018),butTukey'stestrevealedthat onlytheclearanceratesofrotifersfedC.reinhardtii weresignificantlylowerthanthosefeedingon S.acuminatus(Fig.3).Meflnrotiferclearance ratesvariedbetween1.5and7"lmicroliter/rotifer/h (Fig.3).Themeanparticlevolumesofthegreenal— gaeshowedconsiderablevariation(Table2).Be— causethedatadidnotsatisfyassumptionsforANO— VA.separatet.testswererun.Thesetestsshowed thatparticlevolumesofD.quadricaudaand S.pectinatusweresignificantlylargerthanofallthe othergreenalgae,whilethoseofM.minutumand Chlorellavulgariswerethesmallest(Table2). Non—linearregressionanalysesyieldednosignificant relationshipbetweentheparticlevolumeandthealgal disappearancerate(r2di=0.00,F=0.82,P= 0.528),clearancerates(r2adi=0.00,F=0.02,P =0.994)orrotifergrowthrates(r2di=0.00,F= 0.93,P=0.481).Algaldisappearanceratesand rotifergrowthrates(r2di=0.597,F:5.45,P= 0.038)aswellasalgaldisappearanceratesandclear— ancerates(ra2di=0.578,F=5.12,P=0.043) weresignificantlycorrelated,butclearanceratesand populationgrowthratesshowednocorrelation(ad2i :0.190,F=1.70,P=0.265). 74动物52卷 ? 蒸 荟 蓍 善 Greenalgalf0odspecies Fig.1Populationgrowthrate(perday)oftherotiferBrachionuscalyciflorusinshort?chronic assays(2-d)fed withtendifferentgreenalgalspecies Errorbarsindicateonestandarddeviation(n=3).Similarsymbolsabovebarsindicatehomog eneousgroupsthatarenotsignificantly different(Tukey'sposthoccomparison,P<0.05). ? 矗 e 罢 8 l O 一 0.2 - 0-4 - 0.6 - 0.8 c, Greenalgalf0odspecies Fig.2Disappearancerate(perday)oftendifferentgreenalgalspeciesusedasfoodforth