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Ihnttadicnsvftk* ftyalSiwfety vj $ .4fcvr. (WSfc), U6<4j. I»-|32 BRIEF COMMUNICATION A DISSECTION METHOD FOR DETERMINING THE GUT CONTENTS OF CALANOTD COPEPODS loTbheeaexusaemfiunlataiiodnwoniJheresctourddyinogfokt!hgeudtiectosntoefntz*oohpalsanpkrtoovne.d avceirdywwaesllo,nlaygapianrtlayppsaurcecnestslfyulb.eTchauesceopoefpothdesirdildarmge estit/aer. The method cwnnol g>e a complete picture orthe did of a Whenever ».he food boluses could be .seen dearly, anlmnl particularspecie*,assome(boditems«rcmoredelicateman remains (cg. cuticle, setae) were,difficultor uupe'ssible to othersand are more icadity brokenduring masticitioriam! rtcogmseasthey usually wereCrushedandcompactedwithin dissolved by uVgeslivc enzymes. However, many td^ac and the holus The hypochlorite erosion merited was also not animals in the diets or copepodv remain sufficiently emitely SBCCBttfitl. Even though the gut contents could be undamaged, or ha\e adequate identifiable pans that 9fe partlymanipulated,thetact,thatthegutboluseswerenotMty aresssiesstasnmtetnoteon?f)dmiaelticfrbormeagkud?owcno,ntteonattstoawnaalyrseiassoln,~ab?l-\yjijtooids wdiasspefrosuenddmtahdaet baunbibmlael*moatferoixaylrjdeinffpicruoldtutcoedseed.urAisngwetlils,suiet preferabletobasetheanalysisonthecontent*of(hefore-gut ctosion accumulated within Ihe body and ubscuredthegut [wfr 2), hi which much ofthe invested material has been contents,andthaicarehad tobe takentoensurerhai thej*ui lessaffectedbyenzymatic actionandislesscompacted, rhan ixintents themselves were not oxidised. on the compacted and well digested bolus in the Ntnd-gut, In order to overcomethese difficulties wedeveloped the or ihe faecal pellets,. following dissection method, which enables ?hc entire gur 11isnotasimple matter, however, todissectoui (heentire contents, ofboth small and large copepods to be relinked gutcontentsofacopepod, largelybecauseofthemanipulative The contents of both fore- and hund-gutx can be cleanly delicacy required Tl»e contents of the foregul can be extracted withoutinterferencefrommoat surroundingtissues, particularlydifficulttoextractintheirentiretybeumseoftheir dispersed* and permanently mounted. difTuseness. Those whoh«\e us<xla dissectionmethod may Needles ror dissection are made from 2 tm lengths ol thuschoosetoremoveonlyihehindgutbolus5 Themethods 0.5mmtungsten whe, whichisrigidenough toallowsome, . modutted forexaminingeutcontentsaw.iddirectdissection, pressure to be applied during dissection, and may be inthe squashtechnique, thegut contentsare extractedfrom sharpened lo a fine point. For dissecting large copepods a either Uvc or ptescrved specimens by pressing down on a sharpenough point can be produced wiUi a finediamond covcrslipovertheanimal1*f. This method hastheadvantage whetstonete$. "fczetap"). For small coper_odsitisbetterto o(hfabtoitnhdirveildeuaaslinigtetmttsemmaatyerbiealidiennttihfeiegduatnidnoduidmitsapde.rsiAnngotirhesro pNraoNduOcre htehaeteddesoi'.r'eerdapobiunntscbnybuerronseironi,n aeirtrhucciibhlie10moolrtefrny methodistorenderthewholeannua] transparentbyclearing eleetrHysix in 10-20% KOH. Bar electrolysis, the wire is itin lacticacid .orincuparaloreanadabalsamafterpaxvtqg dampedtuernelerminalora6Valtertmlingcurrentelectrical itthrough an alcohol series'* Thedotwhackhere isthatUrn circuit iamicroscope-illuminationtransformerIgsuitable)and gCuMt)cbonetecnlte*aralryenste*endistpheersiendd,ivaninudjieivemnowdheintetmhsegaruetMmuosttelsy dmipapneddoiunttooUfitehKeOflHui1d1,,12a.nIdntehiethdeerptcahsel,otwlh»eicwihretheiswmiorveeids difficult to identify positively or to count. One **ay of insertedgovernswhethertheresultingpointoAhoriandstout overcoming suchdifficulties islo place thespecimenunder orlongand slender Thesharpened nocdk isthenmounted acoversdip and erode away most ofthe tissues with weak' inaholder SaiixfacloryholdersmaybemadefrompHIvices sodium hypochlorite4 Thehypochloriteisthen flushed away tsmall fmgcr^.<peraied drill holders -i\'i !,-nk from model before-thegutcontentsareoxidised-andII isusuallyrxwsible shopslthathavebeenlengthened.IftttCMtoz);bytneaddition 10identirymanyoftheindividualfooditemsbygently moving ora section of brass rod (Fig. 1,cj. The tungsten needle is theuosersliprwhichpartly redistribute*thegutcontents.The hentat a slightangletotheaxisofUieholder (Fig. i.m). tc methodhas.beenusedsuccessfully inAustraliatodetermine aid keeping the needle parallel tothe slide surface .turiog maximumgutmod-particlesitesofthecopepvds idtnffltf'L'LJLl diyseelion. Jeweller's forceps, with finelyihaipcncdprints', tttcaii.Boccktllaminutaandfi rnar/icpfanvinWlcrawang are used for iranvfeTrmg copepods, or their parw, Reservoir*. Diswevtioncan hedone inwater, butir iseasier ifa mote calDaunroiindgcaopsetpuoddysoiJBoctatr&ntiivkoirymabjyort.hrHeeplsaerugdeocnbmtmtviNi:noaunsd mviosucnoiuaanrm|ePdViAujm°iisswursyed$,iiiproabllycvi.-n-.yilt caalncobheolu-steudcttoophmeankoel HRifvmeirbnMeucrkrtalylaflxoeootrlipih)iiin,rowmettermipeodraailrythpeoanbdosvoenmetthehoudpspeorf padedremdanteontthmeoPunVtAstoofstlhaemgucthictounitents. Lignm pink may be gut contents analysis. None Of litem proved entirely satistaciory, particularlv fin revealingthe remainsofanimals •n the gtil contents. This appeared to be OiMinly due to the ^1^^ Urgesue andthickbodiesofthecopepods, andbecause we had availableonly specimenspreservedin49t formalinMid 70% alcohol. The squash method appeared reasonably spaatniiscfualc3tiotrjy tfhorossemaplrlesseprevce!dmienns\,%bfuotrimnallianr)getrheangiurmaclosnt(eantnsd Figa fIi.nAelydipsosienctteidngtnuenegdsltee,ncnoenesdilsetitnigroo.fTahpeincovmimceerhcoiladlilnyg were oftendiiTicult toobserveclearly amongst the massof availablepin \\oc has been extended by the additionofa disrupted esoskeleronand muscle tissue. Clearing in lacric section of brass rod <e), Scale hur - 1cm, 1X1 Dissection isdoneusinnaMetcodissectingmicroscopeat a magnification ofca. 3CI-40X. The copepod is orientated withitsventralsurface,partially inclinedtotheleftandaway from the dissector, and.fora right-handedperson, with its anterior end 10 the left |Hg. 2a). Firstly the Urosome and terminalsegmentoftheprosomeareremovedbycuttingalow dashed line I (Fig. 2a}, andthai theantero-vcntral surface oftheccphalosomeplu>.mourhparts,hycuttingalongdashed line2(Fig.2a). Ifdewed,the6Wmiming limbs<PI-P4)may also be removed (by cutting aloru; dashed line 3, Fig. 2c). This is- not absolutely necessary hut may be useful If the ovariesarewelldeveloped Swollenovariolesmakeremoval ol the gut contents difficult, and removal of(he Hwimming limb?; and remaining ventral surface usually results in Ihe voneotmtant removal ofmuch of the ovary tissue The Ii»stcut ismadewiththeanimalorientated»ashown in Fig. 2a. The body isheld withtheleft needleand thecol made wnh the ri^ht needle by pressing clown firmly along line I, using a forward and backward sawing action ofthe rightneedle ifnecessary. For the second cut, the animal is reorientated lerhcpositionshown in Fije. 2b.TheMimvwi is held with the left needle (near thebaseofthe firstantennae isabailablepoint)andtherightneedlefirmly presseddown overthebody (Fijt 2b), withthepointerftheneedlebetween themaxilltpedsand firstpairofKwimminglimbs. Whilethe right needle is pressed firmly down against ihe slide, die anten>ventral surface and mouthparts are severed by back andforthcuttingmovementsby thetippftheleft needle(Fig. 2b). Theprocedureusually pushesthe fore gutbolusslightly dorso-posteriorly towards the K&fotthe fore gut, and veTy occasionally may result intherear-gutbolusbeingeMftktod figi.*2t.haDtiMrftorrvlaiornigohftguhtancodnetdentpse.rsOorni,entaa.tiLoanteorfalspevciiemwenosf fIfortcheispshaapnpdetnrsa,nstfheerrievdart-ogtuhtebsoelcuosncdadnrobpeorfetPrVieAv.edTwhiethbotdhye BcwkeMo majorshowing the tore gut (fgh)and hind gut shuuld now look as shown in Fig. 2c. If necessary, Ihe <hgb)boluses TheOrxtandseconddissectioncut-lintsarc swimming legs may now be removed by cutting along line shownbydashedlines 1 and2; respectively. B. Orientation 3(Fig. 2cl.pressingdownonthebodywithdierightneedle oftheeopepodanddissectiontechnique lorremovingthe Usingtherorccps,thebody maynowbetransferredtoibe antero-ventralportionoftheprosome. The rightneedlets seconddropofPVA.and heldbytheleftneedlewithventral tpsshelwevaiecsmrelmdi(dileiwn.eigBtaahnlcteegkir*tsoaa-nnpvdoedinftnportrraetlhsbsesmetuodrwvlefeaeiemnreem,nlttywhsehdioomcfwuhntnhiielsalnlpiedufptlehldnceekdealdnaladwgeaatiyfhinrsetstnot sfnioedreeed-lugeuptipFecirogn.mLo2esdnt)l,satanhideeabtnohtedenyricroaorrteaeftnuedldlyf1a8sc0ciarn,agapnredidghtothuet(rFweii*gt.rh-g2tudht)ebroTilghuhest theleft.C.Thebody icadyfortransfertotheseconddmp removed in a similar manner. Finally, the body is removed otPVA. Theoptionalcut-line fo» removingtheremaining with Ihe forceps and discarded. ventral smlace is shownby a dashed line. D, Extraction The food boluses may nowbe carefully leased apart with onwfeheitdlhleeeftohreSeefgnourftcew*bnualtresibol=lWusTIhum>emgbe.ontdlyyipsuhlelleddwciutthwthiethleftthenereidghlte dbtooitarhmeedntueeecrdelcteohsveaeannrsdelaiflptshmiaastlblhealestoevtreortbshehapnseatadhrdecehdse.tdaAnddu1ai0ridmng2m4miomcrrmsomsacsloulpee,er examination. Thegut contentscan befully dispersedby the applicationoflightpressure, andperhapsalsosmallstde-to- side movement*, lo the topofthe cover slip with a needle l.'Mngiheforceps,twodrop*ol PVAarcplacedonaslide. or the forceps. Thecopepodi*pickedupwiththeforcepsandplacedinone Thegutcontentsofbothsmall(eg fit^keltasymmetrica, drop inwhichmostofthedirectiontie. removalofurosome, body lengih ca 1-1.5 mm) and large (e.fj. B. major, body aiiHuo-vcnrralsurfaceandmouthparty)wdone. Vhebody is lengtha. 3-5 mm)freshwatercalnnoidcopepod*;canbeeasily thentransferredtottioseconddropfortheremoval.teasing- extractedusingthisdissectionmethod. Becauseboththegut Ot.it and mounting oflhc gul contents, boUwe*can beextractedand leasedapart we found thaithe Fig. 3. Photomicrographsofanimal remains andalgaeindissected gutcontentsofBoccketto mujvr. A. Duphttta camutta: a, post-abdomen; b, mandible*, c.cuticle and thoracic limbs B. a. ealanoid ropepodite limbs; b. calanuid euoepodiic mandible;c,calunoidntiupliu^;d. Jestu&wJlapatina.C. Ktratnilaprocurva, uuphus atrowed.D AIpo? a.Staurastrwn $p; b, indet. diatom 131 4-* "'* < « *# $- •:% b v •; / .^- •' ->.:' .. , "^ #* ': i \ * .-V^'- '* ' ' ":y-:"" '* ":*!i /' />' '.; // x- c | / '* D 132 method revealsanimalremainsintheguteontentsbetterthan Thedissectedlimbsandotherbody partsremaininginthe thewhole-animalsquashandclearingtechniquesmentioned first drop of PVA can be put to good use. The mouthparts above. It ispossibletopickout bothverysmallanimal remains can bedissectedofftheremnantantero-ventralsurfacemore (e.g. rotifer irophi, Fig. 3). and the diaphanous cuticular readily than theycanbe fromthe wholeanimal. Todothis, remnantsandsetaeot cladoceraasandcopepods(Fig. 3). The the apodemes at the bases ofthe mouthparts are anchored visibilityofcuticularfragmentsisenhanced byiheligninpink solidly against the slide with the left needle, while the staininthePVA. andalsoby theuseofNomarski interference mouthparts are easily dissected off with the right needle. optics. Algae, fungi, detritus and inorganic material in the Moreover, egg sacs removed with the urosome can be used guts are also clearly visible (Fig. 3). It is possible to make forclutch-sizedeterminationsand measurementsofeggsize. quantitative counts of the gut contents. Animals preserved in 70*V alcohol proved to be easierto Wearegrateful toDrI.A.E. Bayly forhishelpfulcomments dissectthanthosein4% formalin. Alcoholpreservationresults on the manuscript and for bringing two recondite papers to in the dissolution of much of the muscle tissue and the our attention. This work was done while JDG was on softeningoftheexoskclcton. 1hebody isthuseasiertosever sabbatical leaveatThe Murray-DarlingFreshwaterResearch and lomanipulatethan whenpreserved in formalin, andthere Centre. Albury. N.S.W.. and the use til facilities there is isless tissue "'rubbish" inthe final gut contents preparation. gratefully acknowledged. 'Fryer, G* (1957) J, Anim. Ecol. 26, 263-268. ''Cannon. H. G. (1941)J. Rov. Microscopical Sec.61,58-59. -Glivtie/., Z. M. (1969) Ekol. Polska. Ser. A. 25, 663-708. "Brady,J. (1965)Bull. World Hlth. Organisation32, 143-144. -'Infante, A. (1978a) Arch. Hvdrobiol. 82, 347-358. ,2Frey, D. G. (1986) Cladoeera analysis. Pp. 667-692 In 4Kobayashi, T. (1991) Aust. I Mar. Freshwater Res. 42. B. F. Berglund (Ed)"HandbookofHoloccncRUacuecology 399-408. and Palaeohydrolugy". (Wiley, London). sHart. R. C. (I9H7> Arch. Hvdrobiol. Ill, 67-82. ^Chapman.JV1. A. & Lewis, M. H. (1976)An introduction "Clarke, N. V. (1978) Freshwal, Biol. 8, 321-326 tothefreshwaterCrustaceaofNewZealand, withachapter 'Yen, J. (19U85) Limnol. Oeeanogr. 30, 577-597. on the Araehnidu bv V. H. Stout. (Collins, Auckland). "Green, J, (1976) Arch Hvdrobiol suppl. 50, 313-400. 'infante,A. (1978b)Trans. Amer. Micros. Soe 97.256-258. JOHN D. GREFN, Dept of Biological Science. University of Waikato, Private Bag 3105. Hamilton, New Zealand RUSSELL J- SHIEL. Murray-Darling Freshwater Research Centre, P.O. Box 921, Albury N.S.W. 2640.