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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
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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.
