Fitness-related attributes and gregarine burden in a non-territorial damselfly Enallagma praevarum Hagen (Zygoptera: Coenagrionidae)

2005·1.5 MB·English

by  Canales-Lazcano, J

Odonalologica34(2):123-130 June1,2005
Fitness-related attributes and
gregarineburdeninanon-territorial
damselfly EnallagmapraevarumHagen
(Zygoptera: Coenagrionidae)
J.Canales-Lazcano¹,J.Contreras-Garduño² andA.Córdoba-Aguilar²*
1CentrodeInvestigacionesBiologicas, Universidad Autonoma del EstadodeHidalgo,
Apdo.Postal69,Pachuca,MX-42001 Pachuca,Hidalgo,Mexico
2*InstitutedeEcologia,Universidad National Autonoma del Mexico,Circuito Exteriors/n,
Apdo.Postal 70-275,MX-04510 Mexico,D.F.,Mexico
[email protected]
ReceivedJanuary20,2004/Revised andAcceptedOctober21,2004
Odon. areusuallyinfected with intestinal gregarines.UsingE.praevarum adults,it
wasinvestigatedwhether:(a)bothsexesdifferedinthedegreeofparasitismandimmune
ability(asshownbymelanization ofartificial,nylon-basedimplantsinthethoracicre-
gion);and,(b)gameteproduction,survival andfatreservescorrelatedwith gregarine
burden. 2 sets ofin-copula(tocontrolforage)animals wereused. One wasused for
estimation ofeggandsperm,and theotherforfatreserves.Survivalwasmonitored as
thetimethatfield-capturedinsectssurvivedunderlaboratoryconditions intheabsence
offood. Gregarineswerecountedbydissection ofthegut.Despitethecasethat 9 9
hadmoreparasitesthan 33,bothsexesdidnotdifferinimmuneability. Eggs,butnei-
ther sperm norfatreservesinboth sexes,correlated negativelywith parasite number.
Survivalin bothsexesalsocorrelated inverselywith gregarineburden. This,however,
heldonlyfor 3 3whentheanalysis wasperformedbysex.Theseresultsarediscussed
intermsofthedetrimentaleffectsofgregarineonZygopterahosts.
INTRODUCTION
Recentstudiesinevolutionary biology havefocussedonthepervasiveaction of
pathogens on differentanimalspecies. Inparticular,studies insome odonatespe-
cieshavefound detrimentaleffects of gregarines on different fitnessrelatedtraits
inbothsexes(e.g. ABRO, 1974,1976;SIVA-JOTHY &PLAISTOW, 1999;COR-
*Towhom correspondenceshouldbe addressed
124 J.Canales-Lazcano,J.Contreras-Garduno &A.Cordoba-Aguilar
DOBA-AGUILAR.2002;HECKER etal.,2002;CORDOBA-AGUILARetal„
2003). Gregarinesareprotozoan,mostlyinsectparasites whoselifecycletakestwo
sequentialroutes; anoninfective(having severalinsects asvectors) andaninfective
stage(within thehost’s intestinetract)(ABRO, 1976). Gregarines havea negative
effectonadultsurvival(e.g. CORDOBA-AGUILAR,2002;CORDOBA-AGUI-
LAR etal.. 2003), eggproduction (CORDOBA-AGUILARetal„ 2003), fat re-
serves inflying muscles (SIVA-JOTHY& PLAISTOW, 1999)andfemalemating
decisions(SIVA-JOTHY. 2000;CORDOBA-AGUILAR etal., 2002).
Hostsare not passive actorsintheinteractionwith parasites. Insectsdevelop an
immuneresponseagainst parasites, whichis basedonamelanin-basedencapsula-
tionof theforeign agent(LAVINE &STRAND, 2002). Thisresponseeffectively
isolatesthe invading agent,preventing its development and killing iteventually.
Theuseofmelaninepossibly affectsotherinsect’sstructures as thiscompound is
alsonecessary forotherfunctions, oneexample beingtheelaborationofmalesex-
ualtraits aimed to increasetheindividual’spotential to attractmates (e.g. SIVA-
JOTHY. 1999,2000; HOOPER etal., 1999).Thehost, therefore,faces thephysi-
ological dilemmaofhowmuchmelanineisdivertedto immuneresponseandhow
muchto otherfunctions. Thissortof tradeoffhas infactbecomeacornerstone
in the evolutionary thinking of the parasite-host interaction(ROLFF& SIVA-
JOTHY,2003).
Intheory, thesexesdifferintheirimmuneability.Asfemalesincreasetheirfitness
by augmenting theirlongevity(to produce moreeggs) whilemalesmaximizetheir
matingsuccess, femalesshouldinvestmoreinimmunitythanmales(ROLFF,2002).
ThispredictionhasbeenconfirmedintheJapanese beetle,Popilliajaponica,inwhich
femalesexhibit higher levels ofimmunity thanmales(TUCKER& STEVENS,
2003). Towhatextentthis applies tootheranimal forms awaitsinvestigation.
Enallagma praevarumisasmalldamselfly.Themalesgatherinlenticwaters, oc-
cupying the plant substratesthatfemalesuse foroviposition. E. praevarummales
donotappear to defendterritories,andtheymate withany femalesthatarriveat
oviposition sites. Similarto otherodonatespecies, a considerablenumberofin-
dividualsare parasitised by gregarines, butnostudy has showntheeffectsofthis
parasitism.Inthispaperwe documentthepossible negativeimpact thatgregarines
bearonmaleandfemaleadultsofE.praevarumexamining threefitnessattributes:
gameteproduction (eggs andsperm), survival, andfat reserves. Furthermore,we
also investigated whether therewas asexualdifferenceinimmuneresponse.
MATERIALANDMETHODS
E.praevarumadultswerecollectedin theVenadosRiverintheMetztitlan reserve, Hidalgo,Mexico
insummersof2002and2003.Fordifferencesinparasitismbetweenthesexes,60malesand69females
ofmiddleage(animalswithshinybodycolour,transparentwingsandnopruinescence)wereused.
Forsexualdifferences in immuneresponse, asampleof69 animals (61 6,8 5)wasrandomlycol-
lected. A 1,5mmlongand0.1 mmwideplasticnylonimplantwasinsertedinthedorsalsideofthetho-
Gregarineburden inEnaUagmapraevarum 125
raxofeachanimal. Previoustoinsertion,theimplantwasdisinfectedbyimmersioninethanol(100%)
for2seconds.The damselflieswereindividuallymaintained insmallvials during28h withL/Dcycles
of 12h. Inusingthenylonimplant,theinsectimmunesystemproceedsrecognizingit asaforeign,po-
tentiallyinfectiveagent,coveringitwithseveralhaemolymphcelllayerswhichgetmelanized aftersome
hours;this melanization isaway toisolate andkill theinvader (LAVINE&STRAND,2002).Given
that immune response in damselfliesis usuallycompletedafter24h (S1VA-JOTHY etal,,2001), 28
h seemsthebest logistic compromiseforthisprotocol.Animalswerehand fedusingoneDrosophila
adult perdamselflyafter 12hofmaintenance. The implantwasthenremoved bygentlydissectingthe
areawhereitwasinsertedbyusingfineforcepsand transferredtoethanol. After2hofre-hydration,
theimplantsalongwiththemelanization werephotographed.Arelative valueofthemelanization area
(which, duetoits darkishnature,appearstotallyvisibleonthetransparentnylonimplant)wasmeas-
uredwith imageanalysis software.
Forgameteproduction,46and 127in-copulafemalesandmales,respectively,werecollected andpre-
servedinethanol(70%;thesameconcentration wasusedfortheotheranimalsdescribed below).These
individuals werelaterdissected and theeggs countedunderastereoscopemicroscope. Thein-copula
condition providedsomereliabilitythat theseanimals hadreached sexual maturityandthushad pro-
ducedgametes.Maleswerecollected andmaintained underlaboratoryconditions (24°CandL/D cy-
cles of12h)during24hto allowforspermproductionincasetheyhadengagedincopulationrecently
andhad,therefore,become spermexhausted.Afterthistime,thesemaleswerepreservedalso inethanol
andthenre-hydratedfor24h.Spermmassesfromtheseanimalsweredissected fromthespermvesicle
andprimarygonopore.These masseswerelaid onaslide,squashedwithonecoverslipheldover two
othercoverslipsateitherside (allcoverslipsof 1mmwide)andvideo captured.Thespermmassareas
displacedwerecalculated onimageanalysissoftware.
Forsurvival, 117(1033, 149) animalswerecollected,taken toaninsectary(1mxlmxl m),and
leftdeprivedoffood.Everytwohours,thenumber ofsurvivinganddeadanimalswascounted.Inevery
checking,deadanimals wereimmediatelypreserved.Countingofgregarineswasmadevia dissection
oftheguttractafterre-hydrationofanimalsfor24hours.
For fatcalculation,49pairswerecaptured.Controllingagein damselfliesforfatcalculation isim-
portant,asrelativelyyoungandold animalsmaybe fatdepletedduetoinitial construction offatre-
servesin theformerandfatdepletionduetosexual activitiesinthelatter(e.g.PLAISTOW& SIVA-
JOTHY, 1996).Fortestingtheeffectofparasitesonfatreserves, therefore,middleagedanimalsshould
be available. We assumed that ouranimalswere ofthis age forthe followingreasons;(a) theywere
sexuallyactive(sothat theywerenotyoung-atleast notinthatstageatwhichfataccumulation isthe
animal’smain function- andnotold- otherwisetheywouldnot copulate);and,(b)thebody colour
(anindicator ofage inthese animals,CORBET, 1999)wassimilaramong individuals Theseanimals
wereimmediatelydecapitated;thelast twoabdominal segmentswerepulledofftoallowforintestine
parasiteextractionandpreserved.Therest ofthebodywasleftinglassinebags for24hinadesiccator,
atwhichtimebodyweightwasrecorded. Thesestructureswereleftagaininchloroformfordryingina
desiccatorfor24h,atwhichtimeanotherweightrecordingwas obtained.Theabsolutedifferencebe-
tweenweightswastakenasthefat quantitytheanimalhadprevious tochloroform extraction(seealso
PLAISTOW&SIVA-JOTHY, 1996).
Spermmassesandmelanization areaswere quantifiedusingSigmaScan Pro 5.6®imageanalysis
software.Whenpossible,parasitedataweretransformedas(rootsquare(value+0.5)) fortheiranalysis
usingparametrical tests.Resultsareshownasmean± STDunless otherwisestated.
RESULTS
There was a differencein the numberof parasites: females(3.05 ± 4.44, N =
69) weremore heavily parasitized thanmales(2.33 ± 4.45, N =60; t=2.12, P=
126 J.Canales-Lazcano,J.Contreras-Garduno &A.Cordoba-Aguilar
0.03, d.f.= 127;Fig. 1).
However, the sexes did
not differintheirdegree
ofmelanization(males,
0.05 ±0.03, N =61; fe-
males,0.04±0.04,N =
8;t =0.69, P> 0.05).
Therewas a negative
relationship between
egg number and para-
siteburden (r =- 0.33,
P < 0.01,N = 46; Fig.
2). Sperm mass, how-
ever,was not relatedto
Fig. 1.Sexual differences ingregarineburden inE.praevarum parasite number (r = -
0.006,P>0.05).
Thesexes didnot differinsurvival(survival formales= 19.89± 8.11, N = 103;
forfemales = 18.42± 4.58,N = 14; MannWhitney U test= 0.71,P> 0.05). Sur-
vivalwas relatedto parasite number(r= -0.32,P< 0.001,N = 117):moreheavily
parasitized animalswereless likely tosurvive. Thisresultheldformales(r=-0.33,
P<0.0006;N= 103;Fig. 3)butnot forfemales(r =0.38,P>0.05, N = 14),when
sexeswere analysed separately.
Gregarinenumberwasnotassociatedto fatreserves inmales(r=0.16, P>0.05,
N =23) orfemales(r =0.02,P>0.05,N =24),andthisresultstillheld whenani-
malswithnoparasites wereeliminated(males, r =-0.14,N = 11;females,r =-0.13,
N=20; bothP>0.05). Evenwhencomparing parasitised(N =31)andnon-para-
sitised(N = 18)animals, theseresultsdidnotchange (parasitized 5.17± 3.69, non
parasitised 4.48 ±3.18; t=0.66,P> 0.05).
Fig. 2.Parasiteburden in relation toeggproductioninE.praevarum
Gregarineburden inEnallagmapraevarum 127
Fig. 3.Parasiteburden in relation tosurvivalin E.praevarum
DISCUSSION
Recentestimates of the evolutionary effects of gregarines in damselflies have
shownaset offitness-relatedsurrogates that are detrimentallyaffected. Wehave
explored someofthesesurrogateswhich,indifferentextents, havebeenalso inves-
tigated inotherspecies.
Immatureistheadultperiod duringwhichtheanimal’smainactivity isfeeding.
This timeis also crucial as theanimal startsbeing infected (ABRO, 1974, 1976).
Whiletheanimalismaturing,itisbecoming moreparasitized. Ifbothsexes share
thesamefeedinghabits(e.g.foraginginthesameplaces,capturingandingestingprey
atthesamerate)and immuneabilities, similarintensity ofparasite infectionmay
befound.Thisis, surprisinglyandunlikeotherstudiesindamselflies(e.g. HECK-
ERetal.,2002)andarthropods in general (SHERIDAN etal.,2000), notthecase
forEnallagma praevarum.Theexplanations why femaleshavemoreparasitesthan
malespossibly are thatfemalesfeedinplaces wheregregarinesare morecommon
orthatfemalesfeedatahigherratethanmalessothattheybecomemorefrequently
infected.Thisis,however,inconsistentwiththefactthatbothsexesapparentlyhave
similarimmuneabilities, shownby thenylon melanization, anotherresultwhich
contradictswhatis theoretically(ROLFF, 2002) andpragmatically (e.g. KURTZ
etal,2000;KURTZ&SAUER,2001;YOURTH etal., 2002)expected. Onepos-
sibility is thatimmuneresponsehadnot beencompleted by thetime theimplant
wasextracted.However,evenifthiswerethecase,stillsomedifferencesshouldhave
becomeapparent.Atpresent, therearenoexplanations forthese divergences.
The fact thatgregarines feedonthedamselflies’ingested foodpossibly signifies
areductionintheenergetic valueofthefoodgathered by theadult.Thismaybe
costly, particularlyforfemales, astheenergetic demandexertedforeggproduction
canbeespecially high.Inlinewiththis,ourresultssuggestthatfemaleshavereduced
eggnumberswhenparasitism incidenceis high.Similarresults havebeenfoundin
128 J.Canales-Lazcano,J.Contreras-Garduno & A.Cordoba-Aguilar
theterritorialdamselfly Calopteryx haemorrhoidalis(CORDOBA-AGUILAR et
al., 2003). Thisimpact mayhavefurthercosts forfemalesif thenumberof eggs
carriedby afemalecanbe detectedby amale.InC. haemorrhoidalis, infact, ithas
beensuggested thatmalesdevotelesstimeforpost-copula guardingwhenfemales
havefeweggs tolay(CORDOBA-AGUILAR etal.,2003).Apparently, malesmay
“evaluate”females’weightandhence adjust howlong they stay withthem.These
observationsshouldalsobe carriedoutinE.praevarum.
Thecostofparasitismobservedinthenumberofeggsmaynot necessarily bethe
consequenceoffoodcompetition betweentheparasiteandthehost.Thereduction
ineggproduction mayalsocomeasaproduct oftheimmuneresponsethatthehost
isproducing againstparasites. Forexample,itmaybethatacompromiseemergesin
usingmelanine, thebasisofthecellularimmuneresponse(LAVINE&STRAND,
2002), fordealing withparasites andnot forotherfunctionsinwhichthis compo-
nentisalsovital(e.g. SIVA-JOTHY, 2000). In mosquitoes, for example,melanine
isthemaincomponentintheeggchorion(JOHNSON etal.,2001). Giventhisone
mayeasily translatethecompromise ofmelaninedestinedforeggproduction and
not immunity. Sucha tradeoffis aninterestingpathway worthofstudy.
Thenegativerelationshipbetweeneggnumberandparasiteburdencontrastswith
thatofmales inE. praevarum,inwhich spermproduction was not apparently af-
fected.Possibly, thefactthateggs seemmoreenergetically costly toproduce leaves
femaleswithmoredifficultphysiological imbalanceswhenfacedwithahigherpara-
siteburden.Asimilarrelationship was observed forfatreserves inbothsexes. This
isparadoxical, given thatparasites havebeen shownto directly affectlipid-based
compounds from invertebratehosts (FOLLY et al,,2003). In fact, thisis exactly
thecost thatdamselflyadultswouldsufferfromparasites ofthelifemodesuch as
thatof gregarines (e.g. SIVA-JOTHY & PLAISTOW, 1999;for anotherexample
withectoparasites seeROLFF etal.,2000). Itwouldbeexpected thatthemaindi-
recteffectisonfatacquisition fromingested food.Asetofexplanations canbeput
forwardfor ournegative results.First, although wetriedto controlfor agediffer-
ences, someanimalswere possibly toooldand hencealready fat-depleted. Ithas
beenshown thatrelatively old damselfly malesare fatexhaustedmostlikely due
to highly costly sexual activities(PLAISTOW &SIVA-JOTHY, 1996). The fact,
however,thattheadultsusedwerecopulating clearly suggeststhatmost,ifnot all,
animalswere far frombeingfat depleted. Second, it maybethatthefateffect due
toparasites hadalreadytakenplacewhentheanimalwascaptured, butgiventhat
damselfliesgetinfectedcontinuously, thecurrent parasite burdenwould nothave
reflectedfat depletion. Thisis not true, as anumberof collecteddamselflieswere
notinfected, andthecomparison oftheseindividualswithinfecteddamselfliesstill
didnotproduceany difference.
Survivalwas negatively correlatedwithgregarine number.Thisresultdoes not
parallelprevious findings inEnallagma boreale, whoseadults survived for longer
periodswhenparasitismwashigher(HECKER etal.,2002). Presumably, intheab-
Gregarineburden inEnallagmapraemrum 129
senceoffood,animalsshouldbecomemorestressedandhencetheeffectsofparasite
startbeclearer.Despite experiencing thesameconditions, ourresultsandthoseof
Heckerandcollaboratorsarestrikinglydifferent.Perhaps onedifferenceisthatthey
monitoredtheanimals’conditionevery 12hours, atimeatwhichmuchmortality
hadalreadyoccurred.Inourexperience,2 hourswas thebest logisticcompromise
toobserve theoccurrence of deadE. praevarumadults.Possibly, intervalsof 12
hoursmaskedthegradual mortalityoccurring withinthisperiod, Interestingly, E.
borealeclearlysurvived forlongerthanE.pmevarum:whiletheformedhadsurviv-
ingrates of8.2± 0.3 daysforfemalesand5.8±0.4 days formales, thelatternever
reacheda single day. Possibly this differencemayreflectpopulation-based adap-
tationsto localchanges instress conditions. Damselfliesin generalare subject to
weatherchanges thatleavethemwithperiods ofnofood(CORBET, 1999).E. bo-
realefromCanadamaybewelladapted to situationslikethisinwhichtheanimal
mayhaveto tolerateseveral daysofbadweather.For E. pmevarumfromcentral
Mexico, inclementperiods ofbadweathermaybeofshorter duration,andthere-
foredamselfliesmaybesubjectto less prolonged intervalswithoutfood.
Interestingly, femalesdidnot showadiminishedsurvival compared to malesin
relationtogregarine burdens.Thisresult canbeinterpretedonthebasisofthepri-
oritiesinenergyallocationthatbothsexeshaveevolved(ROLFF,2002).According
to this, malesshouldinvest inactivities that directlymaximizetheirreproductive
success, whilefemalesshouldinvestineggproduction. Inmales ofnonterritorial
damselflies, animportant componentoflifetimereproductive success islongevity
(CORBET, 1999). This mightbethecase inE. pmevarummalesinwhichthehigh
energetic demanddevotedtosurvivalofwhichmaybeheavilyaffectedby parasites.
Infemales, this shouldnotnecessarilybethecase, aspossibly survivalmaynot be
adirecttargetofselectionwhileeggproduction is.
Insummary, our correlates show only thategg production and male survival
seemaffectedbygregarines, whichisinagreementwithwhatitispredicted byevo-
lutionaryecology theory(ROLFF, 2002).Furthermore, femalesseemedmoreheav-
ily parasitised than males, aresultthatis apparently contradictedby the similar
immuneresponses shownby both sexes. Giventhatthesecontroversialresults are
notunique fornonterritorialdamselflies(e.g.YOURTH etah,2002,HECKER et
ah,2002), thescene isset forfurtherresearch.
ACKNOWLEDGEMENTS
ToSigmaXiforthefinancialsupport.ToGERARDOSANCHEZ-ROJAS, RAULORTIZ-PULIDO
andANAPAOLAMARTINEZ-FALCONforthelogisticandstatisticalhelp.ToMARIOSEGURA-AL-
MARAZforhishelpwithphotography.Fieldworkwasaniceenterprise,giventhehelpofERENDIRA
yAnez-solis.
130 J.Canales-Lazcano,J.Contreras-Garduno &A.Cordoba-Aguilar
REFERENCES
AERO,A., 1974.The gregarineinfectionindifferent speciesofOdonatafromthesamehabitat.Zool.
Scr. 3: 111-120.
ABRO,A., 1976.Themodeofgregarineinfection inZygoptera(Odonata).Zool. Scr. 5:265-275
CORBET,P.S., 1999. Dragonflies:behaviour andecologyofOdonata. HarleyBooks,Essex.
CORDOBA-AGUILAR,A., 2002.Wingpigmentationinterritorialmaledamselflies,Calopteryxhaem-
orrhoidalis:apossiblerelation tosexualselection.Anim. Behav.63: 759-766.
CORDOBA-AGUILAR,A., J.C.SALAMANCA-OCANA & M.LOPEZ-ARAIZA,2003.Female
reproductivedecisions andparasiteburdenincalopterygiddamselflies.Anim. Behav. 66:81-87
FOLLY,E.,N.L.C.E.SILVA,A.H.C.S.LOPES,M.A.C.SILVA-NETO&G.C. ATELLA, 2003. Tri-
panosomarangeliuptakesthemain lipoproteinfromthehemolymphofitsinvertebratehost.
Biochem. Biophys. Res. Comm.310: 555-561.
JOHNSON, J.K.,J.LI& B.M.CHRISTENSEN, 2001, Cloningandcharacterization ofadopach-
romeconversion enzymefromtheyellowfevermosquito,Aedesaegypti.InsectBiochem.molec
Biol. 31: 1125-1135.
HECKER,K.R.,M.R.FORBES&N.J.LEONARD,2002.Parasitismofdamselflies(Enallagmabo-
reale)by gregarines:sexbiasesandrelations toadult survivorship.Cm.J.Zool. 80: 162-168.
HOOPER, R.E.,Y.TSUBAKI& M.T.SIVA-JOTHY, 1999. Expressionofacostly,plasticsecondary
sexualtraitiscorrelatedwithageandconditionin adamselflywithtwomalemorphs.Physiol.
Ent. 24: 364-379.
KURTZ,J.,A.WIESNER,P.GOTZ,& K.P.SAUER,2000.Gender differencesandindividual vari-
ation in the immune systemofthe scorpionflyPanorpavulgaris(Insecta:Mecoptera).Dev.
comp. Immunol. 24: 1-12.
KURTZ, J.&K.P.SAUER, 2001. Gender differencesin phenoloxidaseactivityofPanorpavulgaris
hemocytes.J.Invert.Pathol. 78:53-55.
LAVINE,M.D.&M.R.STRAND,2002. Insecthemocytesandtheirroleinimmunity.Insect Biochem.
moke. Biol. 32; 1295-1309.
PLAISTOW,S.& M.T.SIVA-JOTHY, 1996. Energeticconstraintsandmale matesecuringtacticsin
thedamselflyCalopteryxsplendensxanthostoma (Charpentier),Proc. R. Soc. Land (B)263;
1233-1239.
ROLFF,J.& M.T.SIVA-JOTHY, 2003.Invertebrateecological immunity.Science301: 472-475.
ROLFF,J.,H.ANTVOGEL&I.SCHRIMPF,2000. Nocorrelationbetweenectoparasitism andmale
matingsuccessinadamselfly:whyparasitematters.J.InsectBehav 13:563-571.
ROLFF,J.,2002.Bateman’sprincipleandimmunity.Proc. R.Soc Land. (B)269: 867-872.
SHERIDAN,L.A.D.,R.POULIN,D.EWARD&M.ZUK,2000.Sexdifferences inparasiticinfec-
tionsamongarthropodhosts:isthereamale bias? Oikos88: 327-334.
SIVA-JOTHY,M.T.,1999. Malewingpigmentationmayaffectreproductivesuccessviafemalechoice
inacalopterygiddamselfly.Behaviour 136: 1365-1377,
SIVA-JOTHY,M.T.,2000. Amechanisticlinkbetweenparasiteresistance andexpressionofasexually
selectedtraitin adaraselfly.Proc. R Soc. Land. (B)267:2523-2527.
SIVA-JOTHY,M.T.&S.T.PLAISTOW, 1999.Afitness costofeugregarineparasitism inadamselfly.
Ecol. Ent. 24:465-470.
SIVA-JOTHY,MX,Y.TSUBAKI,R.E.HOOPER&S.J. PLAISTOW,2001.Investment inimmune
function underchronicandacuteimmune challengeinaninsect.Physiol. Ent. 26: 1-5.
TUCKER,T.M.&L.STEVENS,2003.Geographicalvariation andsexualdimorphismofphenoloxidase
levelsin Japanesebeetles(Popilliajaponica).Proc. RSoc. Land. (B)270(Suppl.):S245-S247.
YOURTH, C.R, M.R.FORBES& R.L. BAKER, 2002. Sexdifferences inmelanotic encapsulation
responses (imrnunocompctcncc)in thedamselflyLestesforcipatusRambur. Can. J.Zool. 80:
1578-1583.