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Micron
jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r o n
Sperm
morphology
of
Trichospilus
diatraeae
and
Palmistichus
elaeisis
(Hymenoptera:
Chalcidoidea:
Eulophidae)
Helen
Pinto
Santos
a,
Uyra
Zama
b,
Heide
Dolder
c,
José
Lino-Neto
a,∗aDepartamentodeBiologiaGeral,UniversidadeFederaldeVic¸osa-UFV,MinasGerais,Brazil bDepartamentodeCiênciasBiológicas,UniversidadeFederaldeOuroPreto-UFOP,MinasGerais,Brazil cDepartamentodeBiologiaCelular,UniversidadeEstadualdeCampinas-UNICAMP,SãoPaulo,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received12December2012 Receivedinrevisedform12June2013 Accepted15June2013 Keywords: Spiraledsperm Ultrastructure Parasitoid Insect
a
b
s
t
r
a
c
t
Inthisstudy,thespermmorphologyoftheparasitoidsTrichospilusdiatraeaeandPalmistichuselaeisis (Eulophidae)wasinvestigatedusinglightandtransmissionelectronmicroscopy.Inthetwospecies,the spermarespiralalongtheirentirelengthandmeasureabout130mand195minlength,respectively. Theheadregionconsistsoftheacrosomeandnucleus.Theacrosomeiscomposedofanacrosomalvesicle and,inP.elaeisis,aperforatorium.Inbothspecies,anextracellularlayerinwhichseveralfilamentsare radiatedcoverstheacrosomeandtheanteriornuclearregion.Thenucleiarefilledwithhomogeneous andcompactchromatinandmeasureabout50minlengthinP.elaeisisand20minT.diatraeae.The flagellumconsistsofanaxonemewiththe9+9+2microtubulearrangementspiraledinalonghelix, twomitochondrialderivativescoilingaroundtheaxonemeand,inP.elaeisis,twoaccessorybodies.InT. diatraeaewereobservedtransversestriationsthroughoutthecentralregionoftheaxoneme,whereasthe centralpairofmicrotubuleswasrarelyobserved.InthefinalflagellarregioninT.diatraeae,differentfrom P.elaeisis,onemitochondrialderivativeendswellbeforetheotherandbothendbeforetheaxoneme. Thespermofthesetwospeciesexhibitfeaturesthatdiscriminateonespeciesfromeachother,aswellas characteristicssuggestthatEulophidaeiscloselyrelatedtoTrichogrammatidaeandbothofthesefamilies aremoresimilartoEurytomidaethanAgaonidae.
©2013ElsevierLtd.Allrightsreserved.
1. Introduction
The Chalcidoidea comprise up to a third of parasitic
Hymenopteraspecies(LaSalleand Gauld,1991), formingoneof
themostabundantgroupsofinsects(GrisselandSchauff,1997).
Although some Chalcidoidea are phytophagous or
hyperpara-sitoids, most are parasitoids of other arthropods, thus playing
animportantroleincontrollingthepopulationsofotherinsects.
Therefore,chalcidoidshavebeensuccessfullyusedinmost
biologi-calpestcontrolprograms(Greathead,1986;Neumannetal.,2010;
Polaszeketal.,2012).
Despitetheeconomicandecologicalimportanceofthisgroup
ofinsects,knowledgeofevolutionaryrelationshipsamong
Chal-cidoidearemainsunclear,withisnoconsensusontheplacement
ofseveralfamilies(Heratyetal.,1997;Munroetal.,2011).
There-fore,cleardefinitionsoffamiliesbasedoncharactersthatcanbe
usedincladisticanalysesarenecessary(GrisselandSchauff,1997).
∗ Correspondingauthorat:DepartamentodeBiologiaGeral,UniversidadeFederal deVic¸osa-UFV,Vic¸osa,MinasGerais,CEP36570-000,Brazil.Tel.:+553138993367; fax:+553138992549.
E-mailaddress:linoneto@ufv.br(J.Lino-Neto).
AuthorslikeHeratyetal.(1997,2012)drewattentiontotheuseof
newclassificationsystemstoclarifyrelationshipsbetweenfamilies
andsubfamiliesofChalcidoidea.
Inmanyanimalgroups,includinginsects,spermmorphological
datahavebeencommonlyusedinphylogeneticanalysis(Carcupino
etal.,1995;DallaiandAfzelius,1995;Dallaietal.,2011;Gottardo etal.,2012;Jamieson,1987).Inthis regard,earlierstudieshave
demonstratedthatthestructuraldiversityofspermatozoain
Chal-cidoideacanprovideasystemofcharacters,whichmaybeusedin
combinationwithotherstostudyphylogeneticrelationshipsofthe
groupandresolveuncertaintyatthefamilyandgenuslevels.
Inthispaperwedescribethespermstructureandultrastructure
ofthe parasitoidsTrichospilus diatraeaeand Palmistichuselaeisis
(Chalcidoidea: Eulophidae) to provide spermatological data for
futurephylogeneticanalysesofthisinsectgroup.
2. Materialsandmethods
Adultmale T.diatraeaeandP.elaeisiswerepurchasedinthe
departmentofentomologyattheEscolaSuperiordeAgricultura
LuizQueiroz–ESALQ/USP.
0968-4328/$–seefrontmatter©2013ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.micron.2013.06.006
2.1. Lightmicroscopy
Seminalvesiclesweredissectedandsquashedoncleanglass
microscope slides, followed by spreading, then fixed with 4%
paraformaldehydein0.1Mphosphatebuffer,pH7.2.Afterdrying
atroomtemperature,theslideswereobservedwithanOlympus
BX41 photomicroscopeequippedwith a phase contrastlens to
allowmeasurementofthesperm.Fornucleimeasurements,some
slideswerestainedfor 15minwith0.2g/mlof
4,6-diamino-2-phenilidole(DAPI) and viewedwitha epifluorescenceOlympus
BX60microscopeequippedwithaBP360-370nmexcitationfilter.
TheimageswereanalyzedusingtheImagePro-Plusprogram.
2.2. Transmissionelectronmicroscopy
Seminal vesicles were dissected in 0.1M sodiumcacodylate
buffer,pH7.2andfixedina2.5%glutaraldehydeand0.2%picric
acidsolutionwiththesamebuffer,for24hat4◦C.Thematerial
waspost-fixedin1%osmiumtetroxidesolutioninthesamebuffer.
ThematerialwasdehydratedusingacetoneanembeddedinEpon.
Ultrathinsectionswerestainedwith2%uranylacetateindistilled
waterand0.2%leadcitrateina1Nsodiumhydroxidesolutionand
observedwithatransmissionelectronmicroscope,ZeissLeo906.
Forbasicproteindetection,theethanolicphosphotungsticacid
method(E-PTA)wasapplied.Seminalvesicleswerefixedonlyin
bufferedglutaraldehydesolutionfor24hat4◦C.Thematerialwas
dehydratedinanalcoholicseriesandtreatedenblocbya2%PTA
solutioninabsolutealcoholandembeddedinEpon.
3. Results
ThespermofP.elaeisis(Fig.1A)andT.diatraeae(Fig.2A)are
spiral,longandthin,approximately195mand130minlength,
respectively. They are divided into head and flagellum regions
(Fig.1Aand Fig.2A).Theheadregionconsistsoftheacrosome
andnucleus.Inbothspecies,theacrosomeandtheanteriornuclear
regionarecoveredbyanextracellularlayerinwhichseveral
fila-mentsareradiate(Figs.1C–Eand2C–F).Thisextracellularlayeris
longerinthespermofT.diatraeae(Fig.2C)spermthanP.elaeisis
(Fig.1C),measuringaround2mand0.3m,respectively.Itin
P.elaeisiscontainstworegions,withtheinnerregionbeingmore
electron-dense(Fig.1C).Thislayerandallthefilamentsarisingfrom
itareE-PTApositive,inbothspecies(Fig.2F).
Theacrosomeisverysmallinbothspecies,measuringaround
0.1minlength.Itiscomposedofanacrosomalvesicleand,in
P.elaeisis, a rod-shapedperforatorium. Thislatter hasthebase
insertedintoa smallcavityatthenucleartipandiscoveredby
theacrosomalvesicle(Fig.1C).
Inbothspeciesthenucleusisfilledwithhomogeneously
com-pacted chromatin(Figs.1F–H and2G–J).In P.elaeisisit islong,
measuringapproximately50minlengthand0.2matthebase,
graduallytaperingtowardthetip(Fig.1AandB).InT.diatraeaethe
nucleusmeasures20minlengthand0.4mindiameteratthe
base.Ittapersabruptlyfrombasetonearhalf(Fig.2AandB).
Inthetransitionregionofthenucleus-flagelluminbothspecies,
thenucleusisconnectedtotheflagellarstructuresbyacentriolar
adjunct(Figs.1F,H,Iand2H–K).Thecentriolaradjunctoverlaps
boththebaseofthenucleusandtheaxonemeanteriorregion
(cen-triole).InP.elaeisistheoverlappednuclearareaisgreaterthanin
T.diatraeae(Figs.1Fand2I,however,inT.diatraeaethecentriolar
adjunctsurroundsalmosttheentirenuclearbase(Figs.1Hand2J).
The flagellum consists of an axoneme, two
mitochon-drial derivatives, and in P. elaeisis, two accessory bodies
(Figs. 1J–M and 2H, I, L,N).Theaxoneme shows a
microtubu-lar arrangement of 9+9+2, nine accessory microtubules, nine
peripheralpairsandonecentralpair(Figs.1Nand2N).However,
inT.diatraeaethecentralpairwasdifficultlyobserved(Fig.2L–O),
whiletransversestriationswereobservedthroughoutthecentral
regionoftheaxoneme(Fig.2H,I,PandQ).Thesestriationsare
compoundbydenseandpalelineswithregularspacesbetween
theseones,measuringamongdenselinesabout38nm(Fig.2I).
Theaxonemeisalsocoiled,soallpairscannotsimultaneously
besectionedatrightangles(Figs.1M–Pand2L–O).Attheflagellar
posteriorendtheaxonemeisthelasttofinish,andtheaccessory
microtubulesarethefirsttodisappear(Figs.1O,Pand2L,M).InT.
diatraeaesperm,whentreatedwithethanolicPTA,anEPTA-positive
materialisclearlyobservedaroundandinsidetheaxoneme,butthe
microtubulesareEPTA-negative(Fig.2F).
Themitochondrialderivativesarearrangedinaspiralalongthe
entireaxoneme.Incross-sectiontheyareovalshapedwithequal
areasandmuchsmallerthantheaxoneme(Figs.1L,Mand2H–P).
In the final portionof theflagellum, themitochondrial
deriva-tivesofP.elaeisisendapproximatelytogetherandneartheend
oftheaxoneme,sinceflagellasectionedwithonlyone
mitochon-drialderivativeorjusttheaxonemerarelyareobserved(Fig.1L).
InT.diatraeaeonederivativeendswellbeforetheotherandboth
endbeforetheaxoneme,becauseFig.2Lshowsvariousflagellain
crosssectionwithonlyonemitochondrialderivativeoronlythe
axoneme.
InP.elaeisis,theaccessoriesbodiesareelectron-dense,located
betweenthemitochondrialderivativesandtheaxoneme,andin
cross-sectionsareovalwithaverysmalldiameter(Fig.1JandM).
TheywerenotobservedinT.diatraeae.
4. Discussion
ThespermofP.elaeisisandT.diatraeaeexhibitbasicmorphology
similartothatofotherChalcidoidea,forexample:(1)spiralsperm;
(2)thepresenceofanextracellularlayercoatingtheacrosomeand
partofthenucleusfromwhichseveralfilamentsareirradiated;(3)
thetwomitochondrialderivativeshavereducedandequal
diam-eters;and(4)attheendoftheaxoneme,accessorymicrotubules
finishfirst(Britoetal.,2009;Fiorilloetal.,2008;Lino-Netoetal.,
2000;Quickeetal.,1992;Silva,2010).Meanwhile,thereareother
featuresthatdiscriminatethesetwospeciesofotherschalcidoids.
Althoughthepresenceofanacrosomeis commonin
Chalci-doidea,itsmorphologycanvarywidely.Itmaybeverysmall,e.g.
Mellitobia(Britoetal.,2009),orabsent,asinPegoscapus(Fiorillo
etal.,2008).Also,therearespeciesinwhichthevesicleacrosomal
ispresentbuttheperfuratoriumisnotobserved,asinTrichogramma
pretiosum and Trichogramma dendrolimi (Lino-Neto and Dolder,
2001;Lino-Netoetal.,2000),MellitobiaaustralicaandMellitobia
hawaiiensis(Britoetal.,2009),inIdarnessp.1andsp.2(Silva,2010)
andinT.diatraeaestudiedhere.Likewise,theextracellularlayer
wasnotobservedinsomechalcidoidsas,forexample,Idarnessp.1
andsp.3(Silva,2010).However,thislayer,withfilamentsradiating
fromit,isnotuniquetochalcidoids,asobservedinIchneumonoidea
(Moreiraetal.,2010;Quickeetal.,1992)andCynipoidae(Newman andQuicke,1998).
Thecentriolaradjunct,observedinthesetwoeulophids,occurs
inmany insectordersincludingHymenoptera.In chalcidoids,it
canoffergooddiagnosticcharactersatthefamilyandsometimes
genuslevel.Forexample,inEurytomidae(Lino-Netoetal.,1999),
Trichogrammatidae(Lino-Netoetal.,2000;Lino-NetoandDolder,
2001)andEulophidae(Britoetal.,2009)thisstructureoverlapping
onlythecentrioleregion(about0.5m).However,in
Eurytomi-daeitoverlapsthenucleusbyamuchlargerdistance(about8m)
than in Eulophidae and Trichogrammatidae (lessthan 0.5m).
In theAgaonidae, Pegoscapus (Fiorillo et al., 2008) and Idarnes
Fig.1.Light(AandB)andtransmissionelectron(C–Q)photomicrographsofspermatozoafromP.elaeisisinlongitudinal(C,GandK)andtransversal(D,E,G–JandL–P) sections.(A)Sperminphasecontrast.Thearrowindicatesthelimitbetweenthehead(h)andtail(t).(B)Nucleus(n)stainedwithDAPI.(C–E)Sectionoftheheadregion showingtheacrosomalvesicle(a),theperforatorium(p)insertedinadeepnuclearcavityandthefilaments(f)radiatingfromtheextracellularsheath(s).(F)Sectionof thenucleus–flagellumtransitionregionshowingcentriolaradjunct(ca),centriole(c)andaxoneme(ax).(G)Sectionoftwonuclei.(H–J)Sectionsofthenucleus–flagellum transitionregionatdifferentlevels.Notein(J)theanteriortipofthemitochondrialderivatives(m).(K)Alongitudinallysectionedflagellumshowingthemitochondrial derivativescoilingaroundtheaxoneme.(L)Spermatdifferentlevels.Notethepresenceoftwomitochondrialderivativesinallflagella.(M)Middleregionoftheflagellum showingtheaxonemeformedofdoubleexternalmicrotubules(curvedarrow),acentralpair(smallarrows),andaccessoriestubules(arrowhead).Thelargearrowsindicate thetwoaccessorybodies.(N–P)Sectionsshowingtheflagellatipswhereonemitochondrialderivative(m)terminatesbeforetheotherandbothbeforetheaxoneme.Inthe latter,theaccessorymicrotubules(arrowheads)arefirsttofinish.Bars:AandB=10m;C–JandM–P=0.1m;KandL=0.5m.
ItoverlapsonlythecentrioleregioninPegoscapus,butinIdarnesit
runsparalleltomitochondrialderivativesbyatleast4m.
Althoughtheaxonemewith9+9+2microtubulesisobserved
inmanyinsects,changesinthispatternarecommonlyobserved,
whichmayrepresentimportantcharactersforsystematicsofthese
organisms.Forexample,transversestriationsalongthecenterof
theaxoneme,asoccurinT.diatraeae,haveneverbeenobservedin
anyotherHymenopteraincludingchalcidoids,thisstriationsmay
constituteauniquecharacteristicforthespeciesorgenus.
How-ever,whetherthesestriationsaremodifications ofone existing
flagellarcomponentsasthecentralsheath,andhowtheycombine
withotherelementsinthecentralregionoftheaxoneme,areissues
thatneedfurtherinvestigation.
In most chalcidoids, including these two species, accessory
microtubulesarethefirsttofinish,followedbythecentralpair
andtheperipheraldouble.However,inPegoscapus(Fiorilloetal.,
2008),thecentralpairisthefirsttoend.Thelastperipheral
dou-bleendingdifferentiateschalcidoidsaswellastwoichneumonids
species(Moreiraetal.,2010),oftheAculeata,inwhichthelastto
finishareaccessorymicrotubules(seeZamaetal.,2005).
Inthesetwoeulophidspecies,themitochondrialderivatives,
incross-section,aresymmetrical,slightlyovalandsmallerthan,
andveryclosedto,axoneme. Thesesamefeaturesarethesame
ones showed in Eurytomidae (Lino-Neto et al., 1999) and
Tri-chogrammatidae(Lino-Neto etal., 2000;Lino-Neto and Dolder,
2001),indicatingthatthesethreefamiliesarecloselyrelated.
How-ever,mitochondrialderivativesofM.hawaiiensisandM.australica
(Britoetal.,2009)areasymmetric,possiblyrepresentingaunique
characteristicforthiseulophidgenus.InPegoscapus(Fiorilloetal.,
Fig.2. Light(AandB)andtransmissionelectron(C–Q)photomicrographsofspermatozoafromT.diatraeaeinlongitudinal(C,D,G–I,PandQ)andtransversal(E,FandJ–O) sections.(A)Sperminphasecontrast.Thearrowindicatesthelimitbetweenthehead(h)andtail(t).(B)Nucleus(n)stainedwithDAPI.(C–F)Sectionoftheheadregion showingtheacrosomalvesicle(a)supportedatthenucleartip(arrow)andthefilaments(f)radiatingfromtheextracellularsheath(s).(G–K)Sectionsofanuclearregion (G)andnucleus-flagellumtransitionshowingcentriole(c),centriolaradjunct(ca)andmitochondrialderivatives(m)coilingaroundtheaxoneme(ax).Notethepresenceof transversestriations(arrows)throughoutthecentralregionoftheaxoneme.(L–Q)Sectionsofflagellaatdifferentlevels.Notethatitiscommonflagellawithonlyone(large arrow)orno(arrowheads)mitochondrialderivative.Alsonotethatthecentralpairofmicrotubules(arrowsinO)israrelyobservedandtheaccessorymicrotubulesarefirst tofinish(M).Bars:AandB=10m;C–FandI–O=0.1m;G,H,PandQ=0.5m.
aboveonlybybeingslightlyasymmetrical.InIdarnes(Silva,2010),
theyare larger in diameterand completely surrounded bythe
axoneme,indicatingthatthesetwoagaonidgeneraaredistantly
related.
Accessorybodiesareveryreduced(asinP.elaeisis)orpossibly
absentinsomespecies(asinT.diatraeae),characteristicsthat
dif-ferentiatechalcidoidsfrommostHymenoptera(Zamaetal.,2005;
Araújoetal.,2009;Moreiraetal.,2012).
Inconclusion,themorphologicalcharacteristicsofchalcidoid
spermindicatethatEulophidaeiscloselyrelatedto
Trichogram-matidae,aswasobservedbyHeratyetal.(2012),andbothofthese
familiesareclosertoEurytomidaethanAgaonidae.InAgaonidae,
Pegoscapusspermaremoresimilartothosethreefamiliesabove
thanIdarnes.
Acknowledgments
The authors thank the Nucleus of ElectronMicroscopy and
MicroanalysisoftheUFV,ElectronMicroscopyCentersofthe
UNI-CAMPandtheUniversityofBrasília(UNB),andthetwoanonymous
reviewerswhoprovidevaluablefeedbackonthemanuscript.This
researchwassupportedbyCAPES,CNPqandFAPEMIG.
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