The female seminal receptacle and accessory glands in Pyrrhosoma nymphula (Sulzer) (Zygoptera: Coenagrionidae)

2004·2.8 MB·English

by  Åbro, A

Odonatologica33(3):237-244 September1,2004
Thefemaleseminalreceptacleand
accessory glandsinPyrrhosoma nymphula(Sulzer)
(Zygoptera: Coenagrionidae)
A.Åbro
DivisionofAnatomy,DepartmentsofBiomedicine,University ofBergen,JonasLiesvei91,
N-5009Bergen,Norway
ReceivedJune 12,2003/ RevisedandAcceptedOctober15,2003
Sperm,transmittedtothe2asindividualfilamentous cellssuspendedinaliquidmedium,
aredischargedintoathick-walled pouch,thereceptaculumseminis,onthedorsumofthe
vaginalcanal.Spermatozoasoonappearconcentratedinasingle,smaller,pear-shapedac-
cessory sac,thespermatheca,attachedtothereceptaculum-vaginajunction.Particularcells
in thewall ofthe accessory sacsecrete amaterial thatisthoughttobe addedtothesperm
concentrate.Thepurposeoftheaccessory sacistoserveasastoreofspermatozoaforuse
infertilization. Apairofposterioraccessory glandshas eachanefferentductthatopens
intothe distalregionofthevaginalcanal; theseductsareprovidedwith anelaborate mus-
cularapparatusprobablyservingasapump;infreshmaterial,efferentlydirectedperistaltic
waveshavebeenobserved. Theglandsarepresumedtocontributetotheinvestmentofthe
eggs.Theapicaldomainsoftheglandularepithelialcellscontainintraplasmicassemblages
ofmultiplicatingbacteroids. Theyarelikelytobetransferred totheooplasmandthereby
transmittedtoanewgeneration.
INTRODUCTION
Mostzygopteranfamiliespossess asingle spermatheca attachedtothedistalpartof
abursacopulatrix (SIVA-JOTHY, 1987).Examinationofrepresentatives oftheCoen-
agrionidae havenotrevealedanykindofaggregations (bundling orclustering)oftheir
filamentousspermcellseitherwithinthevasadeferentiaofthemaleorwhendispersed
ininsectsaline(ABRO,2000). Thespermbundlesfoundinthemalepassageways ofthe
anisopteran Aeshnajuncea (L.)havebeenstudiedastostructuralchanges aftertransfer
tothefemale genitaltract (ABRO, 2004).
Basedonfemalespecimens ofthecoenagrionid Pyrrhosoma nymphula (Sulz.),cap-
turedat varioustimes after mating,the present communicationreportson structural
featuresofthefemalegenital tractandthestorageof sperm;includedis a description
oftheposterior accessory glandsofthefemalereproductive system.
238 A.Abro
MATERIALANDMETHODS
Adultfemales,capturedatabreedingsitenearBergen,westernNorway,hadfixativeinjectedintotheab-
domenwithafinehypodermicneedle;the seminalreceptacle,withadditionalstructures, wasexcisedina
largervolume ofthe samefixative.In addition, somespecimensanaesthetizedincarbon dioxide had fresh
seminalreceptacleandaccessoryglandsdissected ininsectRinger'ssolution.
Thefixativewasmadeupof3%glutaraldehydein0.2Mcacodylatebuffer(pH7.3)with0.17Msucrose
added;postfixationtookplaceina 1%solutionofosmiumtetroxideinthesamebuffer.Tissuesdehydrated
throughagradedseriesofethanolandcleared inpropyleneoxide were embedded inepoxy resin, and sec-
tionscutwithdiamond knives.Semithin plasticsections forlightmicroscopy,from material processed, for
electronmicroscopy,werestainedina1%solutionoftoluidineblue.Ultrathinsectionsforelectronmicro-
scopywerecontrastedwith uranylacetateand leadcitrate.
Fig. 1.Distalpartsoftheadultfemalegenitaltract: lateral(left)anddorsal(right)views,basedondissections
ofseveralspecimensfixedandstoredinbufferedglutaraldehydesolution.Surroundingmusculatureisomitted,
—agl:accessorygland;- be:accessorysac; —Gg:positionoftheeighthabdominal ganglion;- od:oviduct;
— st;receptaculumseminis;— v:vaginalcanal;- 7-10:abdominalsegments.[Scalebar2.0mm].
Seminalreceptacleandaccessory glandsin Pyrrhosomanymphula 239
RESULTS
Connectedtothevaginalcanalbyashortbroadjunctionorneck,thereisaratherthick-
walledpouch, thereceptaculum seminis, cuticle-linedandwithinits wallspossessing
muscle-fascicles, running lengthwiseandcrosswise (Figs 1,2).Betweenthereceptacle
andthedorsalvaginal wall,asingleoblong pear-shapedaccessory sac isattached.Both
Figs2-5.Receptaculumseminis(St) anditsaccessory sac (be):(2)crowdedwithsperm,excised fromthe
vaginalwall (atbrokenline).Fasciclesofcontractilemusculaturerunninglengthwiseandcrosswisein the
receptaculumwall appearwhen viewedintransmittedlight.Unstainedwhole-mount ofglutaraldehyde-fixed
tissues.[Scalebar200pm]; — (3)longitudinallycut(approximatemid-sagittalsection),excisedatbroken
line.Notenarrowduct(arrow)tothesperm-repletedaccessorysacandresidualsperminthereceptaclelumen,
c;cuticle.Semithin section/toluidine blue. [Scalebar200pm]; —(4)transversely cut.Noteresidual sperm
inthereceptaclelumen.Semithinsection/toluidine blue. [Scalebar200pm]; — (5)fresh spermcellsfrom
receptaculumseminis,here dispersedininsectRinger’ssolution.Arrows indicatepositionofacrosomein
front ofthenuclearhead.Unstainedwhole-mount/phasecontrastillumination.[Scalebar50pm].
240 A.Abro
receptacle and ac-
cessory sac are
cuticle-lined, and
have beneath the
cuticle a simple
layer of low epi-
theloidcells con-
taining prominent
pigment granules
(Figs 6, 7); their
lateral borders
are strongly inter-
digitated.There is
alsoa broad mus-
cle layer (Figs 6,
7). Outermostare Figs6-7.Survey electronmicrographsdemonstratingthewallof(6)the seminal
investing, adven- receptacle,and(7)theaccessory sac. — ad: adventitiouscell; — c:cuticle; —
titious cells, some mu: layerofmusclefibres; - pe:pigmentedcells. [Scalebars 10pm]:(6)the
of which contain receptaclewall fromafemale specimenjustmated;in the cavityarenumerous
individual spermcells(sp). —tr;tracheole; —(7)theaccessorysachasathicker
small tracheoles.
wall. Beneath the cuticular intirna is alayer ofrecesses (arrows) filled with a
In the accessory homogeneoussubstancethatstainspositivelyaccordingtothePASreaction.Within
sac, just beneath thecuticle,betweenthecuticularintirnaandthepigmentedcells,areseenpartsof
the cuticular inti- flattenedcells(asterisk)which mayproducethissubstance.
mais astratumofrecesses withinwhichasubstancestainsslightly positive according
tothePASreaction.Thismaterialappearstobesecretedby flattenedcellssuperficial
tothe pigmented cells(Fig. 7). Theoverallthickness of the wallis greaterinthe ac-
cessory sac.
Infemalestakenduringmating,spermcells, notenclosedinbundles,werefoundde-
posited intheseminalreceptacle (Fig.6). Individualspermcellsweresuspended ina
flocculent,somewhatviscous,seminalfluid(Fig.5). Quitesoonaftermating,filamentous
spermcellswere concentratedwithintheaccessory sac,whichisconnectedtothesemi-
nalreceptacle viaanelongated narrow duct(Fig. 3).Cell debrisandprecipitates were
leftbehindinthereceptacle (Figs 3,4). Apparently, thespermare storedintheacces-
sory sacuntiltheeggsareready forfertilizationpriortooviposition.
Inadditiontotheseminalreceptacle andits accessoiy sac,apairofflattened,curved
lobulatedposterior accessory glands, situated inthe ninthabdominalsegment lateral
tothehindgut, are connectedtothedistalregionofthevaginal canal(Fig. 1).Eachof
themis provided withashortefferentductthatopensclosetothegenitalaperture(Fig.
8). The glandularcavity is linedwith a cuticlemaintainedby asimple columnarepi-
theliumonabasallaminaandformsacini.Thecuticle, of variablethickness, is lami-
natedand canalized(Fig. 10). Beneaththeepithelium thereare visceralmusclefibres
arranged loosely infascicles, ofteninterwovenwithsurrounding somaticmusculature
SeminalreceptacleandaccessoryglandsinPyrrhosomanymphula 241
Figs8-10. (8)Thefemale
accessory glandwith ef-
ferent duct cut offnear
outlet to vagina (bro-
ken line). Tissues fixed
in buffered glutaralde-
hydesolution, unstained
whole-mount viewed in
transmitted light.[Scale
bar400(tm]; —(9)elec-
tron micrograph of the
epithelial stratum of a
glandularacinusshowing
liningcuticle(c),laminat-
edandcanalizedandwith
surface sockets. Beneath
the epitheliumare some
adventitious cells (mu),
possibly contractile, and
associated fatcells (fc).
— N; epithelialnucleus.
[Scale bar 10 pm]; —
(10) electron micrograph
showingcuticle andapi-
caldomain ofthe glan-
dularepitheliumwith in-
traplasmatic bacteroids
(arrows)residingbeneath
thecuticle(c). — m: mi-
tochondria;— N:epithe-
lial nucleus. [Scalebar 5
pm]; — Inset: bacter-
oidrod,justdividedinthe
hostcell cytoplasm.[Scale
bar 1pm].
andadventitiousfatcells(Fig. 9).
Thecytoplasmoftheglandular epithelialcellscontainsnumerousmitochondriaand
Golgi bodies and,in its apical region, several conspicuous intraplasmatic bacteroids,
whichappeartoundergo multiplication(Fig. 10,inset).Clustersofbacteroidsareoften
seenincloseproximityto Golgi membranes,glycogen depositsandlipidinclusions.
Theglandularefferentductexhibitsacuticle-lined,folded-up lumensurroundedby a
simplelayerofepitheloidal cells(Fig. 11).Thesehaveinterdigitatedlateralbordersand
arefurnishedwithintraplasmatic arraysofmultiplemicrotubulesinvaryingorientations
(Fig. 12).Theapical borderofthesecellsadjoining thecuticleisregularly pleatedwith
membranouspatches resembling hemidesmosomes.Outsidetheepitheloidal cellsis a
sheathoftightly-packed, striatedmuscle cellsarrangedaroundtheduct,constituting a
tunicamuscularis.Muscleandepitheloidcellsarecloselyattachedwithoutabasallamina
between. Outermostisaninvestmentofadventitiouscells,somewithacleartracheolar
242 A.Abro
Figs 1l-12.Electronmicrographs
oftheefferentductofthefemale
accessory gland.Theborderline
betweentheepitheloid(E) and
themusclelayer(mu)isindicated
byanasterisk.—L:lumen:(11)
cross section, showing tissue
coats surrounding the cuticle-
-linedlumen;—ad: adventitious
cells;— E:epitheloidlayer; —
nm: neuromuscular junction.
[Scale bar 20 pm]; - (12)
tissueinvestments.Arrowheads
pointtomembraneous patches
atthetop ofpleats apicallyon
epitheloidcells. — ax: nerve
axon; — bl: basal lamina; —
C:cuticle; —mt:microtubules;
- N:nucleusofepitheloidcell.
(Scalebar5pm].
supply, on abasal lamina.Betweenthislayer andthe musclesheathare seenseveral
nerveaxonsandneuromuscularjunctions (Figs 11, 12).Infreshmaterialdissectedout
ininsectsaline,theductsdisplay tiny waves ofefferently directedperistalsis.
DISCUSSION
Thefemalegenital ducts andaccessory structures of insectsexhibitgreatdiversity
inarrangementofdetails, and thesituationbecomescomplicated by development of
variousout-pocketings (CHAPMAN, 1974).Thereceptaculum seminis andaccessory
sacandaccessory glandsareallectodermalstructures, derivedbyinvagination fromthe
surfaceintegument, andhenceare linedwithcuticle.Libellulidshavepairedaccessory
SeminalreceptacleandaccessoryglandsinPyrrhosomanymphula 243
sacs attached tothe single receptacle dorsal tothe vagina. A corresponding situation
hasbeendescribedfromtheaeshnidAeshnajuncea (ABRO, 2004). However,among
libellulidgenerathereappearstobeconsiderablevariationinthestructureofthefemale
seminalreceptacleanditsaccessory sacs(SFVA-JOTHY, 1987).Theseminalreceptacle
inPyrrhosoma nymphula is consideredhomologous with thereceptacle inA.juncea
and,likewise,thesingleaccessory sacofZygoptera mightbehomologous withthepair
ofaccessory sacs inAnisoptera.
In Pyrrhosoma,thepigmented epithelia ofthereceptacle andtheaccessory sacare
thoughttopromotetheaccumulationofheatfromthesun, andthepigmentation isalso
presumedtoprotectagainst harmfulradiation.Howthespermcellsdischarged intothe
seminalreceptacle are subsequently transferredtotheaccessory sacandconcentrated
withinitisnotknown. Themotilityofthespermatozoaalonecannotexplainthenormal,
ratherfast fillingoftheaccessory sac; theorgansthemselvesare presumedtoplay an
importantrole. Rightly the accessory sac ofPyrrhosoma nymphula couldbetermed
aspermatheca. Sperm deposited inthe femalereceptaculum ofthe mosquito rapidly
locomoteandassembleinthe spermathecae (JONES &WHEELER, 1965).
Intheaccessory sac/spermatheca, thecellssituatedbetweenthepigmented epitheli-
umandthecuticularintimaproduce aPASpositivesecretionwhichislikelytobesup-
plied tothestoredsperm.Thesesecretory cellsprobably functionas unicellularglands
ratherthanasecretory epithelium. Spermatozoa maybestoredintheaccessory sac in
viableconditionforlong periods andexpelledfromitfollowingovulation.InPyrrho-
somaithas not beenpossible,on morphological characters, to decidethecomposition
oftheejaculate andthefemalefluids.Nourishmenttospermcellscouldbesupplied by
the maleintheseminalfluid.
Theintracellularbacteroidsdetectedinthepresentmaterialappearsimilartotheen-
doplasmicbacteroidsfoundinhaemocytesoflarvalEnallagma cyathigerum inconnec-
tion withwoundhealing (ÄBRO, 1999). Judgedby morphological features alone,the
bacteroidbodiesresemblerickettsia. Theyappeartocompriseabacterialendosymbio-
sisand havebeenobservedinotherzygopterancellsas wellas withinthedeutomer-
iteplasmaof eugregarine trophozoites, parasitesinthezygopteranalimentary tract.In
Pyrrhosoma females,itappears thatthebacteroidrods are releasedinto thecavity of
theposterioraccessory glands andeventually reachthevaginalcanalwherethey indue
course can entertheooplasm. This mightrepresentamodeoftransmissionby means
ofwhichthemicro-organisms arepassed fromonegeneration tothenext.Thepresent
bacteroidsresemblecorrespondingendoplasmic rodsobservedinfleas(ROTHSCHILD
etal., 1986). Incockroaches numbersofbacteroidsexistbetweenovarianfolliclecells
andthesurfaceofdevelopingoocytes; theyarebelievedtoentertheeggs (MILBURN,
1966;SMITH, 1968).Intracellularsymbiotic bacteriafoundintermitesandcockroaches
are acquired viatransovarialtransmission (SACCHIetal.,2000).
Bacteroids foundinzygopterans maybelong tothe genusWolbachia, cytoplasmi-
callyinheritedrickettsia-likebacteria,widespreadandcommonininsects, oftenseenin
reproduction tissues(WERREN, 1997;WERREN& WINDSOR, 2000).
244 A.Abro
Thefemaleaccessory glandsininsects probably serveseveralfunctions.Sometimes
theyare calledcolleterialglands, as they addsomething totheegg-shell (WIGGLES-
WORTH,1972).Sincethereisaratherpoorvisceralmuscularsupplyclosetotheglan-
dularaciniinPyrrhosoma,thecomplex muscularapparatusarrangedroundtheefferent
duct,andthe occurrence ofperistaltic movements along thistubule, suggestapump-
ingfunctionforthegland.Theabundanceofmicrotubulesintheepitheloid cellslikely
meetscytoskeletal requirements ofchanges incellshape.
REFERENCES
ÄBRO,A., 1999. Reactionstolegexcision inlarvaeofEnallagmacyathigemm(Charpentier)(Zygoptera:
Coenagrionidae).Odonatologica28:117-125.
ÄBRO,A.,2000. Spermclusters inZygoptera(Coenagrionidae,Lestidae,Calopterygidae).Odonatologica
29:51-56.
ÄBRO,A.,2004.Structureandfunctionofthemalespermductsandfemalesperm-storageorgansinAeshna
juncea(L.)(Anisoptera;Aeshnidae).Odonatologica33: 1-10.
CHAPMAN,R.F., 1974.Theinsects:structure andfunction.[2ndedn],EnglishUniv. Press,London.
JONES,J.C.&R.E.WHEELER, 1965.Studieson spermathecalfillinginAedesaegypti(Linnaeus). 1.De-
scription.Biol.Bull. 129: 134-150.
MILBURN,N.S., 1966.Finestructureofthepleomorphicbacteroidsinmycetocytesandovariesofseveral
generaofcockroaches. J.InsectPhysiol. 12: 1245-1254.
ROTHSCHILD, M„Y.SCHLEIN &S.ITO, 1986.Acoloratlasofinsecttissuesviatheflea.Wolfe,Lon-
don.
SACCHI,L„C.A.NALEPA,M.LENZ,C.BANDI,S.CORONA,A.GRIGOLO&E. BIGLIARDI,2000.
Transovarial transmissionofsymbioticbacteriainMastotermesdarwiniensis(Isoptera;Mastotermin-
itidae)ultrastructural aspectsandphylogeneticimplications.Ann.ent.Soc.Am. 93: 1308-1313.
S1VA-JOTHY,M.T.,1987.Thestructureandfunctionofthefemalespermstorageorgansinlibellulid drag-
onflies.J.InsectPhysiol.33;559-567.
SMITH,D.S., 1968.Insectcells:theirstructureandfunction.Oliver &Boyd,Edinburgh.
WERREN,J.H., 1997.BiologyofWolbachia.A.Rev. Ent. 42:587-609.
WERREN,J.H.&D.M.WINDSOR,2000.Wolbachiainfectionfrequenciesininsects:evidenceofaglobal
equilibrium?Proc. R.Soc. Land.(B)267: 1277-1285.
WIGGLESWORTH,V.B., 1972. Theprinciplesofinsectphysiology.[7thedn].Chapman&Hall,London.