Table Of ContentOdonatologica36(4):373-379 December 1,2007
Ultrastructural organization
of thelarval spiracles inLibellula depressa L.
(Anisoptera: Libellulidae)
E.Gaino*,S.Piersanti andM.Rebora
DipartimentodiBiologiaCellulare eAmbientale,Università degliStudidi Perugia,
ViaElcediSotto,I-06123Perugia,Italy
ReceivedFebruary9,2007/RevisedandAcceptedMarch 17,2007
Inthe last larval instar (F-0)of L. depressa,2paired spiracles, in the formof
elongatedeye-shapedstructures,arelocatedintheanteriorregionofthemesothorax
segment.Afinestructural analysisofthese spiraclesunderthescanningandelectron
microscopesrevealsthateach spiracleconsistsofawell-developedcuticularperitreme
with adorsal-anterior lip bearingathin laminar coat and aventral-posteriorlip
bearingafilterapparatus.Thefilterapparatusderivesfromaseriesoffoldsforming
discrete groupsadheringtooneanothertodelimit emptyspaces and producinga
honeycomb-likestructure.Thisstructureiscoherentwiththeneed toavoidentryof
waterwhenthe larvaissubmerged.The functionofthesespiraclesduringtheinsect
developmentisdiscussed,notingthatin anisopteranlarvae therectal epithelium,
formingthe socalled branchial basket,isthe main respiratory organ.
INTRODUCTION
Inpterygoteinsects, spiracles areimportant structuresinvolvedinthebalance
betweengasexchange andtranspiratorywaterloss. Thegrossmorphology ofthe
spiracles hasbeenstudiedinrepresentatives ofanumberoflepidopteran families
(SCHMITZ & WASSERTHAL, 1999); using electronmicroscopy they showed
important detailsof the organization of the spiracle apparatus in both larvae
andadults. Inthe Diptera, studiesonthe spiracle apparatusof water-living lar-
vaeshowed thatthemorphology ofthissystem differs markedly fromgroupto
group,not only in organization butalso inlocationandinrelationto the devel-
opmental stadiumconsidered(FAUSTO etah, 1999). In addition, inimmature
"CorrespondingAuthor:Prof. Elda Gaino,Tel.: ++390755855729;fax:++390755855733;E-mail
[email protected]
374 E.Gaino,S.Piersanti& M.Rebora
stadiaofDiptera, ithasbeendemonstratedthattheorganisation ofthe spiracle
apparatusvariesaccording toenvironmentalconditions(KHOLE, 1979;NIKAM
& KHOLE, 1989; FAUSTO etal., 1999). Spiracle structure is also a usefultool
for taxonomicpurposes(MAROL1 et al., 1992; FAUSTO et al., 1998;MILL,
1998a).
Anatomicalstudies of the adultand larval spiracles of Odonata have been
carried out (TILLYARD, 1917; WOLF, 1935). Differences in the morphology
of thoracic and abdominalspiracles have beendescribedin the adultsofPan-
talaflavescens (TONAPI, 1960).In certainadultAfrican dragonflies, MILLER
(1962) carried out anexhaustiveanalysis ofspiracle controlandof the factors
altering itsactivity, suchas changes inwater balance (MILLER, 1964a), degree
of hypoxia andchanges intemperature(MILLER, 1964b).
Inanisopteran larvaetherectalepithelium, formingthe socalledbranchialbas-
ket, is themainrespiratoryorgan andthefunctionofthetwomesothoracicspir-
aclesis unclear.
Thegoalofthepresent study is thefinestructuralanalysis ofthespiracles of
the last larval instar of Libellula depressa. Thisspecies is apond-dwelling drag-
onfly, commonandwidespread inItaly(UTZERI, 1995)andthroughout therest
of Europe(CARCHINI, 1983).
MATERIALANDMETHODS
Larvae ofLibellula depressa,attributedtotheultimatestage(F-0)onthebaseoftheirheadwidth
andbody length(D1GIOVANNIetat,2000),werecollected inpondsincentralItaly(Perugia,Um-
bria).Inthelaboratory,thespecimenswerekept inplasticcontainers(60x40x40cm)with water,de-
tritus,floraand faunafromthecollectingsite.Thelarvaewerefedadlibitumwithplankton(Daphnia
sp. and Cyclopssp.).
Forelectron microscopy,the larvaeweredissected underastereomicroscope,afterhavingcutoff
the headtoreducestress, andplacedinto2.5%glutaraldehydeincacodilate buffer(pH,7.2)forfive
hours. Afterwards,the materialwasrinsedin thesamebufferand post-fixedin 1%cacodilate buff-
ered osmium tetroxide for 30minutes. Forscanningelectron microscopy (SEM),the material was
dehydratedinacrescent ethanol seriesupto 100%,critical-pointdriedinaCPD 030Bal-Tec. Speci-
menswerethenmounted onstubswithsilverconductingpaint, sputter-coatedwithgold-palladium
in anEmitech K550Xsputter,andobserved withaPhilips EM 515SEM. Fortransmission electron
microscopy(TEM),dehydratedspecimenswereinfiltratedwith anEpon-Aralditemixture.Ultrathin
sectionswerecut with adiamond knifeonaLeica EMUC6 ultramicrotome,collected onformvar-
coatedcopper grids,stained withuranyl-acetateand lead citrate,and observedin aPhilipsEM208
TEM.
RESULTS
Inthe laststage (F-0)of Libelluladepressa larvae,two paired spiracles, inthe
formofelongated eye-shaped structures,are locatedintheanteriorregionofthe
mesothoraxsegment(Fig. 1).Eachspiraclemeasuresabout800pminlength. The
view fromtheexterior shows thateach spiracle consists ofawell developed cu-
Ultrastructure oflarvalspiraclesin Libellula depressa 375
ticularperitremewitha dorsal-anteriorlipanda ventral-posteriorlip(Figs 1-2).
Along itsexternalborder, theventrallipbearsafilterapparatuswhilethedorsal
lipbearsathinlaminarcoat(Fig. 2).Thefilterapparatusconsistsof aseriesof
longfolds, emerging fromthecuticularperitreme. Thefoldsformdiscretegroups
whereseveralfoldsare incontact.Theoveralleffectis a honeycomb-like surface
(Figs 3-4). Movinginwardstowardstheatrium, itswallsappearcompletely cov-
eredwithshortfoldsthatshowanirregular honeycomb-like patternbothonthe
ventral(Fig. 4)andonthedorsalsurface(Fig. 5).
Theatriummeasuresabout 100pminhighandabout250pmin depth(Fig. 6)
Figs 1-5.Scanningelectronmicrographsofthemesothoracic spiraclesofthelastlarval instar(F-0)
ofLibellula depressa:(1)twomesothoracic spiracles(arrows)in the form ofelongatedeye-shaped
structures;— (2)detail oftheleftspiracleshowingthedorsal-anterior lip(DL),bearingathinlami-
narcoat(arrow),and theventral-posteriorlip(VL)withthefilterapparatus(FA);— (3)detailofthe
filterapparatus(FA)derivingfromaseriesoffoldsformingdiscrete groups (whereeach fold in the
groupadherestotheadjacentones)todelimitemptyspacesproducingahoneycomb-likesurface;—
(4)viewofaspiraclefromtheinsidelookingoutwards(thebottomofthespiraclehasbeenremoved).
Notethesequence offolds formingthefilterapparatus(FA),Theventralwall oftheatriumshows an
irregularhoneycomb-likestructure(arrow);- (5)detailoftheupperwall oftheatriumconsistingof
shortfolds witha similar,irregular,honeycomb-likepattern(arrow);DL, dorsallip.
376 E.Gaino, S.Piersanti & M.Rebora
On itsventral surfacethefolds delimitingthewallsbecomeprogressively shorter
from thefilterapparatusto the innerend ofthe atrium, where they are hardly
visible. Thelaminarcoat fits overthe edge of thefilterapparatus(Fig. 6).
Thinsections, observedundertheTEM,revealthatalltheatriumisbordered
by athin electron-densecuticle (Figs 7-9).The foldsof the filterapparatus are
30-50pm high.Theirinnercore consists ofafibrouscomponentcoveredby thin
cuticle whichexpands at theapex to produce contact points linkingthefolds to
oneanother(Fig. 7).Thedorsallipbearsathinlaminarcoatborderedbycuticle,
whichis thickerontheexternalsurface, andincludesafibrousregion interposed
Figs6-9. A semithin section underthe lightmicroscope(6)and transmission electron micrographs
(Figs7-9) ofthemesothoracic spiraclesofthelastlarvalinstar (F-0)ofLibellula depressa:(6)section
ofaspiracleshowingtheatrium(A), theventralsurfaceofwhich hasfoldsthat becomeprogressively
shorter fromthefilterapparatus(FA)toitsinnerlimit (arrow),where theyarehardlyvisible.Note
that thelaminarcoat(LC)onthedorsal lip(DL)fitsovertheedgeofthefilterapparatus(FA)onthe
ventral lip(VL); —(7)detail ofthe foldsofthefilterapparatuswith theirinnercoreconsistingofa
fibrousmaterialcoveredbythincuticlewhichexpandstoproducecontactpoints linkingthe foldsto
oneanother(arrows); — (8)detailofthethinlaminar coat(LC)ofthedorsallipbordered bycuticle
which isthickerontheexternalsurface(arrow).Thefibrousmaterialinterposedbetween thetwocu-
ticularlayersalso fillsaprotrusion (P)atthebaseofthe laminar coat.Double arrowspointoutshort
folds ontheupperwall oftheatrium; - (9)detailsoftheventralsurfaceoftheatriumshowingthat
thewalls ofitsinnerlimit aredecorated byveryshort cuticular folds(arrows).
Ultrastructureoflarval spiraclesinLibellula depressa 377
betweenthe twocuticularlayers (Fig. 8).Afibrousregion also fillsaprotrusion
(P) atthebaseofthelaminarcoat, betweenthe latterandthefirstfolds (3-5 pm
high)oftheatrium(Fig. 8).Theventralsurfaceoftheatrium, inits innerlimit,
showswallsdecoratedby veryshort cuticularfolds, irregularlyexpanded attheir
apices (Fig. 9).
DISCUSSION
Thetwo mesothoracicspiracles ofAnisoptera larvaeseemtobeopenonly in
the finalstadium, even ifthey are also present in earlierdevelopmental stages
(CORBET, 1999). AccordingtoCORBET(1999), larvaeofmanyspecies ofAni-
soptera,intheirfinalstadium,leavethewaterpartly orwholly andintermittently
or irrevocably, days orevenweeksbeforeemergence.Thisbehaviourmayreflect
theneedtoexpose thethoracicspiracles to theair.Thelarvaeofpond-dwelling
Anisoptera commonly congregateat thepond margin with theirspiracles open
as metamorphosis proceeds (CORBET, 1999).Inparticular, thelarvaeofLibel-
luladepressa, closetothefinalmoult,tendto spend timeinsuchlocations, some-
timesreturninginto the water(personal observation).
Thefinestructureofthe spiraclesofthefinallarvalstadiumofL. depressa is
coherentwiththeneedtoavoidwaterentering when thelarvaissubmerged. In-
deed,eachspiracle hasa dorsallaminarlip fitting over thefilterapparatuswith
afibrousprotrusion onitsbaseontheinternalside.Thisprotrusion could help
to seal the lip to the edgeof thefilter. Thehoneycomb-like surfaceofthefilter
andoftheatriumwallsmayfunctionas a hydrofuge structure,abletoresist wa-
terentryintothetrachealsystem. A honeycomb-like structure withthisfunction
hasbeendescribedin thespiracles ofPeriplaneta americana(MILLER, 1981;
MILL, 1998a).
InAnisoptera larvae the mainrespiratory organ is represented by the rectal
epithelium (MILL, 1998b). Infact, by comparing variousstructural modelsfor
gasexchange intheinternalgillsof theanisopteran Aeshnacyanea,KOHNERT
etal.(2004) concludedthatrectal gillsalonecan satisfy themetabolicdemandof
this active carnivorousinsect. In addition, CORBET (1999) reports thatlarval
Anisoptera, withtheirthick integument, probably rely almost entirely on their
rectal gills,except perhaps in veryearly stadia, when theintegument isrelatively
unsclerotizedandtranspiration viathecuticleis possible.
Previousinvestigations onthelarvaeofL. depressa, belonging to thefinalsta-
dium, gaveevidencethattheseinsects areableto survive forseveral days outof
water,ifkept inairwithhighrelative humidityvalues(PIERSANTI etah,2007;
REBORA et ah, 2007). In such conditionswe can hypothesize that the larvae
couldtakeoxygenfromtheairthroughtheirrectalepitelium. Indeed,asreported
by CORBET(1999), terrestrialorsemiterrestriallarvaecommonly employrectal
respiration and larvaeofaquatic species, in response to lowoxygen concentra-
378 E.Gaino,S.Piersanti& M.Rebora
tion,climbbackward tothewatersurfaceandplace theiranalpyramid intheair
(seereviewin CORBET, 1999).
Eventhough during theexperiments withL. depressa larvaeit was not detect-
ed ifthethoracicspiracles were involvedinoxygen uptake, wecannot excludea
possibleroleofthesestructuresin gasexchange. Aninvolvementofthespiracles
in dragonfly larvalbreathing has neverbeenshown, neverthelessithasbeenhy-
pothesized insomeprimitive semiterrestrialspecies, like Uropetala carovei(Pet-
aluridae)(GREEN, 1977).Inaddition, thefinallarvalstadiumofthelivingfossil
anisozygopteran Epiophlebia superstes leavesthewaterfourorfivemonthsbefore
moultingand,during this period,gasexchange probably occurs through theme-
sothoracictrachealspiracles (see reviewinWICHARD etah, 2002).
Thepresentworkrepresentsafirstapproach tothefinemorphologyofthespir-
acles in larvalanisopteran dragonflies andgives anessentialbaseforphysiologi-
cal studiesof therolethatthesestructures mayperform during development.
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