Journal of the American Mosquito Control Association, 14(4):363-368, 1998Copyright O 1998 by the American Mosquito Control Association, Inc.

ULTRASTRUCTURE OF THE EGGS OF CULICOIDES MOLESTUS(DIPTERA: CERATOPOGONIDAE)

BRONWEN W. CRIBB'2 eNO ERIC CHITRA3

ABSTRACT, The eggs of Culicoides molestus (Skuse) are described and illustrated by scanning and trans-

mission electron microscopy. Eggs are elongate with a slight dorsoventral curvature. No outer chorionic tuberclesare present. Aeropyles are present in large numbers at the anterior end and in lower numbers at the posterior

end and lateral regions. The chorion has 5 layers. An outer, rough, proteinaceous layer covers a smoother innersurface, which in turn encloses a layer of columns and meshwork that appears capable of containing air. Thesecolumns are underlain by an additional 2 layers. The aeropylar region, in combination with the chorionicmeshwork, appqus to provide a plastron that may aid in the survival and development of inundated eggs.

KEY WORDS Culicoides molestus, egg morphology, chorion, aeropyle, biting midge, electron microscopy

INTRODUCTION

Culicoides molestus (Skuse) is a major pest incoastal southeastern Queensland and northern NewSouth Wales, Australia (Kettle et al. 1979, Kay andLennon 1982). Larvae have been recovered fromsandy beaches and requi-re fairly clean sand, suchas is found in canal estates (a constructed labyrinthof coastal canals, usually lined with beach). Theselarvae do not have the association with surface-tun-neling crabs (Mictyris livingstonei McNeill) that ispresent for larvae Culicoides subimmaculatus Leeand Reye and the 2 species are rarely sympatric(Kettle et al. 1979, Marks and Reye 1982). Ovi-position has been assumed to occur where larvaehave been found and this assumption is strength-ened by the poor dispersal of the species as adults(Kettle et al. 1979); however, no published data ex-ist for egg recovery and the description of the eggsis lacking. We investigated 3 canals on the GoldCoast, a major residential, tourist, and recreationarea of southeastern Queensland, for presence of C.molestus eggs.

A number of studies address the ultrasffucture ofeggs of Culicoides species but none have looked atthe structure of the chorion using transmission elec-tron microscopy (TEM). Using scanning electronmicroscopy (SEM), eggs of Culicoides circwn-scriptus Kieffer, Culicoides imicola Kieffer, andCulicoides gejgelensis Dzhafarov have been de-scribed by Day et al. (1997); Culicoides brevitarsisKieffer by Campbell and Kettle (1975) and Kariyaet al. (1989); Culicoides arakawae (Arakavta), Cul-icoides oxystoma Kieffer, Culicoides punctatus(Meigen), Culicoides sumatrae Macfie, Culicoidesactoni Sfiitl;^, and. Culicoides maculatas (Shiraki)by Kariya et al. (1989): and Culicoides variipennis(Coquillett) by Nunamaker et al. (1987). The sur-

lDepartment of Entomology, The University ofQueensland, Brisbane, 4072, Australia.

2 Centre for Microscopy & Microanalysis, The Univer-sity of Queensland, Brisbane, 4072, Australia.

3 School of Applied Science, Griffith University GoldCoast Campus, PMB 50 Gold Coast Mail Centre, Bundall9726, Australia.

face structure of the eggs of all 11 species displaytubercles. In our study we report a different surfacestructure for the eggs of C. molestus and investigatethe ultrastructure of the chorion usins both SEMand TEM.

MATERIALS AND METHODS

Eggs of C. molestus were collected from sandbanks along a canal estate on the Gold Coast(Southport, Queensland, Australia: 27"59'5,I53"25'E). Sand banks were sampled for C. moles-rus in April and May of 1997, 5 days after a fullor a new moon phase. Layers of sand were removedfrom various locations across the beach, near mid-tide level, packed into separate containers, andtransported into the laboratory for egg extraction.Particles lighter than sand were flooded out of thesamples using a water jet that suspended them with-in a volumetric cylinder. Then, the water was runinto a tube fitted with flne stainless steel mesh (100-pm apertures) at the base. The contents were trans-ferred into a petri dish to be sifted for eggs undera dissecting microscope. Larvae and pupae wereextracted using the same protocol. Eggs were al-lowed to eclose in saline agar medium (7-8Vo agarin autoclaved canal water from the same site as thesample) and reared to adult for identification. Usinglight microscopy, eclosed larvae were compared toextracted larvae. Pupae were allowed to emergeand adults were identified, also under the light mi-croscope. Adult midges were collected on thebeach, above the sites of egg collection, using hu-mans as bait. All samples were identified as Culi-coides molestus.

Two techrdques were used to prepare extractedeggs and eggshells for SEM. Eggs were fixed inmodified Karnovsky's fixative (2.5Vo glutaralde-hyde and 2.OVo formaldehyde in 0.1 M sodium cac-odylate buffer), washed in buffer, and postfixed in17o osmium tetroxide in 0.1 M cacodylate bufferbefore being dehydrated in an acetone series andcritical point dried. Alternatively, eggs were dehy-drated in acetone to lOOVo and air dried. Forty-nineeggs were prepared according to this 2nd protocol

363

JouRNll op rns AuenrcAN Moseurro CoNrnol AssocrerroN Vor-

and the best-preserved ones were selected for mea-surement. Egg size was unaffected by technique.Determination of the chemistry of the outer layersof the chorion was achieved using 2 further tech-niques: eggs were placed in 57o aqueous proteinaseK at 37oC for 45 min (technique modified fromBodley and Wood 1996), washed in about IOVo De-con 90 for 30 min, then sonicated for 30 sec whilein the detergent, or eggs were placed directly intoabout IOVo Decon 90 (Decon Laboratories Limited,Hove, East Sussex, United Kingdom) detergent forI h and sonicated for 30 sec. Eggs from both treat-ments were then fixed in 3Vo glutaraldehyde in 0.1M cacodylate buffer, washed in buffer, and post-fixed in 17o osmium tetroxide prior to dehydrationin ethanol and critical point drying. All sampleswere mounted on double-sided adhesive tape andcoated with gold to a thickness of 7-10 nm beforeviewing using a JEOL 6300 or 6400F SEM (JapanElectron Optics Laboratory, Tokyo, Japan) at 5 kV.

Eggs were prepared for TEM by fixing in mod-ified Karnovsky's fixative in 0.1 M cacodylate buff-er, washing in buffer, postfixing in l% osmium te-troxide in 0.1 M cacodylate buffer, dehydrating ina series of acetone, and embedding in Spurr resin.Sections were cut with glass and diamond knives,stained with uranyl acetate and lead citrate, andviewed using a JEOL 1010 TEM (JEOL USA, pea-body, MA) at 8O kV.

Terminology used follows Day er al. (1997), Hin-ton (1981), Linley et al. (1991), and Harbach andKnight (1980).

RESULTS

The eggs of C. molestus are dark brown to black,elongate structures with a slight dorsoventral cur-vature (Figs. la, 1b). Egg dimensions are given inTable 1. Freshly extracted eggs were occasionallyembedded in a clear, jellylike substance that, whenfixed in place, obliterated surface detail. Bacteriawere seen in this layer (Fig. lc). Eggs without thislayer showed a rough surface structure (layer 1)that appeared to peel back in some regions to ex-pose a smooth, continuous layer, marked by a pat-tern of low-relief, domelike papillae (Fig. ld). Inplaces this smooth layer was seen to be brokenaway from the papillae (Fig. 1d). A thin layer (layer2) joins the tops of the papillae, which are therounded tops of columns.

Eggs treated with detergent (Decon 90) aloneshowed no change to the outer layers of the chorionbut when eggs were pretreated with proteinase Kand then detergent, the outer, rough layer was re-moved (Fig. 1e). This indicates that the outer layeris proteinaceous in nature. The inner, smoother lay-er that was revealed was unaffected by the treat-ments and therefore has a different chemical con-struction from the outer layer.

A poorly developed anterior micropylar dome ispresent and the surface at this end contains many

aeropyles and is often fenestrated (Figs.2a,2b). Asmaller number of aeropyles occurs along the eggand at the posterior end (Fig. 2c). Eclosion occursat the anterior end, below the micropylar cap,which remains unbroken, and proceeds through aslit along about one third of the length of the egg(Fig. 2d). Eclosion of eggs was observed under thelight microscope so this split is not a preparationartifact. Using SEM, no obvious hatching line isseen in intact eggs.

The cross section of the egg, viewed with TEM(Fig. 2e), shows the rough, scalelike outer layer,which may be absent but when present can be upto 2.4 pm thick (layer 1). A smooth, thin (20- to140-nm) layer (layer 2) lies beneath this outer layerand a row of columnlike struts (layer 3) supportslayer 2. The columns sometimes appear overlain bythe thin layer 2 (Fig. 2e) and in other regions thelayer is not distinct from the columns. The columnsare about 380 nm high (Figs. 2e,2f). They sit upona finely lamellate layer (layer 4) that is usuallyabout 4O nm thick but is sometimes seen to be con-structed of 2 such layers and is about 80 nm thick(Fig. 2f). This lamellate layer covers an inner, ho-mogeneous, layer (layer 5) that is about 405 nmthick (data obtained from 10 measurements). Thelarva lies adjacent to this inner layer. In all the cho-rion has 5 layers.

DISCUSSION

The egg surface structure of C. molestus is dis-tinctly different from the 11 other species that havebeen studied by SEM (Table 1). All previouslystudied species have a covering of tubercles (alsoreferred to as ansulae, hairlike papillae, granularpapillae, or outer chorionic tubercles). Outer cho-rionic tubercles are prominences of the outer cho-rion (Harbach and Knight 1980). Sometimes theseare arranged in rows, as in C. circumscriptus, C.gejgelensis, and C. imicola (Day et al. 1997); andC. arakawae, C. orystoma, C. punctatus, C. su-matrae, and C. actoni (Kariya et al. 1989). In othercases the tubercles are scattered over the entire sur-face, as in C. brevitarsls (Campbell and KettleL975), C. maculatus (Kariya et al. 1989), and C.variipennis (Nunamaker et al. 1987). No outer cho-rionic tubercles are presentin C. molestas. The sur-face of the chorion, beneath the rough outer layer,is relatively smooth, depending on the degree ofcollapse of the layer overlying the chorionic mesh-work. Where papillae are seen, they are evident asthe tops of the inner chorionic meshwork/columnsand are not a separate structure on the outer cho-rion. Tubercular projections have generally been re-garded as a plastron, to aid in air exchange andtheir absence in C. molestus raises the question asto how these eggs survive inundation.

No TEM studies of the chorion of a Culicoidesspecies have been reported, although hints as to thestructure for C. variipennis are available from SEM

Dscnt'lsBn 1998 Eccs op Cuucotozs uotgsrus 367

Table l. Egg dimensions (pm) for Culicoides species for which scanning electron microscopy data are available.

Length Width Length to width ratio

Speciesr (number)Mean Mean+ SE Range + SE Range Reference

Mean+ SE Range

Culicoides brevitarsis

C ulicoide s c irc umsc rip tus (lO)

Culicoides gej gelensis (ll)

Culicoides imicola (lO)

Culicoides nolestus (13')

C ulic oi de s v ariip enni s

? 7 3 + 2 ? Campbell andKettle (1975)

Day et al. (1997)Day et al. (1997)Day et al. (1997)This paperNunamaker

et a l . (1987)

2 9 0 + 2

4 0 3 + 33 2 1 + 53 6 3 + 53 3 3 + 2

400

384-413285-34r334191322-356

,|

54 + | 47-6047 ! | 4l-5265 r I 63-6962 ! 2 47-75

6 8 7

7.4 + O.2 6.8-8.66 .9 + 0 .1 6 .1J .55.6 + 0.1 5.3-6.05.5 + O.2 4.5J.O

- 5 ?

I Culicoides arakawae, C. orystoru, C. punctatus, C. sumtrae, C. actoni, and C. mculatus are reported as having eggs 300-5O0

pm long (Kariya et al. 1989).

study (see Nunamaker et al. 1987). We have shownthat in C. molestus a chorionic network and its as-sociated spaces exist below the outer chorion. Ev-idence exists of a layer of tubercles seen to underliethe base upon which the outer layer of tubercles sitin C. variipennis, but a separation of the chorioniclayers was not unequivocally demonstrated in thiscase (Nunamaker et al. 1987). Demonstration ofsuch a space in C. molestus adds support to thetheory that this separation also exists in C. vari-ipennis and it may exist in eggs of other Culicoidesspecies as well. Hinton (1981) describes such col-umns within the chorion as serving as an air-con-taining meshwork. The lack of an obvious externalplastron in C. molestus, coupled with our obser-vation that the eggs can survive, grow, and eclosewhen constantly inundated, leads us to the conclu-sion that the inner network of columns and theirconnection to aeropyles, particularly the extensiveanterior aeropylar region, enables efficient respira-tion. Aeropyles are usually found to be hydrofugeand resist water entry and so can act as a plastronwhen covered with water (Hinton 1981). However,aeroplyes have to be sufflciently numerous to pro-vide a large area for effective gas exchange. Webelieve that the anterior aeroplyar region in eggs ofC. molestus may well be large enough to function,in combination with the chorionic air spaces, as aplastron.

Eggs of C. molestus are similar in size to thoseof other species (see Table 1). Because egg sizes ofspecies overlap, size is not particularly useful fordistinguishing eggs of different species. Day et al.(1997) were hopeful that surface morphology ofeggs could be used to separate Culicoides speciesbut concluded, as a result of their study, that thiswould not be possible for most Culicoides species.In contrast, Kariya et al. (1989) found that the sur-face of the eggs from each of the 7 species theystudied had a specific pattern that could be usefulin species identification. Evidence from our studysupports the view that surface morphology of eggsmay be extremely useful in determination of par-ticular species. Culicoides eggs are now known tofall into 3 broad types (and many more refined cat-

egories) on the basis of external structure: tuberclesin rows along the eCg (C. circumscriptus, C. imi-cola, alnd C. gejgelensis [Day et al. 1997f; C. ar-alcatvae, C. orystoma, C. punctatus, C. sumatrae,and C. actoni fKariya et al. 19891); scattered tu-bercles with no pattern (C. brevitarsls [Campbelland Kettle 1975, Kariya et al. 19891 and C. vari-ipennis lNunamaker et d. 1987]) or with variablepatterns (C. maculatus [Kariya et al. 1989]); andeggs with no tubercles (C. molestus). Morphologymay be used to discriminate species within thesetypes; however, more quantification is needed.

Culicoides molestus occurs in a different habitatfrom the other species discussed here. It is marineand eggs are found in sand that is often inundated,whereas eggs of the other 11 species occtu eitherin open muddy areas, dung, or damp organic soil,depending on the species (Day et al. 1997, Kariyaet al. 1989, Kettle 1995). Kariya et al. (1989) pro-posed that habitat may be linked to egg morphol-ogy, specifically that eggs covered with hairlike pa-pillae (scattered tubercles) may be laid in drier ar-eas, such as cow dung, than those with tubercles inrows. These authors related surface structure to ad-herence of the eggs rather than to respiration. Thistrend has one contradiction: C. imicola has rowsrather than scattered tubercles (Day et al. 1997) andis found in dung (Kettle 1995). However, on thebasis of structure of eggs of C. molestus, morpho-logic differences may reflect terrestrial versus ma-rine habitats and further studies of eggs of marineor esturine Culicoides species are warranted. Culi-coides molestrrs often occurs alone but one specieswith which it can overlap is C. subimmaculatus artdwe intend to investigate the surface morphology ofthe eggs of this species for comparison with thoseof C, molestus.

ACKNOWLEDGMENTS

We gratefully acknowledge the Gold Coast CityCouncil for allowing access to canal beaches. Wealso wish to thank W. M. and N. R. St. George forunlimited access to their canal frontage and enthu-siastic interest in our work. We are indebted to the

368 Jouruar oF THE AMERTCnN Mosqurro Coxrnol AssocrarroN Vor . 14 , No.4

Centre for Microscopy & Microanalysis for accessto facilities.

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