HISTOLOGICAL STUDIES ON EGG DEVELOPMENT IN PAINTED GRASSHOPPER, POEKILOCERUS PICTUS

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HISTOLOGICAL STUDIES ON EGG DEVELOPMENT IN PAINTEDGRASSHOPPER, POEKILOCERUS PICTUS FABRICIUS (ORTHOPTERA:ACRIDOIDEA: PYRGOMORPHIDAE)

Somenath Deya and M. Raziuddin b

a Department of Zoology, P.G. Building, Durgapur Government College, J.N. Avenue, Durgapur- 14, Burdwan, WestBengal, Indiab University Department of Zoology, Vinoba Bhave University, Hazaribag- 01, Jharkhand, IndiaEmail: [email protected] / [email protected]

Received 18 May 2011,Accepted 26 August 2011

ABSTRACTHistological features of developing egg of Poekilocerus pictus Fabricius under light and transmission electronmicroscope have been described. In freshly moulted females the follicular epithelium is low columnar whichduring the pre vitellogenic and vitellogenic phases becomes cuboidal and columnar respectively. Follicles withmature eggs have flattened epithelium. In vitellogenic oocytes, fissures appear between the epithelial cellswhich expectedly help in the transport of extraovrial proteins. Ooplasm of mature egg has a less dense corticalpart and more dense medullary part.

Key words: Poekilocerus pictus, Ovary, Oocyte, Histology, LM, TEM

201155-60Columban J. Life Sci. Vol.12 No. 1 & 2

ISSN-0972-0847

INTRODUCTIONPoekilocerus pictus (Aak or painted grasshopper)common throughout the planes of India (Raziuddinand Anwar, 1997) is primarily a defoliator of Aakplants (Calotropis procera). This insect is a prolificbreeder and besides C. procera, it feeds upon anumber of alternative host plants, many of whichare of economic value (Pruthi and Nigam, 1939;Pruthi, 1954; Parihar, 1974; Khurana, 1975; Raziuddinet al., 1991).The insect ovary consists of variable number ofovarioles which in many cases, including Orthoptera,are connected with the lateral oviduct in linearsequence (Wigglesworth, 1965; Karim, 1979;Chapman, 2000; Tembhare, 2006). Insect eggs arecovered externally by a complex multilayer egg shellconsisting basically of outer chorion and inner vitellinemembrane formed by the follicle cells while the eggsare inside the ovariole. The present paper deals withthe histological structure of egg during developmentas revealed under light microscope (LM) andtransmission electron microscope (TEM).

MATERIAL AND METHODSLive Poekilocerus pictus adults and nymphs werecollected from wild fields and reared in an insect

rearing cage in the Department of Zoology, VinobaBhave University. Immature and mature females ofknown ages were dissected in orthopteran saline(Clayton et al., 1958) under a stereoscopic binocularmicroscope and eggs were obtained. Morphometryhas been done with the aid of slide callipers.For light microscopy (LM) the aqueous Bouin’s fixedeggs were washed in distilled water and thendehydrated in ascending grade of ethyl alcohol. Thespecimens were cleaned with xylene and paraffinblocks were prepared. 7µ thick sections were cutusing a rotatory microtome (1090A, WESWOX,DPTIK) and were stained with haematoxylene andeosine.For transmission electron microscopy (TEM) eggswere primarily fixed in 2.5% glutaraldehyde in 0.12M Millonig’s phosphate buffer for four hours at 4oC.These were post fixed in 1% aqueous osmiumtetroxide for 15 minutes at 4oC and dehydrated byascending grades of alcohols (30%, 50%, 70%, 90%and absolute). Before embedding samples wereimmersed in transitional fluid (epoxypropane).Embedding was done in araldite. Rough trimmingwere made by glass knives and then ultra thin sections(60nm) were obtained from the ultramicrotome (LKB

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Bromma with Olympus microscope). Ultra thinsections were collected on golden grids and the gridscontaining the sections were stained by uranyl acetateand lead citrate. The grids (both unstained andstained) were dried in desicator and then viewedunder transmission electron microscope (FEI FP5018/40 TECNAI G2 Spirit Bio Twin).

RESULTSIn Poekilocerus pictus there are two ovaries ofunequal size (Dey and Raziuddin, 2008 a), eachconsisting of many ovarioles. Each ovariole containsa linear series of oocytes in different stages ofdevelopment; however, it is only the basal oocytewhich incorporates yolk and gradually develops intoan egg. The number of egg rudiments per ovariolevaries from 8 to 11 and does not appear to increaseafter day 5 of final ecdysis. Table 1 shows gradualchanges in the length and breadth of the basal oocyte.In the following description we intend to describethe histological changes in the basal oocyte.

In freshly moulted specimens the basal oocytemeasures 0.856 ± 0.013 mm X 0.198 ± 0.07 mm(mean ± SD). At this stage the epithelium consistsof closely packed low columnar cells (6.0 – 7.0 µ inlength) and the ooplasm of the oocytes is finelygranular without any trace of yolk (Fig. 2). The shapeof the basal egg follicle is elliptical (Figs. 1 and 6). Itcontains a centrally placed nucleus with loosegranular chromatin. On the third day of fledging thebasal oocyte attains a length of 1.308 ± 0.007 mm(mean ± SD). The follicle cells surrounding the

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oocytes are by now slightly larger, 8.5 – 9.5 µ inlength, and appear low-cuboidal. The ooplasm is stillfinely granular without yolk granules and it basicallyresembles that of day one specimens. On day 5 (Fig.3) histology of the basal egg follicle is similar to thatof the three-day-old females except that a slightincrease in the dimension of follicular cells has beennoted. In this species vitellogenesis starts on day 7when small yolk globules make their appearance inthe peripheral ooplasm (Fig. 4). Thus duringprevitellogenic phase the basal oocyte attains criticalsize and becomes ready for yolk incorporation. Inthese females the basal oocytes measure 2.206 ±0.008 mm (mean ± SD) in length and 0.294 ± 0.004mm (mean ± SD) in breadth. As soon as the basaloocyte becomes vitellogenic, the follicular cellsincrease in dimension and become distinctlycolumnar; these cells are now 20 – 26 µ in lengthwith distinct large nuclei, 9.5 – 10.5 µ in diameter,occupying a major part of the cell. The nuclei containdense chromatin.In the vitellogenic oocytes fissures between thefollicular cells make their appearance, and theseappear to form channels for the movement ofextraovarial proteins from the haemolymph into theooplasm. This is also evident from the fact that anumber of protein yolk spheres appear in the ooplasmquite adjacent to these channels. From day 7 onwardthe basal oocyte is actively engaged in theincorporation of yolk and as such there is an enormousincrease in the amount of yolk globules in the ooplasm(Fig. 5). The oocyte rapidly grows in size. The basichistological feature of the oocyte remains the sameas described above for the day 7 specimen.However, from day 15 (oocyte size 5.808 ± 0.007mm X 0.908 ± 0.007 mm) onward the epithelial cellsgradually decrease in height and finally in the maturefollicle (7.99 ± 0.02 mm X 1.504 ± 0.01 mm) thesebecome almost flattened. With the incorporation ofyolk into the ooplasm the oocyte nucleus gradually

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becomes eccentric and finally in the mature egg itlies at the posterior pole of the egg (Fig. 6).TEM studies of mature egg of P. pictus are in linewith those of the light microscopic studies. However,interestingly, the ooplasm has been found to be dividedinto two distinct regions, lighter cortical part anddenser medullary part. The cortical part varies inthickness from 0.25 to 0.45ì

(Figs. 7, 8, 9 and 10). At higher magnification also(X 60000) the two regions are quite distinct, thecortical part containing low electron contrast granulesand the medullary part containing electron densegranules (Fig. 11).In mature oocytes at lower magnification (X 18500and X 20500) the two parts of the ooplasm appear

to be separated by an extremely thin membrane;however, at higher magnifications (X 60000) a distinctmembrane separating the cortical and medullary partof ooplasm is not discernable (Fig. 11). Inprevitellogenic oocytes the ooplasm appearshomogenous. But as the oocytes become vitellogenic,cortical and medullary layers become distinct (Figs.7 to 11).Eggs in which vitellogenesis has almost beencompleted has a thin deeply stained follicularepithelium with granules of high electron density.These granules, concentrated in the follicularepithelium appear to migrate into the ooplasm wherethey are scattered into smaller fragments rangingfrom 10 to 25 nm in diameter (Figs. 8 and 12). Theseelectron-dense granules, despite appearing tooriginate in the follicular cells, may be post-fixationartifacts.

DISCUSSIONThe observations on this aspect confirm the earlierobservations made by Karim (1979) in this species.In P. pictus the basal oocytes passes through theprevitellogenic growth phase during which theseattain the length of just over 2.0 mm. This is the so-called “critical size” of the oocytes which iscompetent for yolk deposition. In this insect,vitellogenesis in the basal oocytes commenced onthe 7th day after fledging which lasted for 14 to 16days in the first ovarian cycle. These observationsare in line with those of Raziuddin and Ghose (1987)and Anwar and Raziuddin (2002) made earlier inthis insect and are also in agreement with those ofHill et al. (1968) and Tobe and Pratt (1975) on thedesert locust S. gregaria.The terminology used by previous workers inconnection with the ovariole sheath is both conflictingand confusing. Many workers reported the presenceof two ovariole sheaths in insects. The outer sheathhas been described by Gross (1903) as a peritonealsheath and Nelson (1934) referred to an outer thinconnective tissue layer as membrana propria.Snodgrass (1935) mentioned an epithelial sheath.Riede (1912) and Wigglesworth (1950) havedescribed a connective tissue sheath and Bonhag(1959) referred to an external ovariole sheath. Justbelow it lies the second sheath which is amembranous and non-cellular basement membranecovering each ovariole and is termed the tunicapropria. In P. pictus the ovariole has two thin covers,the outer ovariole sheath and the inner basement

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membrane lying immediately beneath the sheath. Asusual in this insect the follicular epithelia are initiallyformed of small cuboidal cells which, duringvitellogenesis, become columnar and, in oocyteswhich are nearing completion of vitellogenesis,become flattened.In the present study fissures have been observedbetween the follicular cells which facilitate passageof extraovarial proteins in the growing basal oocyte.SEM study of ovariole surface in previtellogenic stageof P. pictus (Dey and Raziuddin, 2008 b) hasrevealed deep longitudinal convolutions, but asvitellogenesis commenced and proceeded in the basaloocytes, the surface folds distinctly decreased indimension and pores and cracks over the surfaceappeared in patches which probably facilitatevitellogenin entry into the ooplasm.Cytoplasmic qualities in the insect egg have beenstudied by a few workers through ultrastructuralanalysis (see, Buning, 1994). TEM study of matureegg of P. pictus shows that the ooplasm of the eggis distinctly divided into a lighter cortical partcontaining granules of low electron density, and adenser medullary part containing granules with highelectron density. In the flea, Hystrichopsylla talpae,Buning and Sohst (1988, 1989) have reported that inearly previtellogenic stage there is a homogenouscytoplasm which is very poor in free ribosomes butgradually and slowly a belt of ribosome-richcytoplasm appears around the oocyte nucleusindicating a low rate of synthesis of rRNA andribosome; however, later on ribosome-rich cytoplasmcontributes to more than 90 percent of the finaleuplasm at the end of previtellogenic growth. Inmosquitoes the cortical layer of ooplasm beneath theplasma membrane is almost free of ribosome andmitochondria and consists of filamentous matrix(Clements, 1992). Further, coated vesicles of 120-140 nm diameters are the main constituents of thecortical layer (Raikhel and Lea, 1985). We expect asimilar situation as described above to exist in thedeveloping eggs of P. pictus, in which the denserpart of the ooplasm appears to be very rich in freeribosomes.

ACKNOWLEDGEMENTSAuthors are thankful to the Director, Indian Instituteof Chemical Biology, Jadavpur, Kolkata, India forproviding facilities of TEM. One of the authors (SD)is thankful to Dr. D. R. Mandal, Principal, DurgapurGovernment College, Durgapur, Burdwan, WestBengal, India for his interest in the work.

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HISTOLOGICAL STUDIES ON EGG DEVELOPMENT IN PAINTED GRASSHOPPER, POEKILOCERUS PICTUS

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