Proc. Zool. Soc. lndia. 7 l2l = 27 - 39 : (2008)

OOCY'TE RESORPTION IN THEGRASSHOPPER POEKILOCERUS

PYRGOMORPHIDPICTUS FABRICIUS

I

SOMENATH DEY* AND M. RAZIUDDINP.G. Department of Zoology,

Vinoba Bhave University, Hazaribag 825 30'1, Jharkhand* Department of Zoology, Krishnagar Government College,

Krishnagar, Nadia 741101, West BengalEmail: somenath0B@yahoo.co. in

Received - 10.04.2008 Accepted - 24.07.2008

ABSTRACT

This study was undertaken to investigate the phenomenon of oocyte resorption(oosorption) in the vitellogenic oocytes of Poekilocerus picfus. An appreciabty highpercentage (45%) of the growing basal oocytes undergoes resorpflo n in both laboratory.reared and field-collected females. Light microscopy, scanning electron microsicopy andfransmissio n electron microscopy have been used to study the morphology and hi_stologyof the resorption body.

Keywords : Oocyte, Resorptive bodles, Oosorption,Grasshopper, SEM, TEM

INTRODUCTION

Resorption of oocyte has been reported in a number of insect orders (seeEngelmann, 1970; Chapman,2001). This phenomenon is more pronounced in insectsfacing adverse conditions (Verma and Raziuddin, 1993, 1993a, 1g93b; Anwar andRaziuddin, 2002,2003). However, varying degree of resorption occurs in insects evenunder apparently optimal conditions. ln some acridids, a variable number of eggs whichhave grown to various sizes undergo degeneration while oocytes in the neighboringovarioles continue to incorporate yolk and mature (Phipps, 1949, 1966; Singh, 195g;Highnam and Lusis, 1962; Lusis, 1963; Tobe and Pratt, 1975; Raziuddin et at., 1gg7;Karim, 1989). This phenomenon is also reported for many other insects (see Engelmann,1970; Verma, 1991 ; Sharma, 1992).

A review of literature reveals that in the pyrgomorphid grasshoppe r, Poekilocerus

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DEYAND RAZIUDDIN

pictus the phenomenon of oocyte resorption has been briefly described in the past by'Raziuddin et at. (1987) and Anwar and Raziuddiil (2002, 2003). The present paper deals

with a comparative description of oocyte resorption in field reared and laboratory reared

Poekilocerus pictus as well as their morp,hshistology as revealed through light microscopy

and electron microscopy.

MATERIALAND METHODS

Live P pictus were collected from wild fields of both Jharkhand and West Bengal

and reared in insect rearing cages at P.G. Department of Zoology, Vinoba Bhave University,

Hazaribag. The insects were maintained on fresh Calotropis procera leaves. The rearing

techniques described earlier by Raziuddin et al., (1977) and Raziuddin and Anwar (1996)

have been followed. The female grasshoppers of known ages were dissected in insectRinger solution at selected intervals and ovaries were examined after short fixation in

formalcalcium (Highnam etal., 1963; Raziuddin ef a/., 1987) and numbers of resorptivebodies were noted. For the purpose of comparison of age between laboratory-reared and

field-collected specimens, size of the vitellogenic basal oocytes was considered.

Resorptive bodies were fixed in aqueous Bouin's fluid for light microscopy. Paraffin(melting point 56-5BoC) sections of 6p thickness were cut by a rotary microtome (1090A,

WESWOX, DPTIK). The sections were double stained by Delafield's haematoxylin and

eosin (alcoholic).

For scanning electron microscopy (SEM) resorptive bodies were fixed in 2.5 per

centglutaraldehydein0.l MphosphatebufferatpHT.4at4oCandthendehydratedbyascending grades of alcohols (30%, 50o/o,70o/o,90% and absolute). These were thenimmersed in a mixture of alcohol and acetone of various grades (3:1 ,2:1, 1:1 , and 1:2)

and finally dehydrated in anhydrous acetone at room temperature and proceeded.forcritical point drying (CPD). After CPD samples were coated by gold by sputter (30 min)and then viewed under scanning electron microscope (JEOL JSM 6700F).

For transmission electrorr microscopy (TEM) resorptive bodies were primarilyfixed in 2.5o/o glularaldehyde in 0.12 M Millonig's phosphate buffer for four hours at 4oC.

Then they were secondarily fixed (post fixation) in 1% aquas osmium tetroxide for 15

minutes at 4oC and dehydrated by ascending grades of alcohols (30%, 50o/o,70o/o, 90o/o

and absolute). Before embedding samples were immersed in transitional fluid(epoxypropane). Embeddings were done in araldite. Rough trimming were made by glassknives and then ultra thin sections (60nm) were obtained from the ultramicrotome (LKBBromma with Olympqg,microscope). Ultra thin sections were collected on golden gridsand the grids containing the sections were stained by uranyl acetate and lead citrate.

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PROC. ZOOL. SOC. tNDtA

The grids were dried in desicator and then viewed under transmission electron'mlcroscope(FEl FP 5018/40 TECNAI G2 Spirit Bio Twin).

RESULTS

ln Poekitocerus pictus paired ovaries lie dorsolaterally in the abdomen on eitherside of the alimentary canal. Generally the two ovaries of a specimen vary in length, theleft ovary being larger than the right ovary and hence the former contained more numberof ovario'les (105.66 t 9.6) than the right ovary (75.0 t 7.7) [Table l].

ln P. pictus 3 to 5 percent of the previtellogenic basal oocytes fail to incorporateyolk from the very beginning and remain as such throughout the ovarian cycle, while inthe neighboring ovarioles yolk deposition had started and developed normally. After theprocess of vitellogenesis had started and oocytes had grown to various sub-mature sizes,some of the oocytes undergo oosorption or resorption and were finally resorbed. lt was,therefore, many resorptive bodies could be found in each ovary of the females in whichoocyte maturation was nearing completion.

Oosorption of growing vitellogenic oocytes has been observed in both field-collectedas well as well fed and properly mated labbratory-reared females. The record of vitellogenicbasaloocytes of P pictus undergoing oosorption is presented in Table ll. The observationsshow that in both field-collected as well as laboratory-reared females' almost similarnumberof oocytes underwentoosorption (average 81.16 in laboratory-reared and 82.8 infield-collected specimens) (Fig. 1). On the whole in P. pictus about 45 percent of basaloocytes do not mature and undergo resorption. Further, the percentage of vitellogenic

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basal oocytes which i.rnderwent resorption increased as the vitellogenesis proceeded. lt

was found that the growing oocytes may

TABLE NO. I: NUMBER OF OVARIOLES IN LEFT AND RIGHT SIDE OVARY OF

LABORATO RY-REARED P O EKI LOCERUS P I CT U S

enter oosorption at any stage during their maturation but majority of the oocytes entered

oosorpti6n when they were 3.0 to 5.5 mm long and only about 5 percent of the oocytes

underwent oosorption when they were nearing maturation. lt was, therefore, the basal

oocytes undergoing oosorption were in different stages of oosorption.

Number of ovarioles

Left Ovary Right Ovary

SI No. of ovarioles Mean SD S! No. of ovarioles Mean SD

01 126

{05.66 9.6032

01 71

75.0 7.6539

02 109 02 62

03 95 03 59

u 115 u B5

05 105 05 7B

06 % 06 61

07 103 07 82

08 101 08 81

09 117 09 86

10 110 10 85

11 97 11 66

12 96 12 u

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sr. Laboratory-reared female Field-collected femate

Left Ovary RightOvary LeftOvary RightOvary

01 57 32 59 35

V2 49 28 47 30

03 43 27 45 26

M 52 38 50 40

05 47 35 49 37

06 42 27 45 31

07 6 37 45 30

08 45 36 47 36

09 53 39 52 37

10 49 38 51 40

11 44 30 46 31

12 42 3B 45 40

RangeMean t SD

42 -5747.41 !4.53

27 -3933.75!4.47

45-5948.41 r 3.89

26 -4034.41!4.49

PROC. ZOOL. SOC. INDIA

Table-ll: showing the number of resorptive body in taboratory-reared ^

and field-collected female poekilocerus picfus.

Fig. 1: Comparison of oocytes undergoingoosorption in left (LO) & right(RO)ovaries of P.

pictus

BLab. rcared i

rE Field collected i.--l

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SURFACESTRUCTURE

Scanning electron microscopic studies of a few basal oocytes in different stages ofoosorption revealed that the surface of such oocytes gets enormously folded (Fig. 2).

The folds are of various dimensions and are mainly longitudinal, some taking spiral course(Fig 3).

Fig. 2: Scanning electron photomicrograph of resorptive body (RB) of P. pictus Showingfolds over the surface. TR - trachea.

Fig. 3: Enlarged surface area of resorptive body showing spiral fold (SR).

The oocytes in advanced stage of oosorption clearly show that their surfaces are

degenerated and damaged with the presence of a number of broken areas (Fig. 4). The

surface bears a'number of blunt warts of various dimensions some of which have theirsurface cracked (Fig. 5). lt also appeared that the oosorbed materials are exuded-out in

the form of granules of various shapes and dimensions (Fig. 6, 7).

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PROC. ZOOL. SOC. tNDtA

Fig. 4: SEM of resorptive body showing degenerated and damaged surface (.). Note thewarts (W)over the surface.

Fig. 5: Highly magnified SEM photogiaph showing the cracks (c) over the resorptivebody surface.

Fig. 6: SEM showing exudations of various dimensions over the surface of resorptive body.

Fig. 7: Magnified SEM showing blunt warts O over the surface of resorptive body which ultimately

break to release material.

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HISTOLOGY

Sections of the basal oocytes in advanced stage of oosorption when studiedunder light microscope revealed the following features:

i) The follicular epithelium is disorganized but many of the cells still remainattached to tunica propria,

ii) tunica propria is considerably folded,

iii) oocyte nucleus disorganized,

iv) decreased size of yolk spheres obviously because of their breakdown, and

v) the cytoplasm is extensively vacuolated.

Studies by transmission electron microscopy also revealed extensive vacuolationof cytoplasm and nuclearfragmentation (Fig.8,9 and 10).

Fig.8: Transmission electron micrograph of resorption body showlng many vacuoles (V)and nuclear fragments (.) in the cytoplasm.

Fig. 9: TEM photomicrograph of resorption body showing vacuoles (V) in the cytoplasm(highly magnified).

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PROC. ZOOL. SOC. tNDtA

Fig. 10: Transmission electron micrograph of resorption body (highly-magnified) showingnuclear fragments (NF) scattered in the cytoplasm.

DISCUSSION

Vitellogenic oocytes undergo resorption in many groups of insects. The oocytesenter resorption at any stage of their growth and thus resorption is not time and statebound event in the ovarioles. ln Poekilocerus plcfus the average number of ovarioles in

the paired ovaries is 180 but a female has never been found to lay these numbers ofeggs. Raziuddin ef al., 1977 has reported 66 to 136 eggs / pod in this insect. LateronAnwar and Raziuddin (2003) have reported 114.60 t 3.81 eggs /pod in field-collectedgravid females and 83.87 t 3.78 eggs / pod in laboratory-reared P. pictus females. Almostsimilar observations have been made by other workers viz., Pruthi and Nigam (1939),Menon (1952), Parihar (1974) in this insect. Variations in the number of eggs /poddescribed by different authors is'most likely due to difference in rearing conditions indifferent laboratories as well as difference in the environmental conditions prevailing inthe fields in different regions. ln all the observations the number of eggs/ pod in P.pictus.is much less than the total number of ovarioles in the ovaries in a female. The differencein the total number of ovanoles in the ovaries of femalesand number of eggs laid by themis obviously due to the fact that many of the vitellogenic basal oocytes in the ovaries donot mature and are resorbed sometime during their development even though apparentlyoptimal conditions prevailed in the laboratory or in the field. Under the most favourableconditions, in the desert locust, with about 50 ovarioles / ovary, nearly 22o/o of oocytesunciergo oosorption (Highnam and Hill, 1977). Similarly in females of Locusfa about 25%oocytes were resorbed even when these were reared on high quality food (see Chapman,

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2OO1). tn autogenous females 'of Chrysomya (Screw-worm fly) 30% of the first batch of

oocytes was resorbed and even if these were reared on additional protein, 10% of the

oocytes underwent oosorption (Spradberry and Schweizer, 1981).

The present results clearly demonstrate that abq$t- 45% of the basal oocytes

underwent oosorption during maturation and thus only about 55% of the total ovarioles

were able to mature and were subsequently laid by the females. Thus the percentage of

oocyte resorption is appreciably high in P picfus in comparison to other insects described

above. lt appears that the high percentage of oosorption i:n P. pictus which is almost

similar in size to the desert locust is because of the presence of much higher number of

., ovarioles in the ovaries competing for nutrients and other factors during oocyte

development.

ln Locusfa the percentage of resorbed oocytes is inversely proportional to the

quantity of the amount of grass eaten by the female (McCaffery, 1975). ln Cimex low

levels of protein lead to resorption of oocytes (see Chapman,2001). ln the egg

development of Aedes aegyptiresorption of oocytes depends on the quantity of blood

and the interval between blood-meals (Lea et a|.,1978). Besides food, adverse conditions

such as parasitisation of the insect (Liu, 1992), absence of proper mating (Willis ef a/.,

1958; Highnam ef a/., 1963; Anwar and Raziuddin; 2003), extremes of photoperiods (Verma

and Raziuddin, 1993a, 1993b; Anwar and Raziuddin, 2002), age of the insect and inability

to produce or lay fertile eggs (see Chapman, 2OO1), unfavourable conditions for egg

laying (Phipps, 1966) etc. also lead to resorption of oocytes.

According to Highnam ef a/ (1963) in Schistocerca gregaria competition between

the vitellogenic oocytes for available proteins and corpus allatum hormone play a key

role in oocyte resorption, Resorption of a certain proportion of vitellogenic oocytes thus

appears to provide a mechanism for increasing availability of nutrients and hormone (which

facilitate vitellogenesis) to the remaining growing oocytes to ensure formation of eggs

with adequate quantity qf yolk (Be!l and Bohm, 1975; Highnam and Hill, 1977).

ln P. pictus marked morphological and histological changes occur in the oocytes

undergoing oosorption. Light microscopical findings of resorption bodies of P. pictus

revealthat the follicular epithelium becomes folded and disorganized and the cytoplasm

becomes extensively vacuolated resulting in the shrinkage and collapse of the oocyte to

form a resorption body. SEM and TEM studies confirm the above facts. ln the SEM study

shrinkage, damage and di6oiganization of the surface are clearly revealed.'lt also appears

that the materials fioh the resorbed oocyte are returned back to the haemolymph pool in

the form of droplets which are to be utilized by other developing ogcytes.

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AGKNOWLEDGEMENTS

The authors wish to express theiigratitude to the Director, lndian Association forthe Cultivation of Science, Jadavpur, Kolkata and the Director, lndian lnstitute of ChemicalBiology, Jadavpur, Kolkata for providing facilities of electron microscopy.

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