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Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.



J.P. Pandey and R.K. Tiwari
 
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ABSTRACT

The red cotton bug, Dysdercus cingulatus, an important polyphagous pest, causes heavy loss to cotton and lady’s finger (bhindi) crops which badly affects the economy of poor farmers. In the present study, the impact of different concentrations of neem based insecticides (NBIs) on coupling, moulting, development, hemocyte, fecundity and egg-hatching of D. cingulatus was evaluated. Various concentrations (0.25, 0.5, 1.0 and 2.5%) and doses (2.5 to 20 μL) of NBIs viz. neemark, multineem and neemazal were prepared by diluting them in acetone were applied topically on the dorsum of the body of nymphs and adults and eggs of different age groups. NBIs causes developmental abnormalities such as prolongation of nymphal periods, ecdysial stasis, formation of adultoids and adults with varied degrees of wing deformities. The delay in moulting of treated nymphs is found concentration, dose and stage dependent. The application of NBIs on cephalic and thoracic regions and on ventral surface of insects showed more sensitivity than on abdominal region and dorsal surface. The NBIs application interrupted the coupling and cause lessening of fecundity and egg-hatching also. Among aforesaid NBIs, neemazal treatment was somewhat more effective. In addition, significant reduction in Total Hemocyte Count (THC) and deformities in hemocyte morphology were also observed in NBIs treated insects. It is assumed that the phenomena like metamorphic developments, coupling and fecundity are controlled by Juvenile Hormone (JH) via regulating the release of allatotropic factor from the brain and all these effects of NBIs are stage specific and concentration and dose dependent. NBIs induced changes in hemocyte contour might be also regulated by the brain hormone.

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J.P. Pandey and R.K. Tiwari, 2011. Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.. International Journal of Agricultural Research, 6: 335-346.

DOI: 10.3923/ijar.2011.335.346

URL: https://scialert.net/abstract/?doi=ijar.2011.335.346
 
Received: January 27, 2011; Accepted: March 10, 2011; Published: May 21, 2011



INTRODUCTION

The synthetic pesticides have been a menace to human health and are hazardous to our environment causing phytotoxicity, pollution, development of insecticide resistance and negative impact on non-target organisms (Ogbuewu et al., 2011; Boursier et al., 2011; Jadeja et al., 2011). Considering all these aspects, many chemical insecticides have been barred in most of the advanced countries. Now-a-days, eco-friendly methods are being adopted that are rather precise for the target species without harming the other organisms to a great extent. The neem tree Azadirachta indica, thus, has emerged as a most important source of eco-friendly insecticide. Neem contains various biological functions viz. feeding and oviposition deterrent, repellent, sterilant, larvicidal, growth regulatory agent and ovicidal (Singh, 1996; Kumar et al., 2003; Tiwari et al., 2006; Khan et al., 2007; Sadeghian and Mortazaienezhad, 2007; Gunasekaran et al., 2009; Depieri and Martinez, 2010) and showing many effective qualities for insect pest control. More than 300 insect species have now been tested with neem (Singh, 1996). The much varied physiological effects of NBIs on various insect species at different doses are toxicity, change in hemocyte profile, prolongation of larval-pupal durations and developmental deformities (Saxena et al., 1981a, b; Singh, 1996; Medina et al., 2004; Efil et al., 2005; Pandey et al., 2008b; Sharma et al., 2010). It is reported (Koul, 1996) that developmental aberrations caused by azadirachtin are due to the modulation of ecdysteroid titres. Tiwari et al. (2006) proposed that azadirachtin blocks release of neurosecretory material from the corpora cardiaca and others reported (Salehzadeh et al., 2003) anti-mitotic effect of the neem-terpenoid azadirachtin on cultured insect cells. Impact of azadirachtin on alterations of acetyl-cholinesterase and electrical activity in the nervous system has been also reported by Shafeek et al. (2004) and Senthil et al. (2008). The studies related with azadirachtin effects, thus, reveal that the endocrine system alone may not be entirely the target of this compound; it has direct effects on a whole variety of tissues and organs as suggested by Koul (1996). Most of the studies regarding NBIs effect on insects are not unanimous and are curbed to few instars of their life only. Exceptionally, a very few studies have been made during entire postembryonic development. It was, therefore, thought enviable to explore this aspect throughout nymphal-adult transformation to link biological changes with the developmental measures and hemocyte morphology.

MATERIALS AND METHODS

Insect culture: The insect species employed in the present study is the red cotton bug, Dysdercus cingulatus (Fabricius) belonging to order Hemiptera. Its life cycle includes five nymphal instars and one imaginal instar. The various nymphal instars and adult insects collected from okra fields in the vicinity of Gyanpur town were reared in glass jars (4.5x3.5 inches) and glass troughs (8.0x4 inches) in a BOD incubator maintained at 28±1°C, 70% RH and 16L: 8 D photoperiod. The mouth of the glass jars and glass troughs were covered with muslin cloth pieces bound with rubber band or wax coated cotton threads. The insects were fed on soaked cotton seeds and water was provided in glass vials plugged with small cotton balls. The containers, cotton seeds and water were changed twice a week. A strip of folded blotting paper was kept in culture jars to provide spaces for hiding and egg laying and to facilitate insect’s movements. The cream colored oval eggs deposited in heaps were separated and kept in fresh glass jars. The eggs hatched into first instar nymphs in 6-9 days. There are five nymphal instars of which the first lasts for 7-9 days and each of the remaining instars take 4-5 days to moult into the next stage. The ultimate (fifth) instar nymphs take 6 days to become adults and thus a total of 35-40 days were required from egg to adult. One generation of insect culture was acclimatized at aforesaid controlled condition. Newly emerged V instar nymphs or adults were regarded as 0 h old V instar nymphs or adults and different ages (viz. 24, 48, 72, 96 and so on) of these two instars were reckoned from 0 h stage. Different stage and age group insects from the said lab culture were used. Research work was carried out during the year 2003-2004 at Department of Zoology, K.N. Govt. P.G. College, Gyanpur, Bhadohi, India.

Neem based insecticides (NBIs): Neem based insesticides viz. neemark (neem oil based E.C. containing azadirachtin 0.03% EC 300 PPM minimum, West Coast Herbochem Ltd. Mumbai, India), multineem (seed extract containing 0.03% azadirachtin; Multiplex Fertilizers Pvt. Ltd., Bangalore, India) and neemazal T/S (azadirachtin 1%, other limonoids 3%, oil fatty acids glycerol esters 46.3%, polyethylene monosorbitol oleate 49.7%; EID Parry, India Ltd.) procured from city markets of Varanasi and Allahabad were used in present study.

Neem based insecticides treatment: Various concentrations of NBIs (0.25, 0.5, 1.0 and 2.5%) were prepared by diluting them in acetone and different doses were applied topically with the help of a glass micropipette on the dorsum of the body of nymphs and adults and eggs of different age groups. Various age group insects were treated with required concentrations and doses of NBIs whereas control insects received a treatment with acetone only. The effects of above treatments on survival, sensitivity, hemocyte morphology moulting, coupling, degrees of development, wing shape, fecundity and egg hatching of test insects were observed.

Hemocyte study: The fresh hemolymph samples from control and NBIs treated insects were collected individually by cutting the antennae on a slide and mixed well with anti-coagulant. A thin uniform smear of hemolymph was spread on the slide by rubbing the edge of an inclined slide backward. Stock solution of Giemsa stain was prepared by employing the method of Yeager (1945). For total hemocyte count (THC), the hemolymph was drawn into a thoma blood-cell pipette up to its graduated mark of 0.5 and diluted up to the 11th mark with Tauber-Yeager’s fluid (Tauber and Yeager, 1935). The number of circulating hemocytes per cubic millimetre (mm3) was calculated using the formula of Jones (1962). The methods of smear formation, staining, THC and hemocyte categorization were similar to those applied earlier (Tiwari et al., 2006; Pandey et al., 2010).

Statistical analysis: The Impact of NBIs on survival, moulting, coupling, fecundity and egg hatching of test and control insects were observed and experiment was repeated three times. Data from thirty insect were taken from each category and analysed statistically using Students‘t’ test and presented as Mean±SD.

RESULTS

The interactions of NBIs in different concentrations and doses with sensitivity, moulting, coupling, fecundity, egg hatching and hemocyte morphology of D. cingulatus were described here briefly.

NBIs interaction with sensitivity and morphogenetic development: The application of NBIs on cephalic and thoracic regions and on ventral surface of insects showed more sensitivity than on abdominal region and dorsal surface. Immediately after treatment with a lower dose, the insects showed a swift movement hither and thither but their movement is immediately stopped for a while before getting them normal at higher doses. The different concentrations and doses of three NBIs used in present study affect the morphogenetic development and each NBI effect is stage and age specific. Among three NBIs, neemazal treatment was much effective than neemark and multineem. While 5 μL dose of neemark at 0.5% concentration inhibited the moulting of late III instar nymphs into IV instar and caused their mortality after surviving for 3 days in majority of the treated insects, it also led into the death of those IV instars which emerged from treated III instars after 3 days of survival. Application of the said dose and concentration on late IV nymphal instar though prolonged their survival period up to 9 days showing ecdysial stasis (Fig. 1a) but could not avert their mortality after this duration whereas a dose of 30 μL at the same concentration caused immediate mortality.

Image for - Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.
Fig. 1: (A-J) Showing effect of NBIs on morphogenetic development of red cotton bug, Dysdercus cingulatus (a) IV nymphal instar showing complete ecdysial stasis following application of 5 μL dose of 0.5 % neemark. (b) an adult bug with less deformed left hindwing (arrow) emerging from 3 day old V instar nymph treated with 5 μL dose of 0.5% neemark. (c) adult bug with deformed right forewing (arrow) emerging from 3 day old V instar nymph treated with 10 μL dose of 0.5% neemark. (d) An adultoid with little larger wings and sunken abdomen as adult and black abdominal spots as nymphal characters following application of 15 μL dose of 0.5% neemark on 3 day old V instar nymph (e) An adultoid with a little larger forewing (thin arrows) and left hindwing (thick arrow) after application of 5 μL dose of 0.5% neemazal on 3 day old V instar nymph. (f) The adult bug with much reduced and deformed right forewing (arrow) and without right hindwing emerging from 3 day old V instar nymph treated with 10 μL dose of 0.5% multineem. (g) The adult bug with much deformed right forewing (thin arrow) and exuviae attached to posterior body end (thick arrow) emerging from 3 day old V instar nymph treated with 10 μL dose of 0.5% neemark. (h) An adultoid with exuviae at posterior body end (thin arrow) and small wing (thick arrow) as nymphal character and disappearance of black abdominal spots as adult characters following application of 8 μL dose of 0.25% neemazal on 3 day old V instar nymph. (i) A normal V instar nymph. (j) A normal adult

The application of 5μL dose of 0.5% neemark on 3 day old V instar nymph resulted adult bug with less deformed left hind wing (Fig. 1b). Treatment of 10 μL dose of 0.5% neemark on 3 day old V instar nymph yielded adult bug with deformed right forewing (Fig. 1c) but when 3 day old V instar nymphs were treated with 15μL dose of 0.5% neemark, it formed adultoids with little larger wings and sunken abdomen as adult and black abdominal spots as nymphal characters (Fig. 1d). The 5 μL dose of 0.5% neemazal application on 3 day old V instar nymphs resulted adultoids with a little larger forewing and left hindwing (Fig. 1e) but while treatment of 3 day old V instar nymphs with 10 μL dose of 0.5% multineem formed adult bug with much reduced and deformed right forewing and without right hindwing (Fig. 1f). Treatment of 3 day old V instar nymph with 10 μL dose of 0.5% neemark resulted adult bug with much deformed right forewing and exuviae attached to posterior body end (Fig. 1g) but 8 μL dose of 0.25% neemazal on 3 day old V instar nymph formed adultoid with exuviae at posterior body end and small wing as nymphal character and disappearance of black abdominal spots as adult characters (Fig. 1h). There was no molting deformity in acetone treated respective control insects and all produced nymphs (Fig. 1i) and adults (Fig. 1j).

NBIs interaction with coupling, fecundity and egg hatching: The copulation occurs on the second day of the adult emergence in control insects and continues up to few hours before egg laying. It has been normally observed that the females have six egg cycles, the first taking 7-8 days and remaining ones 4-5 days each. In case of newly emerged adults, a considerable reduction in coupling period (only for ½ h) was observed if the four day old virgin females were treated with NBIs and allowed to couple with normal males. It was further observed that the total number of eggs laid during the first egg laying cycle and their hatchability were very much reduced in experimental insects. The 1% concentration and 10 μL dose of neemark proved fatal for adult bugs but a lesser concentration of 0.5% interrupted the continued and prolonged coupling resulting in a loss of fecundity and hatchability of eggs. Application of 0.5% concentration of neemark with 5 μL dose on 3 day old V instar nymphs could not stop the moulting but episodic coupling, little deformity in wings and reduction in fecundity and egg-hatching % were recorded. Similarly, equal concentration of same NBI with 10 μL dose caused episodic coupling, much deformity in wings and heavy reduction in fecundity and egg-hatching percentage. The 0.5% concentration of neemark with 20 μL dose was lethal but 15 μL dose caused only 40% mortality and 60% adultoids. Moulting deformities, episodic coupling, reduction in fecundity and decrease in egg-hatching percentage were also observed in multineem treated insects. In case of neemazal treated insects, even less concentration (0.25%) and dose (10 μL) was effectual than 0.5% concentration and 10 μL dose of neemark and multineem individually (Table 1). Likewise, various concentrations (0.5 and 1%) and doses (10 and 20 μL) of NBIs application on different age groups of adult caused episodic coupling, mortality, reduction in fecundity and sharp decline in hatching percentage and these effects were age specific (Table 2). Similarly, topical application of 2.5 and 0.5% concentration and 10μL dose of NBIs caused decrease in egg-hatching percentage. In contrast to 90% egg hatching in control, only 15, 16 and 5% eggs were hatched when 4 day old eggs were treated with of 2.5% concentration and 10 μL dose of neemark, multineem and neemazal respectively. Interestingly, eggs of 0.5% (10 μL) NBIs treated bugs were much sensitive to NBIs than eggs from normal (untreated) bugs. Among three NBIs, neemazal treatment was much effective than neemark and multineem (Table 3).

Table 1: Impact of topical application of azadirachtin containing NBIs on three day old fifth instar nymphs of D. cingulatus
Image for - Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.
- = Insect died prior to adult stage. Values represent Mean±SD for 30 insects

Table 2: Impact of topical application of azdirachtin containing NBIs on adult D. cingulatus egg-hatching
Image for - Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.
-: Adult Insect died prior to coupling and egg laying, therefore, no fecundity and egg-hatching. Values represent Mean±SD for 30 insects

Table 3: Impact of topical application of azdirachtin containing NBIs on D. cingulatus eggs
Image for - Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.
Values represent Mean±SD for 30 insects

NBIs interaction with THC and hemocyte morphology: Significant reduction in THC was observed in all NBIs treated nymphs and adults in comparison to control insects (data not shown). Five types of hemocyte have been recognized in both V instar nymphs and adults. They are prohaemocytes (PRs), plasmatocytes (PLs), granulocytes (GRs), adipohaemocytes (ADs) and oenocytoids (OEs) (Fig. 2A-F). Control insect GR showed distinct cytoplasmic granules (Fig. 2F) and application of 5 μL dose of 2.5% neemark on 3 day old V instar nymph caused breakdown of GRs (Fig. 2G) some GRs devoid of plasma membrane (Fig. 2H). In treated insects, mostly PLs and GRs are seen to participate in capsule formation by fusing with each other. While 5μL dose of 0.5% neemazal on 3 day old V instar nymph resulted PL without pseudopods (Fig. 2I) and PLs lost their pseudopods, their consistency and became transparent after application of 5 μL dose of 2.5% multineem on 3 day old V instar nymph (Fig. 2J), it also resulted PLs without pseudopods (Fig. 2K). Vacuolization of the cells mostly of GRs and PLs occurred frequently was observed in hemolymph smear of neemazal treated (5 μL dose of 0.5%) 3 day old V instar nymph (Fig. 2L). A large number of nodules (arrows) seen in the blood smear following 5 μL dose of 2.5 neemark on 3 day old V instar nymph (Fig. 2M). Application of 5μL dose of 0.5% neemazal on 1 day old adult bug induced aggregation of hemocytes (Fig. 2N). The 5μL dose of 0.5% neemazal on 3 day old V instar nymph caused GRs with many peripheral vesicles/vacuoles (Fig. 2O) and GRs nuclei digitations leading ultimately into disintegration of cells (Fig. 2P). Similarly broken hemocytes, devoid of plasma membrane (Fig. 2Q) and disintegrating GRs with lysed cellular contents (Fig. 2R) were seen when in 3 day old nymphs were treated with 5 μL dose of 2.5% neemark.

Image for - Neem Based Insecticides Interaction with Development and Fecundity of Red Cotton Bug, Dysdercus cingulatus Fab.
Fig. 2: A-R Showing impact of topical application of NBIs on hemocyte morphology of D. cingulatus (A) different types of hemocytes of control insect x 675. (B) PLs morpho-types in control insect x 675. (C) control PL x 1000. (D) control insect PL showing mitotic division (arrow). x 675. (E) PL variant with a much elongated pseudopod. x 1500. (F) control insect GR with distinct cytoplasmic granules.x 1000. (G) broken GR (arrow) devoid of complete plasma membrane following 5μL dose of 2.5% neemark on 3 day old V instar nymph.x 1000. (H) half broken GRs without plasma membrane following 5 μL dose of 2.5% multineem on 3 day old V instar nymph. x 1000. (I) PL (arrow) without pseudopods following 5 μL dose of 0.5% neemazal on 3 day old V instar nymph. x 1000. (J) PL (arrow) seen with losing cellular compactness following 5 μL dose of 2.5% multineem on 3 day old V instar nymph. x 1500. (K) PL (arrow) without pseudopods following 5 μL dose of 2.5% multineem on 3 day old V instar nymph. x 1500. (L) much vacuolisation of PLs (thick arrows) and GRs (thin arrows) following 5 μL dose of 0.5% neemazal on 3 day old V instar nymph. x 675. (M) A large number of nodules (arrows) seen in the blood smear following 5 μL dose of 2.5 neemark on 3 day old V instar nymph. x 675. (N) hemocyte aggregation following application of 5 μL dose of 0.5% neemazal on 1 day old adult bug. x 675. (O) GRs with many peripheral vesicles/vacuoles following 5 μL dose of 0.5% neemazal on 3 day old V instar nymph.x 1000. (P) GRs nuclei showing digitations leading ultimately into disintegration of cells following 5 μL dose of 0.5% neemazal on 3 day old nymph. x 1000. (Q) broken and disintegrating GRs following 5 μL dose of 2.5% neemark on 3 day old nymph. x 1000. (R) disintegration of cells following 5 μL dose of 2.5% neemark on 3 day old V instar nymph. x 1000

DISCUSSION

It has been observed that the sensitivity of the insects in present investigation against NBIs varies greatly depending upon region and surface of the body. The NBIs applied topically on ventral surface of the cephalo-thoracic region showed more sensitivity than if they were applied on dorsal surface and abdominal region. This variation in their response seems to lie in the fact that the NBIs could have acted directly upon central nervous system lying on the ventral side and ultimately on the brain which lies at the anterior end of the body. A large number of developmental aberrations caused by NBIs treatments observed in present study are in conformity with those of Schmutterer and Ascher (1984), Schmutterer (1988), Singh (1996), Khan and Kumar (2003), Tiwari et al. (2006), Al-Fifi (2006), Gunasekaran et al. (2009), Depieri and Martinez (2010) and Sharma et al. (2010). The factors for these developmental aberrations are reported to lie in the endocrine system and the metamorphosis hormones secreted by them which support the finding of Sieberand Rembold (1983), Schluter et al. (1985), Mordue et al. (1986) and Koul and Isman (1991).

NBI induced prolongation of D. cingulatus nymphal period without moulting to next instar or ecdysial stasis seem to be caused by a change in intrinsic JH titre as reported by Khan and Kumar (2003) in Earias vitella. Production of adultoids or deformed adults by V instar nymphs treated with NBIs, showed their juvenilising effects probably by acting at the level of Corpus Allatum (CA). A similar effect was found by Katiyar and Srivastava (1982) in Callistemon lanceolatus oil treated nymphs of D. koenigii also supports present findings. This indicated their juvenoid or JH-mimick behaviour. In the present study, the failures of hatching of multineem treated eggs indicated its ovicidal effects and substantiate the finding of Sharma et al. (2010).

Interruptions in coupling and loss of fecundity have also been observed in NBIs treated V instar nymphs and newly emerged adults. The role of CA or its hormone (JH) in regulation of coupling and egg maturation has been reported earlier by Srivastava and Tiwari (1978) and Tiwari and Srivastava (1979). It is, therefore, assumed that the reductions in coupling period and in egg number are caused by interruption of JH release. The decline in CA hormone decreases the rate of vitellogenin accumulation in the haemolymph, on one hand and its incorporation in the developing oocytes, on the other, with the help of brain hormone.

Further, the lesser percentage of egg hatching in newly emerged adults in comparison to V instar nymphs following topical application of these NBIs is explained on the basis of the fact that since the oogenesis starts in adult bugs, the insecticides are more effective in this developmental stage rather than the latter one. Present findings are thus in contradicted the reports of Tanzubil and McCaffery (1990) and Medina et al. (2004) who did not find the effect of azadirachtin on the percentages of egg-hatch. Since NBIs contains numerous active ingredients to affect the embryonic development of eggs hence, present observations are in contradiction with these authors. Effects of neem products on reproduction have been observed earlier in insects belonging to various orders viz Orthoptera, Hemiptera, Hymenoptera, Lepidoptera and Diptera (Schmutterer, 1995; Tiwari et al., 2006). Vinuela et al. (1999) and Sharma et al. (2010) also found inhibition in egg laying/hatching after azadirachtin containing insecticides application and thus our observations are in agreement with these authors.

The hemocyte response to biological agents as well as toxins has been studied in many insects and the resulting phenomena are phagocytosis, encapsulation and distortion of cell contour or cellular disintegration (Wago and Kitano, 1985). The drastic reduction in THC in present investigation following NBI treatment is also reported by Saxena and Tikku (1990) in D. koenigii, Azambuja et al. (1991) in R. prolixus and Sharma et al. (2003) in S. litura supports present findings. The decrease in hemocyte count may be attributed to the formation of nodules comprising groups of hemocytes or to the inhibition of endocrine glands and their secretions substantiate the finding of Tiwari et al. (2006) where decrease in THC and variation in hemocyte profile was observed in D. koenigii. The damages thus caused to PRs, PLs and GRs as well as the loss of pseudopodial processes of PLs suggested that the defence reactions in present insect have been reduced following NBI application. Furthermore, the changes in hemocyte morphology leading into cell death following NBIs treatments in present study have also been reported earlier by Saxena and Tikku (1990), Sharma et al. (2003), Tiwari et al. (2006) and Pandey et al. (2008b). It is assumed that metamorphic developments, coupling and fecundity are controlled by JH via regulating the release of allatotropic factor from the brain and all these effects of NBIs are stage specific and concentration and dose (volume applied topically) dependent. Since Mini and Prabhu (1986), Mala et al. (1987), Tiwari and Shukla (2000), Pandey and Tiwari (2005) and Pandey et al. (2008a) reported that the stresses like starvation, chilling, heating, injury etc., exert their effects directly upon neurosecretory cells of the brain.

CONCLUSION

On the basis of present experiment with NBIs, it is suggested that the phenomena like metamorphic developments, coupling and fecundity are controlled by JH via regulating the release of allatotropic factor from the brain and all these effects of NBIs are stage specific and concentration and dose (volume applied topically) dependent. Since a number of workers reported that the stresses like starvation, chilling, heating, injury etc. exert their effects directly upon neurosecretory cells of the brain, it is postulated that cell damages/deaths induced by these physical factors as well as NBIs application in present investigation are probably regulated by the brain hormone.

ACKNOWLEDGMENTS

The authors are thankful to Prof. G. C. Maurya, Principal of the College for laboratory facilities.

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