pesticidal effects of annona squamosa l. on male oryctes ... · pesticidal effects of annona...

Current Biotica 6(1): 8-21, 2012 ISSN 0973-4031

8 ________________________________________________________________________

www.currentbiotica.com

Pesticidal effects of Annona squamosa L. on male Oryctes rhinoceros Linn. (Coleoptera : Scarabaeidae) in relation to reproduction

C. Sreeletha* and P.R. Geetha

Post Graduate Department of Zoology and Research Center, M. G. College,

Thiruvananthapuram – 695 012, Kerala, India *E- mail : [email protected]

ABSTRACT

Pesticidal effect of Annona squamosa L. leaves on the fat body and testis of Oryctes rhinoceros Linn. was studied by incorporating the leaf powder along with the culture medium, cowdung. The grubs were reared in the Annona leaf powder-cow dung mixture, in different proportions (5%, 10%, 20%, w /w). The beetles grown in cow dung alone served as control. Larval mortality, pupal mortality and abnormal adult formation of O. rhinoceros were 50%, 20% and 20%, and 30%, 20% and 20% in 20% and 10% w/w proportion of leaf powder cow dung mixture, respectively while only 10% larval and pupal mortality was recorded in 5% leaf powder cow dung mixture thus exhibiting maximum survival rate and hence this particular dosage was employed for rearing adult O. rhinoceros for studying the effect of A. squamosa on the development of fat body and testis. The fat body and testis were subjected to morphological, histological, biochemical and Electrophoretic (SDS-PAGE) analysis. In the experimental groups certain morpohological changes like enlargement of lobes in parietal fat body, failure of lobe formation in visceral fat body and reduction in the size of testis were observed. Histological changes like disintegration of cell membrane and pycnotic nucleus in the fat body cells and reduction in number and size of spermatids in testis were observed. Biochemical changes like reduction in protein content were observed in fat body as well as testis. SDS-PAGE analysis revealed the failure of transfer of certain protein bands from fat body to testis. As these changes were observed only in those beetles reared in plant powder culture medium, the active components of A. squamosa may be explored for its insecticidal activity and its utilization in pest management programs. KEY WORDS: Annona squamosa, fat bodies, Oryctes rhinoceros, pesticide, SDS-PAGE INTRODUCTION

Pests posed grave concern to mankind, since the days he started farming. Oryctes rhinoceros Linn. is one of the serious pests of coconut palm Cocos nucifera L. The adult beetle bores into the unopened fronds and inflorescence of the palm. Decaying organic matter and cattle dung are the breeding sites of the adult beetle. The emerging grub grows by feeding on the organic matter and takes

about 100-250 days to become an adult (Nirula, 1955). Treatment of breeding sites with insecticides and application of pesticides on the infested region of coconut is the usual practice to control this pest. This control method is problematic not only because of development of resistance to insecticides by the grubs but also the negative effect of pesticides on the environment. Botanical insecticides, which contain plant extracts as active components are safer as well as


9 ________________________________________________________________________


environmental friendly than synthetic insecticides. Botanical products are useful tools in many pest management programes, as they are effective against pests and safer to natural enemies (Isman, 2006). Plant extracts are capable of producing multiple effects in insects such as antifeedancy, growth regulation fecundity suppression, sterilization, ovipositional changes, repellency or attractancy and change in the biological fitness like reduced life span, loss of flying ability, low absorption of nutrients, high mortality, immune depression, enzyme inhibition and disruption of biological synthesis (Samidurai et al., 2009). Botanicals are reported to be safer than synthetic insecticides, easily degradable, environmentally safe, broad spectrum in action non-persistent and easily processed (Solsoloy and Solsoloy, 1995: Talukadar and Howse, 1995). Thus, the botanicals offer desirable alternatives to use of synthetic chemicals in the agro ecosystem where protection of the environment and preservation of beneficial organism are important (Weathersbee and Tang, 2002). More than 2000 plant species have been known to produce chemical factors and metabolites of value in pest control programme (Ahmed, 1984; Emara et al., 2002; Talukader, 2006). The present investigation aims to find out the possibility of ecofriendly management of the pest, Oryctes rhinoceros by incorporating leaf powder of a commonly available plant Annona squamosa L. in its natural breeding site. Annonaceae (custard apple family) is a large family of tropical trees and shrubs comprising about 130 genera and 2300 species. Some plants of this family have been used traditionally as insecticides (Castillo et al., 2010). The phytochemicals like flavonoids (Seetharaman,1980) have been reported to isolate from the leaves of

A. squamosa. The seeds of A.squamosa are known to show insecticidal property and germicidal activity. Brady et al. (1978) reported that the seeds of A. squamosa contain chemicals known as acetogenins, which are toxic to insects. Hemalatha et al. (2001) tested foliar extracts and reported antimicrobial activity against common microbial infestants of pulses and insecticidal activity against pulse beetle, Callasobruchus chinensis. Mehra (1988) has reported the efficacy of A.squamosa against larvae and pupae of Culex quinquefasciatus. The biowash (washings of vermicompost) prepared from the foliage of Annona and Jatropha were shown to have high level of inhibition against Sclerotium rolfsii, a pathogen of chickpea and sorghum (Subramanian et al., 2010). The objective of this study is to determine the destructive effects exercised by A. squamosa on the fat body and testis of adult male of O. rhinoceros MATERIALS AND METHODS Insect rearing Adult male O.rhinoceros beetles reared in the laboratory, from the third instar grubs, collected from local manure pits in the vicinity of Mahathma Gandhi College campus, Thiruvananthapuram during 2008-2010. Rearing methods described by Mini and Prabhu (1986) was adopted in the present study. The beetles were maintained at temperature 23 °C – 33 °C and relative humidity 70%-90%. The adult which attain sexual maturity at 30-35 days (Mini, 1993; Sreelatha 2008) were used for the experiment. The beetles were fed on slices of banana.


10 ________________________________________________________________________


Preparation of culture medium Tender leaves of A. squamosa were collected from nearby places. The leaves were allowed to be shade dried for one hour and chopped into small pieces and mixed with sun dried cow dung in three different proportions. Cow dung without leaf powder served as control. Experiment with grubs The plant powder [5%, 10% and 20% (w/w)] was thoroughly mixed with sterilized cow dung. Late third instar grubs were introduced individually in small containers of size 10x5 cm, with 125 g cow-dung plant mixture. The containers were covered with perforated plastic lids. The culture medium was changed by week until the grubs became pupae. Daily observations were made on the development of grubs. Larval and pupal mortality and formation of any abnormal adults were recorded. Experiment with adult beetles Morphological, histological and biochemical studies

Insects were anaesthetized by cooling in a freezer (4 °C) and dissected under binocular dissection microscope. Thorax and abdomen were opened and the fat body lying beneath the body wall and around the gut was taken out after severing it from reproductive organs. Similarly testis lobes were taken out. Microscopic observation of fresh unfixed fat body and testis was done. Photographs were taken using Kodak m1033 digital camera. The fat body and testis were removed and fixed overnight in acqueous bouins fluid and processed for histological studies (Gurr, 1962). Paraffin blocks were made.

Sections were cut at 5µ thickness and stained in Ehrlich’s Hematoxylene and Eosine, mounted in DPX and examined under the light microscope.

Total protein content was estimated following the method of Lowry et al. (1951) using bovine serum albumin as standard. The tissue extracts were mixed with the protein reagent (alkaline copper sulphate solution) incubated for ten minutes at room temperature followed by addition of Folin-phenol reagent. The reaction mixture was further incubated at room temperature for thirty minutes. The absorbance of blue colour was monitored at 580 nm using spectrophotometer. Simultaneously a blank was also prepared containing all the reagents except the protein. Electrophoretic separation of the proteins present in parietal fat body, visceral fat body and testis of beetles grown in control and plant powder media were performed, under denaturing and discontinuous conditions on 10% SDS-PAGE using vertical gel unit as per Laemmli (1970) with slight modifications. RESULTS AND DISCUSSION

The data presented in the Table 1 shows that larval mortality was 50% in 20% plant powder cow dung mixture. 30% and 10% larval mortality was observed in 10% and 5% leaf powder mixture, respectively. Pupal mortality was 20% in 20% and 10% leaf powder medium and 10% in 5% leaf powder mixture. Hence, total mortality was 70%, 50% and 20% in 20%, 10% and 5% leaf powder medium, respectively, while no mortality was observed in control. Percentage of deformed adults (Fig.1) were 20% in 20% and 10% leaf powder medium and 10% in 5% leaf powder medium. No deformed adults were formed in control (Fig. 2). The


11 ________________________________________________________________________


present work demonstrated the potential of A. squamosa to induce larval and pupal mortality and formation of deformed adults in a dose dependent manner. Maximum mortality was observed in 20% A. squamosa cow dung medium followed by 10% and 5% plant powder culture medium. Musca domestica larvae exposed to the ethanol extract of A. squamosa is reported to cause significant mortality in a dose dependent manner. Morphological changes like darkening of the puparium, condensed appendages and failure to metamorphose were also recorded (Nighat Begum et al., 2010). Sandhya Jadhav (2009) has reported that Annona seed extract was an effective toxin to the larvae of Corcyra cephalonica.

Morphological, Histological and Biochemical observations were done in 35 day old beetles reared in 5% leaf powder cow dung medium. The fat body is divided into a peripheral portion located underneath the body wall is termed as parietal fat body and central mass which exist as a compact mesh work of anastomotic lobes in the space between body wall and gut referred here as visceral fat body. The control beetles exhibited similar morphological features as that was seen in normal beetles. The parietal fat body was composed of lobes attached by tracheal stands to main lateral trachea (Fig. 3a). The visceral fat body was composed of globular lobes attached by thin strands to visceral trachea around gut (Fig.4a). The morphological observations of the treated beetle exhibited significant differences from the control. The parietal fat body lobes were unusually enlarged and highly porous. There was a considerable reduction in the number of fat body lobes (Fig. 3b). The visceral fat body also showed reduction in lobe formation. Due to the failure of lobe formation the gut was seen surrounded by poorly developed

fat body .Hence lengthy white coloured ducts remained as such devoid of lobes (Fig 4b). Visceral fat body of control was seen to be empty of their contents. Abnormal enlargement of lobes of parietal fat body and reduction in the lobe formation of visceral fat body of treated O.rhinoceros may be due to the impact of Annona leaf powder.

In an adult beetle the essential parts of reproductive system include a pair of testis and accessory gland seminal vesicle bundle. Each testis is formed of six bun shaped lobes of almost equal size having wavy margin. The reproductive organ is characterized by rich supply of lobes from parietal and visceral fat body (Fig. 5a ). The controls showed similar structural features as that of the normal beetles while the beetles reared on A.squamosa cow dung medium exhibited isolated reproductive system from fat body connections (Fig. 5b). The testis in the treated beetle (Fig.6a) was comparatively smaller than that in the control (Fig. 6b). The weight of the testis in control and treated insect were 115.88±1.84 and 18.25 ±1.06 mg/beetle, respectively. A significant decrease in the testis weight (t=45.99.p<0.0001) was observed in treated beetles with respect to control .Reduction in size of the ovary was reported in Oryctes rhinoceros on treatment with methanolic extract of Annona squamosa (Sreelatha, 2008). The histological observations showed that in normal beetles the parietal fat body and visceral fat body is characterized by the presence of nucleated and vacuolated cells. In controls similar structures were observed (Fig 7a). In beetles grown in plant powder medium, the fat body cells were found disintegrated and nuclei seen scattered and shrunken


12 ________________________________________________________________________


(Fig. 7b). Similar observations have been recorded in Eriopis conexa (Sarmento et al., 2004) and in Brontocoris tabidus (Walkymario et al., 2010). Histological observations noticed changes like disintegration of cell membranes, shrunken and pycnotic nucleus, in treated insects suggest the adverse effect of Annona squamosa. Histology of the testis of controls present definitely arranged bunches of well developed spermatids (Fig. 8a). But in beetles which were reared in A. squamosa leaf powder mixture, a reduction in number and size of the spermatids was observed. The orderly arrangement of spermatids was not found and spermatid bunches were found reduced in size (Fig.8b). These observations suggest the efficacy of A. squamosa to interfere in the reproductive process. Arrested spermatogenic meiosis in Azadirachtin treated deserst locust, Schistocerca gregaria was observed by Linton et al. (1977) which is in conformity with the present study.

Biochemical studies (Table 2) showed that there were significant differences between mean protein content of parietal fat body, visceral fat body and testis of experimental groups and the corresponding controls. The mean protein content in the parietal fat body was 12.09± 0.41 and 5.16± 0.44 mg/g wet tissue, in beetles reared in control and A. squamosa medium respectively. A significant decrease (t=11.5, p<0.0001) in protein content was observed in beetles grown in A.squamosa medium in comparison to control. The mean protein content in the visceral fat body was 45.68 ± 1.17 and 186.2 ± 3.9 mg/g wet tissue in control and treated groups respectively. A significant increase (t=34.5,p<0.0001) in protein content was observed in beetles grown in plant powder medium with respect to

control. In testis the mean protein content was 37.80±0.59 and 9.98±0.43mg/pair respectively in control and treated beetles. A significant reduction (t=38.38, p<0.0001) in protein content was observed in treated beetles in comparison with control. Reduction in protein content observed in the parietal fatbody and testis of the experimental groups may be due to the interference of plant materials in the culture medium. The hypertrophy observed in the visceral fat body of beetles grown in plant powder culture medium may be due to the failure of lobe formation in the visceral fat body. Results of this kind are rarely reported.

SDS-PAGE analysis showed that

all protein bands that were present in normal beetles were present in control (Fig.9a). The Electropherogram of beetles grown in plant powder cow dung medium exhibited two additional bands in testis and one each in parietal fat body and hemolymph (Fig. 9b). The bands V2(43KDa) and V3(97.4KDa) were present in the fat body of beetles grown in plant powder medium. In normal beetles these bands are seen in immature stages but disappear when they mature. This may be attributed to the effect of Annona on reproductive structures. The hypertrophic condition of visceral fat body in the treated beetles may be due to this failure of migration of visceral fat body proteins. Observations of the present study is supported by the reports of Kuruvilla (1993) that on treatment with Neem and Azadirachtin, additional bands were appeared in hemolymph of Eligma narcissus. Biju et al. (2004) reported that after topical and oral administration of the extract of seaweed Bryopsis plumosa in Hyblae puera a reduction in number of protein bands in the hemolymph and fat body was observed.


13 ________________________________________________________________________


CONCLUSION

The morphological changes like enlargement of parietal fatbody lobes, failure of lobe formation in visceral fatbody and reduction in size of testis were observed in beetles grown in Annona leaf powder cow dung medium. Histological changes like disintegration of cell membrane, occurrence of pycnotic nucleus in fatbody, reduction in number and size of spermatids and biochemical changes like reduction in protein content in parietal fatbody and testis, hypertrophic condition of visceral fatbody and failure of migration in protein bands from visceral fatbody to testis were also observed in these plant treated beetles. All these abnormal

changes point towards the insecticidal effects of A. squamosa on O. rhinoceros. The results of the present study suggest that the leaves of A. squamosa are potential sources of biopesticides for managing economically important pests like O. rhinoceros in an ecofriendly manner.

ACKNOWLEDGEMENTS The fellowship granted by the UGC 1s gratefully acknowledged .The author is also thankful for the facilities afforded by the Head of the Research centre.

Table 1: Effect of Annona squamosa leaf powder on the development of O. rhinoceros

Table 2: Effect of A. squamosa, on the protein content of parietal fatbody ( PFB), visceral fatbody (VFB) and testis (T) of adult O.rhinoceros with respect to the control group

Variable Group Mean ± SE t df P

PFB Control 12.09 ± 0.41 - - -

Annona 5.16 ± 0.44 11.55** 14 <0.0001

VFB Control 45.68 ± 1.17 - - -

Annona 186.20 ± 3.90 34.50*** 14 <0.0001

T Control 37.80 ± 0.59 - - -

Annona 9.98 ± 0.43 38.38*** 14 <0.0001 *** Significant at 0.0001; ** Significant at 0.01 level

Dosage (% plant powder)

Initial number of

larva

% of larval mortality

% of pupal mortality

% of total mortality

% Deformed

adults 5 20 10 10 20 10

10 20 30 20 50 20

20 20 50 20 70 20

control 20 00 00 00 00


14 ________________________________________________________________________


Fig. 1: Deformed adult beetles emerged from plant powder cow dung culture medium

Fig. 2: Normal adult beetle emerged from control medium


15 ________________________________________________________________________


Fig. 3a: Normal parietal fat body lobes in control Fig. 3b: Enlarged fat body lobes of

beetles grown in A. squamosa medium. FBL-Fat body lobes

Fig. 4a: Visceral fat body; Densely packed fat body lobes around the gut in control Fig. 4b: Failure

of lobe formation in beetlesgrown in A. squamosa medium FBL-Fat body lobe, G-Gut, MWD-

Milky white colored duct

3a 3b

4a 4b


16 ________________________________________________________________________


Fig. 5 a: Reproductive system; Packed within normal type of fat body in control

Fig. 5b: Isolated and fluffy reproductive system in beetles grown in A. squamosa FBL- Fat body

lobe, TL-Testis lobe

Fig. 6a: Reduced testis in beetles grown in A.squamosa medium

Fig. 6b: Fig. Normal testis in control

5 a

5 b

6 a 6 b

5 b


17 ________________________________________________________________________


Fig. 7 a: Definitely arranged nucleated cells in control. Fig. 7 b: Cells showing shrunken and

pycnotic nucleus and disintegrated cell membrane inbeetles grown in

A. squamosa. n-nucleus, Cy- cytoplasm, Pyn–Pycnotic nucleus

Fig . 8a: Mature testis with orderly arranged spermatid clusters and normal spermatid in control

Fig. 8b : Scattered spermatid clusters with reduced abnormal spermatids in beetles grown in A.

squamosa; Spd- spermatid, Asd-Abnormal spermatid

8 a 8b

7 a 7b


18 ________________________________________________________________________


Fig. 9a: Protein profile in the Parietal Fat Body (PFB), Visceral Fat Body (VFB), Hemolymph (HL) and Testis (T) of beetles grown in control medium

Fig. 9b: Protein profile in the Parietal Fat Body (PFB), Visceral Fat Body (VFB),

Hemolymph (HL) and Testis (T) of beetles grown in A. squamosa


19 ________________________________________________________________________


REFERENCES

Ahmed, S.,Graivge, M., Hylin, J.W., Mitchell, W. C. and Listinger, J. A. (1984). Some promising plant species for use as pest control agents under traditional farming system. In: Proceedings of Second International Neem Conference. ((Eds.Schmutteer,H.and Ascher. K.R.S.) Germany. 565-80.

Brady, N. C, Khush, G. S. and Heinrichs, E. A. (1978). Visit of the IRRI team to the socialist Republic of Vietnam. April. 13- May 19. IRRI. Annual Report: 49.

Biju, B., Mariamma Jacob., Padmakumar and Muraleedharan, D. (2004) Effect of extract of the sea weed Bryopsis plumose (Huds.) Ag. on the feeding rate and protein profile of haemolymph and fat body of Hyblaea puera (Cramer) (Lepidoptera : Hyblacidac) Entomon. 29; 271-276.

Castillo, L. E, Jimenez, J. J, Delgado, M. A(2010). Secondary metabolites of the Annonaceae, Solanaceae and Meliaceae families used as biological control of the insects. Tropical and subtropical Agroecosystems. 12; 445-462

Emara, S., Bakr, F.R., El- Bersmawy, S., Abulyazid, I. and Abdel-Wahab. (2002) Biological effects of four botanical extracts on the different developmental stages of cotton leaf worm Spodoptera litoralis. Second International Conference. Plant Protection Research Institute, Cairo, 1; 904-916.

Gurr, E.(1962) Staining animal tissues, Practical and Theoretical.

Leonard Hill (Books) Ltd, London.

Hemalatha, M. K Prashant, S. M, Sangeeta,V.G. S. S, Shipra R.J, Shripad, M.U. and Maheshwar, V. L. (2001). Antimicrobial and pesticidal activity of partially purified flavonoids of Annona squamosa. Pest Management Science. 58; 33-37.

Isman, M.B.(2006). Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annual Review of Entomology. 51; 45-66.

Kuruvilla Sarasherly.(1993). Studies on the influence of phytochemicals on the spermatogenesis of certain insect pests. Ph.D Thesis, University of Kerala, Thiruvananthapuram.

Laemmli, U. K.(1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227; 680-689.

Linton, Y. M., Nisbet, A. J., Mordue (Luntz), A. J. (1997). The effects of Azadirachtin on the testis of the desert locust Schistocerca gregaria (Forskal ). J. Insect Physiol. 43; 1077-1084.

Lowry, O.H, Rosen brough, N.J Farr, A.L, Randall, R. J.(1951). Protein measurement with Folin phenol reagent. J. Bio. Chem. 193; 265-275

Mehra, B. K, Hiradhar, P. K.(2000). Effect of crude acetone extract of seeds of Annona squamosa Linn.


20 ________________________________________________________________________


(Family: Annonaceae) on possible control potential against larvae of Culex quinquefasciatus say. J. Entomol Res. 24 (2); 141-6.

Mini, A and Prabhu, V. K. K.(1986).Significance of critical developmental stages on starvation induced endocrine mediated precocious metamorphosis in Oryctes rhinoceros (Coleoptera: Scarabaeidae). Proc. Indian Acad. Sci. (Anim.Sci). 95(4); 379-385

Mini, A. (1993).Some aspects of reproductive behavior in the coconut rhinoceros beetle Oryctes rhinoceros. Ph. D Thesis, Kerala University, Thiruvananthapuram.

Nigat Begum, Bechan Sharma and Ravi S. Pandey. (2010). Evaluation of insecticidal Efficacy of Calotropis procera and Annona squamosa ethanol extracts against Musca domestica. J. Biofertil Biopestici.1; 1-6.

Nirula, K. K.(1955). Investigations on the pest of coconut palm. Part 2. Oryctes rhinoceros. L. The Indian Coconut Journal. 8; 161-179.

Samidurai, K., Jebanesan, A., Saravana Kumar, A., Govindarajan, M. and Pushpanathan, T.(2009) Larvicidal, ovicidal and repellent activities of Pemphis acidula forst against filarial and dengue vector mosquitoes. Academic Journal of Entomology., 2 (2);62- 66.

Sandhya Jadhav. (2009) Relative toxicity of certain plant extracts against Corcyra cephalonica under laboratory condition. J. Appl. Biosci. 35; 89-90.

Sarmento, R. A., Oliveira H. G., Holtz , A. M., Silva, S. M., Serrao, J. E. And Pallini, A.(2004). Fatbody morphology of Eriopis connexa (Coleoptera: Coccinellidae) in function of two alimentary canal sources. Braz. Arch. Biol. Tech., 47; 407-411.

Seetharaman, T. R.(1986). Flavonoids from the leaves of Annona squamosa and Polyalthia longifolia. Fitoterapia. 57; 189-198.

Solsoloy, A.D. and Solsoloy, J.S.(1995). A safe and effective pesticide. ILEIA News letter, 11 (4); 31.

Sreelatha, K.B.(2008). Studies on some aspects of vitellogenesis in Oryctes rhinoceros (Coleoptera: Scarabaeidae). Ph.D Thesis. Kerala University, Thiruvananthapuram.

Subramanian Gopalakrishnan, Iyer Girish Kumar Kannan, Gottu mukkala Alekhya, Pagidi, Humayun, Sree Vidya, Meesala and Deepti Kanala.(2010). Efficacy of Jatropha, Annona and Parthenium biowash on Sclerotium rolfsii, Fusarium Oxy sporum f. sp. ciceri and Macrophomina phaseolina, pathogens of chickpea and sorghum. African Journal of Biochemistry, 9 (47); 8048- 8057.


21 ________________________________________________________________________


Talukadar, F.A. and Howse, P. E. (1995).Evaluation of Aphanamixis polystachya as a source of repellants, antifeedants, toxicants and protectant in storage against Tribolium castaneum (Herbst). Journal of Stored Products Research, 31; 55-61.

Talukader, F. A. (2006).Plant products as potential stored product insect management agent. A mini review. Emir. Journal of Agricultural Sciences., 18; 17-32.

Walkymario Paulo Lemos, Jose Cola Zanuncio, Mabio Chrisley Lacerda, Rafael Coelho Ribeiro and Jose Eduardo serrao.(2010). Morphology aspects of the fat

body and the hemolymph of males of the predator Brontocoris tabidus (Pentatomidae): Effect of the phytophagy and age. ARPN Journal of Agricultural and Biological Science. 5; 32-40.

Weathersbee, A. A and Tang, Y.Q. Effect of neem seed extract on feeding, growth, survival and reproduction of Diaprepes abbreviatus (Coleoptera: Curculionidae). Journal of Ecological Entomology., 95; 661–667.

[MS received 05 May 2012; MS accepted 21 June 2012]

Disclaimer: Statements, information, scientific names, spellings, inferences, products, style, etc. mentioned in Current Biotica are attributed to the authors

and do in no way imply endorsement/concurrence by Current Biotica. Queries related to articles should be directed to authors and not to editorial board.

pesticidal effects of annona squamosa l. on male oryctes ... · pesticidal effects of annona...

Documents