ABSTRACT- Present investigations evaluated the effect of Argemone mexicana leaves extract on gut of Heliothis armigera (Hub.) at different solvents. The effect of leaf extract of Argemone mexicana in ethanol and acetone solvent after 24 and 96 hours of treatment on Heliothis armigera shows severity of the damage of epithelial lining, epithelial cells showed vacuoles at certain places. The gut lining was also found to be damaged and the lumen became wider after the effect of ethanol extract of A. mexicana. In acetone extract of A. mexicana, the thickness of the fore gut wall has been increased due to clumping of the tissue and hence the diameter of the foregut was reduced. The lumen therefore became narrower and columnar epithelial cells showed the vacuoles.
Key-words- Heliothis armigera, Argemone mexicana, Ethanol, acetone, Epithelial lining, Epithelial cells, vacuoles, Gut lining, Gut wall

INTRODUCTION
Heliothis armigera is a cosmopolitan, polyphagous insect causing serious damage to cultivated crops in India such as cotton, tomato, chickpea, pigeon pea, maize, sorghum etc. It is distributed throughout the India and is reported from Andhra Pradesh, Kerala, Madhya Pradesh, Maharashtra, Meghalaya, New Delhi, Orissa, Punjab, Rajasthan, Tamil Nadu, Uttar Pradesh, West Bengal etc. Chemical pesticides are commonly used to protect crop from the H. armigera infestation, however chemical pesticides are very toxic to us and domestic animals and as they have high residual value, they are not suitable to spray on the vegetables and fruits which are to be harvested after short period of spray. The plant Argemone mexicana contains many alkaloids ^[1-2] and was found to possess larvicidal and growth inhibiting activity against the second instar larvae of Aedes aegypti.
These extracts have also been shown to induce behavioral and morphological modifications in the larvae of A. aegypti. Researchers have also reported the larvicidal and chemosterilant activity of phytochemicals derived from A. mexicana seeds against A. aegypti ^[3].
Weeds, the so-called nuisance plants, belong to one such group, which has attracted researchers’ attention as eco-friendly substitutes to chemical insecticides for the mosquito management ^[4]. The larvicidal potential of the extracts prepared from the different parts of Mexican prickly poppy, Argemone mexicana, has been reported against the early fourth instars of A. aegypti ^[5].

MATERIALS AND METHODS
The larvae of Heliothis armigera were collected from the field of tur (Cajanus cajan), tomato (Lycopersicon esculentum) and gram (Cicer arietinum) etc. near Aurangabad, India. Each larva was reared in different plastic bottles in department of zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, India in 2010 on artificial diet to avoid cannibalism and food was changed every day (http://www.cicr.gov.in).
Fresh leaves of Argemone mexicana was collected from the field near Aurangabad and were dried in the shade and then in the oven at 55⁰C. The dried leaves were powdered in the grinder and powder was stored in the airtight polyethylene bags. The powder was packed in filter paper and extract was extracted with acetone and ethanol in Soxhlet apparatus in 1:10 ratio i.e. 10 gm powder in 100 ml solvent. After eight hours of continuous extraction the final extract was kept open to evaporate the solvent and obtained extract was stored at 4ºC in a refrigerator until use. The artificial diet prepared was mixed with each of the extract so as to prepare various concentrations of the extract and was poured in the vials. 10 vials for each concentration of each extract were prepared and the larvae were released one in each vial. There is cannibalism in larvae of H. armigera and hence one larva was reared in each vial. Ten larvae one in each vial with food without extract was allowed to grow as control.
Mortality of the larvae was recorded for four days. The mortality data in the extracts showing considerable toxic effect on the larvae was used to calculate the LD₁₀ and LD₅₀ values.
Calculation of regression equation for LD₁₀ and LD₅₀ values of Heliothis armigera after the treatment of acetone and ethanol extract of Argemone mexicana for 24 and 96 hrs.
After 24 and 96 hours of exposure, the larvae were dissected in 0.6 % saline and the fore gut was fixed in the Bouin’s fluid for 24 hours. The tissues were washed and processed as usual and sections of 5 micron thickness were affixed on the slide. The sections were stained by Mallory’s triple stain.

RESULTS
Table 1: comparison of LD10 and LD₅₀ values of leaf extracts of Argemone mexicana to Heliothis armigera

Name of plant and Solvent	Time of exposure	Regression equation Y =y¯ +b(x-x¯ )	LD10 value in ml/Kg	LD50 value in ml/Kg
Leaf extract of A. mexicana in ethanol	24	Y = 4.8633x + 2.7013	2.618	2.969
	96	Y = 2.3775x + 1.9715	1.842	1.878
Leaf extract of A. mexicana in acetone	24	Y = 4.4982x + 2.9413	1.488	2.868
	96	Y = 1.7098x + 3.1430	0.4608	1.219

Fig 1: Normal histological structure of the fore gut

Fig. 1 shows the normal histological structure of the fore gut of the larva of Heliothis armigera. The fore gut consists of six layers, the innermost lining towards the lumen is cuticular layer outside to which is single layered columnar epithelium. The columnar epithelial cells are comparatively very tall. The outer side of the epithelium rests on thin basement membrane. The epithelium consists of some goblet cells which secretes the mucopolysaccharides. Outer side of basement membrane is surrounded by the circular muscles and then the longitudinal muscle fibers. Outer most layer is the peritoneal membrane. The fore gut on its outer side shows the secretary glands which may be the salivary glands.

                                                    
              Fig. 2 Effect of ethanol extract at 24 hrs                                                                                                      Fig. 3 Effect of ethanol extract at 96 hrs

Fig. 2 and 3 shows the effect of ethanol extract of A. mexicana after 24 and 96 hours of treatment. The severity of the damage of epithelial lining is more after 96 hours as compared to that of 24 hours, epithelial cells shows vacuoles at certain places. Cuticular lining is also found to be damaged and the lumen became wider.

                                                    
              Fig. 4 Effect of acetone extract at 24 hrs                                                                                                      Fig. 5 Effect of acetone extract at 96 hrs

Fig. 4 and 5 shows the effect of acetone extract of A. mexicana after 24 and 96 hours of treatment. The thickness of the wall has been increased due to clumping of the tissue and hence the diameter of the foregut was reduced. The lumen therefore became narrower. The columnar epithelial cells show the vacuoles.

DISCUSSION
Effect of A. mexicana alone as well as its synergy with other plants revealed less pupation at moderate doses, when applied alone, where as its efficacy was more at lower dose when applied in combination with Dononae aangustifolia, Monactis dubia and Castanae dentata. Similar results were recorded in H. armigera with the Eucalyptus ground leaf powder (2 %) with significant reduction in pupation (12 %) and mean larval weight ^[6]. The insect pests have developed resistance to a variety of insecticides due to the indiscriminate use of chemical pesticides. Insecticides affect the non-target organisms and human beings, directly or indirectly. Plant extracts or pure compounds manifest their effect on insects in several ways including toxicity, mortality, antifeedancy, growth inhibition, suppression of reproductive behavior and reduction of fecundity and fertility ^[7].
Ethyl acetate leaf extract of A. tagala at 5.0% showed higher larval mortality of 40.66%. These results support the earlier findings of ^[7] who observed potential insecticidal agents for the control of the larvae of Anticarsia gemmatalis in acetone and ethanol extracts of A. pubescens. A. albida plant had acidic metabolites like aristolic acid, aristolochic acid, aristoloctam and aristolone which exhibited larval mortality against S. zeamais ^{[8]. [9]} reported that ethyl acetate leaf extract of Acorus calamus at 5.0% exhibited maximum larvicidal activity of 40.24% against S. litura. The root extract of Tageteserecta proved more toxic to the lesser grain borer and red flour beetle than malathion ^[10]. A. mexicana is a source of medicines as it is a reservoir of chemical agents with therapeutic properties. It provides a good source of anti-infective agents, for example emetine, quinine and berberine which still remain to be highly effective in the fight against microbial infections. Various publications have documented the antimicrobial activity of plant extracts ^[11-13].

CONCLUSIONS
The severity of damage of epithelial lining was more in larvae of 96 hours of exposure as compared to those of 24 hours, epithelial cells showed vacuoles at certain places. The gut lining was also found to be damaged and the lumen became wider after the effect of ethanol extract of A. mexicana.
In acetone extract of A. mexicana after 24 and 96 hours of treatment, the thickness of the fore gut wall has been increased due to clumping of the tissue and hence the diameter of the foregut was reduced. The lumen therefore became narrower. The columnar epithelial cells showed the vacuoles.

ACKNOWLEDGMENT
The authors are highly grateful to the respective Universities and Principals of relevant Institutions to carry out the present investigations.

REFERENCES

1. Sangwan N.K. and Malik M.S. (1998): A long-chain alcohol from Argemone mexicana. Phytochemistry. 49. 755 – 756.
2. Yuh Chwen C., Hseih P.W., Chang F.R., Wu R.R., Liaw C.C., Lee K.H. and Wu Y.C. (2003): Two new protopines argemexicaines A and B and the anti HIV alkaloid 6 acetonyldihydrochelerythrine from Formosan Argemone mexicana. Planta Medica, 69, 148-152.
3. Sakthivadivel M. and Thilagavathy D. (2003): Larvicidal and chemosterilants activity of the acetone fraction of petroleum ether extract from Argemone mexicana L. seed. Bioresource Technology, 89, 213-216.
4. Kumar S, Nair G, Singh AP, Batra S, Wahab N, Warikoo R. (2012): Evaluation of the larvicidal efficiency of stem, roots and leaves of the weed, Parthenium hysterophorus (Family: Asteraceae) against Aedes aegypti L. Asian Pac J Trop Dis.;2:395–400.
5. Warikoo R, Kumar S (2013): Impact of Argemone mexicana extracts on the cidal, morphological and behavioural response of dengue vector, Aedes aegypti L. (Diptera: Culicidae). Parasitol Res; 112:3477–3484.
6. Kaushik N. (2001): Pesticidal activity of Eucalyptus leaf extracts against Helicoverpa armigera larvae. In: 88th Session of Indian Science Congress, Women and Science Forum, 3-7 January 2001. IARAI, New Delhi, India.
7. Jbilou R., Ennabili A., Sayah F. (2006): Insecticidal activity of four medicinal plant extracts against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) African J. Biotechnol; 5:936–940.
8. Arannilewa S.T., Ekrakene T., Akinneye J.O. (2006): Laboratory evaluation of four medicinal plants as protectants against the maize weevil, Sitophilus zeamais (Mots) African J. Biotech; 5:2032–2036.
9. Elumalai K., Jayasankar A., Raja N., Ignacimuthu S. (2004): Ovicidal and larvicidal activity of certain plant extracts against the tobacco armyworm, Spodoptera litura Fab. J. Curr. Sci.; 5:291–294.
10. Morallo-Rejesus B. and Decena A. (1982): The activity, isolation, purification and identification of the insecticidal principles from Tagetes. Phillip. J. Crop Sci, 7, 31-36.
11. Hoffman D. L. (1987): The Herb Users Guide. Thomsonius Publishing Group, Welling borough, London.
12. Nasar-Abbas S. M. and A. K. Halkman (2004): Antimicrobial effect of water extract of sumac (Rhus coriaria L.) on the growth of some food borne bacteria including pathogens. Int. J. Food Microbiol, 97: 63-69.
13. Rahman A., Parvez M.A.K., Parvin T., Paul D.K. and Sattar M.A. (2004): Antimicrobial activity of three bioactive compounds from the stream bark of Piper chaba hunter. Bioscie. Res., 1: 16-20.

International Journal of Life-Sciences Scientific Research (IJLSSR) Open Access Policy Authors/Contributors are responsible for originality, contents, correct references, and ethical issues. IJLSSR publishes all articles under Creative Commons Attribution- Non-Commercial 4.0 International License (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/legalcode