In Vidarbha region of Maharashtra, the wild silk, Antheraea mylitta (D) Eco..race ... larvae at an early stageor would latent form and express at late stage with ...
Proceedlngs of UGC Sponsored National Level Conference on "Environmental Biology & Biodiversity"
EB-FLP-Ol BIOLOGY AND EFFECTS OF ENVIORNMENTAL FACTORS AND PATHOGENS ON THE VANY A TASAR SILKWORM, ANTHERAEA' MYLITTA (D). ECO~RACE BHANDARA D.D. Barsagade, M. N. Kadwey*, S. A. Gharde, M.P.Thakare H.M. Meshram, G. B. Gathaikar, l\1. MJJn~ And R. J. Andrew* P.G.Department of Zoology, RTM, Nagpur University, Amrawati road, Nagpur- 440033, India *P. G. Department of Zoology ,Hislop College Nagpur , ~
ABSTRACT In Vidarbha region of Maharashtra, the wild silk, Antheraea mylitta (D) Eco .. race Bhandara is trivoltine tropical tasar silkworm inhabit in three main ranges of hills in Chandrapur, Gadchiroli Bhandvd Gondia districts. The post embryonic, development of A. mylitta is drastically influenced by the environmental factor i.e. temperature, humidity, rainfall and food plants and the diseases flacherie and grasserie. The haemolymph, fat body and silk gland during bacterial and viral(CPV) infection shows the quantitative changes in the total Protein, Carbohydrate, DNA and RNA concentration, reflects on the absorption and positive transportation of major biomolecules in the body of diseased larvae of tasar silkworm. Physiological anomalies and infection are responsible for altered metabolism 'and result showed that the amount of all biomolecules decreased significantly in the larvae. The ultrastructural studies confrrmed the pathological effects of CPVon in midgut cell cytoplasm. The multiplication of polyhedralinclusion bodies took place into the vacuoles andform virogenic stromata in the cytoplasm of cells. However,the encapsulations of polyhedral inclusion bodiesinto the polyhedrin protein occurred and polyhedrawere released into the lumen. At the late stage of infection, cells showed the regressed cytoplasmic organelleswith large vacuoles and elongated mitochondria, Hence,the horizontal transmission of CPV causing the midgut cells disintegration in the tasarsilkworm, Antheraea mylitta (D) confirmed during infection. •
Key words: - Antheraea mylitta, haemolymph, fat body, silk gland, biomolecules, microbial infection.
Introduction The tropical tasar silkworm, Antheraea mylitta (D) eco-race Bhandara is wild in nature and feeds on the leaves of primary' food plants like Terminalia tomentosa (Yen), Terminalia arjuna (Arjun) and dozens of secondary food plants (Jolly et al., 1979). The larvae of Antheraea mylitta can tolerate a very wide rang of temperature and humidity because of their wild habitat. Temperature and humidity influence the larval life span and vigour, where as in the first brood (July-Aug 24-34°C and 75- 85% RH), the larval period is 30-35 days. In second brood (Sept- Oct' 20-30°C and 60-80% RH) and in third brood (Nov-Jan 18-26°C and 4 -60 %RH), it lasts for 40-45 days and 60-70 days respectively. This variation makes the larvae suscepti Ie to grasserie and flacherie diseases (Sen and Jolly, 1967). Mortality from bacterial diseases is aggravated below temperature 20-25°C combined with low humidity (55-60%) and from viral diseases by the cumulative effect of high temperature (30°C) and high humidity (95-95%). Pathogens are reported to induce several biochemical and physiological alteration in insect tissue (Martignoni, 1964; Etebari et al., 2007). Most of the pathophysiological studies associated with BmNPV infection are concerned with metabolism and level of biomolecules in the infected B. mori tissue (Benz, 1963; Bhosle and Kallapur, 1990 and Khurad et al., 2006). Information on the cytomorphological changes and effect of microbial pathogen in Antheraea mylitta is perhaps, very meager (Jolly and Sen 1972; Bansal et al., 1997; Sivaprasad et al., 2003; Tembhare and Barsagade, 2003 and Biabani et al., 2005 a, b). The vertical transmission of TnCPV in Trichoplusia ni population were observed dtiringrearing (Fuxa et al., 1992, 2002). The virus transmitted inthe progeny either may kill the host NCEBB 2011
Proceedings ofUGC Sponsored National Level.Conference on "Environmental Biology & Biodivenity"
larvae at an early stageor would latent form and express at late stage with favorable fluctuation in the environmental conditions. . Ultrastructural studies confirmed the pathological effects of CPVon in midgut cell cytoplasm. The multiplication of polyhedral inclusion bodies took place into the' vacuoles and form virogenic stromata .in the cytoplasm of cells. However, the encapsulations of polyhedral inclusion bodiesinto the polyhedrin protein occurred and polyhedra were released into the lumen. At the late stage of infection, cells showed the regressed cytoplasmic organelleswith large vacuoles and elongated mitochondria, Hence,the horizontal transmission of CPV causing the midgutcells disintegration in the tasar silkworm, Antheraea mylitta (D) confirmed during infection The present study is undertaken to explore pathogenic effect on the cell organelles and their total protein, carbohydrate, DNA and RNA concentration, during the various microbial infections in haemolymph, fat body and silk gland and CPV infection on midgut show the pathogen specific metabolic variation in the infected haemolymph and tissue. Materials and Methods The healthy and infected fifth instar larvae of trivoltine tropical tasar silkworm, Antheraea mylitta (D) eco-race Bhandara, were brought to the laboratory from the field. The larvae suffering from bacteriosis and virosis were recognized from their external characteristic (Jolly et al., 1979) and reared separately in the laboratory. The isolation and identification of bacteria, Staphylloccocus aureus at the Department of Microbiology, RTM Nagpur university Campus, Nagpur. The grasserie infected fifth instar larvae were collected from the rearing field and were triturated individually. The larvae were crushed in the pestle and mortar and the homogenate .was filtered through muslin cloth. The filtrate was observed under the microscope for presence 9(PIBs in it. The homogenate was contaminated with other microbes and it was purified by repeated i centrifugation until clear layer of PIBs was obtained. These isolated PIBs were stored in refrigerator . until their use. The serial ten fold dilutions in distilled water were prepared initially. The 60 larvae starved for six hours and distributed in four trays. First two trays containing equal number oflarvae were inoculated individually by providing 1 sq. em piece of Terminalia arjuna leaves coated with 20 J.Llsuspension (about 2000 ·PIBs)lxI05PIBs Iml. The larvae of third and forth tray were also fed with 1 sq. em piece of leaves dipped in distilled water and thereafter they were provided with fresh leaves. The larvae in the third and forth tray were treated as control and were placed for away from the inoculated ones. Both the inoculated and control larvae were scarified at varying intervals of 24 h, 48 h and 74 h and termed them as early, mid and late stage of infection. Transmission. Electron Microscopy (TEM) The transmission electron microscopy was carried out atAll India Institute of Medical Sciences, New Delhi. Haemolymph was collected by pricking the prolegs of infected and healthy fifth instar larvae in small vials precoated with phenylthiourea to prevent malonization. Silk gland and fat body was transferred in to ice cold phosphate buffer (PH-7.2, 0.2M). To study the biochemical parameters, the homogenized samples were centrifuge at 15000·rpm for 10 min at 0 °C. The supernatant after centrifugation was used for estimation of total concentration of protein according to the method of Lowry et al.,(1951), total carbohydrate by Phenol- Sulphuric acid method of Dubois et al., (1951), DNA and RNA by Bruton's Diphenylamine and Dische-Orcinol methods of Searcy and Maclnnis (1970 a and b).
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Results: Biochemical analysis:' , , .'The data presented, in table 1, 2 and' 3 reveals, the extent of change in biomolecules 'of .haemolymph and tissue of silkworm, Antheraea mylitta (D) infected with grassari and flacherie diseases. The biomolecules viz, protein, carbohydrate, DNA and RNA were significantly decreased P