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Journal of Entomology and Zoology Studies 2016; 4(6): 46-49 E-ISSN: 2320-7078 P-ISSN: 2349-6800 JEZS 2016; 4(6): 46-49 © 2016 JEZS Received: 08-09-2016 Accepted: 09-10-2016

Bhavna Prishnee Baishya Department of Biotechnology, Gauhati University, Guwahati, Assam, India Sunayan Bardoloi Department of Zoology, B. Borooah College, Guwahati, Assam, India Rupjyoti Bharali Department of Biotechnology, Gauhati University, Guwahati, Assam, India Correspondence Bhavna Prishnee Baishya Department of Biotechnology, Gauhati University, Guwahati, Assam, India

Morphological changes in the hemocytes of Antheraea assama (Lepidoptera: Saturniidae) upon

bacterial infection

Bhavna Prishnee Baishya, Sunayan Bardoloi and Rupjyoti Bharali Abstract Giemsa stained hemocytes of bacteria-infected 5th instar larvae of Antheraea assama were studied under 100X magnification by light microscopy. Bacterial infection was observed to induce distinct morphological changes in the various hemocyte types, including enlarged or swollen cells, highly vacuolated cytoplasm, severe nuclear fragmentation, loss of cytoplasmic compactness or cytoplasmic lysis, blebbing of plasma membrane or completely lysed plasma membrane, and sometimes aggregation of hemocytes to one another leading to encapsulation of the bacterial products Keywords: Cytoplasmic extensions, hemocytes, plasmatocytes, granulocytes, phagocytosis, vacuolization 1. Introduction Hemocytes are the cellular inclusions present in the hemolymph of insects and other invertebrate groups. They play important role in the physiology of the organism to which they belong [1]; thus, they are responsible for coagulation of hemolymph [2, 3], connective tissue synthesis [4-6], wound healing, self-recognition, general and specific immune response, including opsonisation, phagocytosis, encapsulation and nodulation [7-11]. Therefore, any stress condition resulting in changes in hemocyte population, morphology and function would ultimately have an adverse effect on the overall physiology and survival of the affected insect or organism. This study aims to investigate the changes in hemocyte morphology of Muga silkworm Antheraea assama upon induced infection with the bacteria E. coli with the help of light microscopy. A. assama has been reported to have basically five distinct hemocyte types, viz. Prohemocytes (PR), Plasmatocyte (PL), Granulocyte (GR), Spherulocyte (SP) and Oenocytoid (OE) [1]. Insects do not have an acquired immune system but possess well-developed innate immune responses, comprising of both cellular and humoral immune reactions; the cellular immune defense being provided by the hemocytes. Infection itself is a stress condition whereby hemocytes, along with other components of the insect immune system, work to eliminate the stress. In doing so, they undergo various morphological changes as evident from works of various workers. These structural alterations sometimes include excessive changes in hemocyte cell contour leading to cytoplasmic lysis and finally cell death which decreases the circulating hemocyte population in the hemolymph available for eliminating the stress condition. 2. Materials and methods 2.1 Insects: 5th instar larvae (48-hr post moult) reared on some plants were directly collected from the State Sericulture farm, Khanapara, and brought to the laboratory for conduction of the experiments. 2.2 Infection of larvae: 5th instar larvae were infected with the bacteria E. coli, cultured in the laboratory in Luria Broth, incubated overnight at 28 ºC, centrifuged and the pellet suspended in physiological saline (final concentration of bacteria 1.9x108 cells/ml approx.). 10 µl of this bacterial suspension was injected sub-dorsally between the prolegs at the seventh segment of the larvae using a Hamilton microsyringe.

 

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2.3 Slide preparation: Hemolymph drops were obtained on clean and dry glass slides by severing the tip of the proleg of larvae, 12 hrs after infection and smears were made. Slides with uniform hemolymph smears were air-dried, fixed in methanol, stained with Giemsa, mounted with DPX and observed under light microscope (make: Labomed) at 100X magnification. 3. Results The structural alterations and malformations of the A. assama hemocytes upon induced bacterial infection are quite evident from figure 1. Hemocytes, particularly PLs and GRs, were observed to adhere to one another forming aggregates in the smears (Fig 1a, 1b, 1c, 1d). This aggregation of hemocytes could enclose bacterial products present in the hemolymph, which may later lead to encapsulation and melanization reactions. Most hemocytes were seen with distorted cell contour with swollen or enlarged cells with loss of

cytoplasmic as well as organelle compactness. The cytoplasm in most hemocytes had extensive vacuolization, leading to cytoplasmic lysis and disintegration of overall hemocyte morphology. Vacuolization was also observed in both PRs and GRs (Fig. 1e, 1f, 1g, 1h); their vacuoles probably enclosing the invading bacteria or products of bacterial degradation, thereby suggesting their role in phagocytosis. The participation of these two hemocytes, viz. PLs and GRs in phagocytic activity was confirmed by the presence of extended cytoplasmic processes arising from their cell surface (Fig. 1e, 1f, 1g). Moreover, nuclear disintegration was observed to be more pronounced in the GRs (Fig. 1g, 1h). PRs generally have distinctive large nucleus with a thin band of cytoplasm surrounding it, however, in the altered morphology they appeared to be much enlarged and swollen (Fig. 1i). Few malformed SPs (Fig. 1i) were also observed; however, disintegrated oenocytoids were not seen.

 

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Fig 1: (a, b, c, d, e, f, g, h, i, j) showing changes in hemocyte morphology upon bacterial infection- swollen enlarged hemocytes with cell membrane lysis, hemocyte aggregation, extensive cytoplasmic vacuolization,

cytoplasmic degradation and cell lysis, extended cytoplasmic processes in plasmatocytes and granulocytes citing phagocytic behaviour.

4. Discussion The altered hemocyte morphology observed in this study were found to be in accordance with those of other workers, viz. Silva, et al, 2002; Sahayarij, et al, 2007; Pandey et al, 2010; Awad, 2012 etc [12-15]. In this study, hemocyte aggregation was a common observation in A. assama hemocytes upon bacterial infection, suggesting that encapsulation of bacterial product may be a subsequent step in this reaction, which was also reported in hemocytes of parasitized Anastrepha obliqua larvae which surround its endoparasite in layers forming capsules, which later lead to encapsulation/ nodule formation and elimination of the target [12]. Moreover, structural malformations of hemocytes evident in this study were similar to that reported in Helicoverpa armigera hemocytes which showed cytotoxic responses, together with cell lysis or aggregation, on treatment with fern phytoecdysterone [13]. Hemocytes of Agrotis epsilon larvae when treated with Bacillus thuringiensis showed numerous structural alterations including swollen and enlarged PRs, extensive vacuolization of the cytoplasm of PLs and GRs, deformed or lysed cell membrane leading to cytoplasmic lysis and cell death [15] which were also evident in A. assama hemocytes. Moreover, Awad (2012) [15] clearly stated that vacuolization of cytoplasm upon bacterial infection as well as presence of cell surface projections may be indicative of phagocytosis of the

bacterial products by the hemocytes, as clearly evident in the cytoplasm of PLs and GRs in this study. Thus, structural alterations in the hemocytes of Antheraea assama during bacterial infection or stress were compared to available literature in this regard and the results were found to be similar to those of other insects. Such studies provide an insight into the physiological functions of the various cell types, specially the phagocytic activity as well as encapsulation by the PLs and GRs in A. assama, which in turn confirm their role as immunocytes in such insects. 5. Acknowledgement We would like to thank the Institutional Biotech Hub, B. Borooah College for permitting us to utilize their instruments during this work. 6. References 1. Baishya BP, Bardoloi S, Bharali R. Ultrastructure of the

hemocytes of Muga Silkworm larva Antheraea assama Westwood (Lepidoptera; Saturniidae): a phase-contrast and electron microscopic study. International Journal of Pure and Applied Biosciences. 2015; 3:234-240.

2. Gregoire C. Blood coagulation in arthropods V. Studies on hemolymph coagulation on 420 species of insects. Archives of Biology. 1955; 66:104-148.

 

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3. Gregoire C. Studies by phase-contrast microscopy on distribution patterns of hemolymph coagulation in insects. Smithsonian Institution Miscellaneous Collections. 1957; 134:1-35.

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7. Salt G. The cellular defence reactions in insects. Cambridge Monographs in Experimental Biology, No. 16. New York, N.Y.: Cambridge University Press, 1970.

8. Gupta AP. Cellular elements in the haemolymph. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, eds- G.A. Kerkut and L.I. Gilbert. Pergamon Press, Oxford, 1985, 402-451.

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10. Xylander WER. Immune defence reactions of Myriapoda- A brief presentation of recent results. In: Thaler K, Meyer E, Schedl W (eds). Advances in Myriapodology (Proceedings of the 8th International Congress of Myriapodology). Ber. Nat-Med. Verein Innsbruck. Suppl. 1992; 10:101-110.

11. Xylander WER. Hemocytes in Myriapoda (Arthropoda): a review. Invertebrate Survival Journal. 2009; 6:114-124.

12. Silva JEB, Boleli IC, Simoes ZLP. Hemocyte types and total and differential counts in unparasitized and parasitized Anastrepha oblique (Diptera, Tephritidae) larvae. Brazilian Journal of Biology. 2002; 62:689-699.

13. Sahayarij K, Selvarj P, Balasubramanian R. Cell mediated immune response of Helicoverpa armigera Hubner and Spodoptera litura Fabricius to fern phytoecdysterone. Journal of Entomology. 2007; 4:289-298.

14. Pandey JP, Mishra PK, Kumar D, Singh BMK, Prasad BC. Effects of temperature on hemocytic immune responses of tropical tasar silkworm Antheraea mylitta D. Research Journal of Immunology. 2010; 3:169-177.

15. Effects of Bacillus thuringiensis and farnesol on hemocytes response and lysozymal activity of the Black Cut Worm Agrotis epsilon larvae. Asian Journal of Biological Sciences, 5, 157-170.