INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, Copyright 2010 All rights reserved Integrated Publishing Association

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, 2011 Copyright 2010 All rights reserved Integrated Publishing Association Research article ISSN Haematology and Haemochemistry of Silkworm, Bombyx Mori L. Infected with Bacillus Thurungenses Anandakumar, M. D 1, Ann Sandhya Michael 2 1- Department of Biochemistry, Adarsh Institute of research & postgraduate studies, SRN Adarsh college, Chamarajpet, Bangalore , India. 2- Department of Life Sciences, Bangalore University, Jnanabharathi, Bangalore , India anupol2004@yahoo.co.in doi: /ijes ABSTRACT Total haemocyte count, differential haemocyte count, changes in nucleus/nucleolar ratios of various haemocytes, as well as haemolymph protein and reducing sugar of fifth instar larvae of Bombyx mori L. inoculated with flacherie were studied. The inoculated worms received spores/ml on the third day of fifth instar. The infected larvae showed 15.3% decrease in total haemocyte count (THC) when compared to healthy worms. Prohaemocytes and granular haemocytes, which form the bulk of the total population, were not influenced by Bacillus thurungienses, where as significant increase was observed in differential haemocyte count (DHC) of plasmocytes alone. Bacterial infection also influenced nucleus/nucleolar (N/Nu) ratio of plasmocytes and oenocytoids indicating their role in larval immunity process. Plasma protein and reducing sugars were marginally reduced on infection. The above results were discussed in the light of pathophysiology of silkworm on bacterial infection. Key words: Silkworm, haemocytes, flacherie, Bacillus thurungienses, plasma. 1. Introduction Flacherie is the most common disease that inflicts the maximum damage to Sericulture. Due to their variations in the symptoms it is often called by different names like flacherie, sotto disease, shrinking disease, softening disease, fecal disease etc. When the larvae affected with flacherie, the nature of haemolymph and digestive fluid also changes and as a result the cells of the gut wall gets affected. Since haemolymph is a circulatory fluid, with several functions, such as storage and transportation of nutrients and also plays an important role in excretion, defense, moulting and metamorphosis (Mullins, 1985). Nucleated haemocytes number varies with the insects; these haemocytes are classified as prohaemocytes, plasmocytes, granular haemocytes, oenocytes or oenocytoid and spherule cells (Arnold & Hinks, 1976). Major function of haemocytes includes phagocytosis of small particles, encapsulation of large foreign materials, haemolymph coagulation and storage and distribution of nutritive materials. Despite several studies made on bacterial disease of silkworm, information regarding the exact biochemical and physiological changes occurring inside the body of silkworm throughout the progress of disease is scanty. Here we present investigate on this disease with a view to find out the changes in haemotology and haemochemistry of silkworm, Bombyx mori infected with Bacillus. Received on September 2011 Published on November

2 2. Materials and Methods The disease free layings of silkworm hybrid PM X NB 4 D 2 were procured from private agency Sri Gurukrupa grainage, Ramanagaram, Bangalore. The silkworm hybrid was reared in environmental chamber at Silkworm physiology research laboratory, Dept of studies in Sericulture, Bangalore University, Bangalore, under the standard rearing conditions at temperature 26 1 C with 75 5% relative humidity and photoperiod of 16 L: 8D as per the recommended rearing practice (Raja Ram, 2000). During inoculation time silkworm larvae were divided in to two batches one batch for inoculation and one for normal/with out any treatment. The Bacillus spores were obtained from the KSSR & DI, Thalaghattapura, Bangalore. Bacillus infection was made orally to the silkworm larvae on 3 rd day of fifth instar for multiplication and the supplement had spores/ml. After 48 hours of inoculation the haemolymph smear was made according to Shapiro (1979). The commonly used parameters to describe the blood profile of an insect are total haemocyte count (THC) and differential haemocyte count (DHC). Total haemocyte count was made according to Tauber and Yeager (1934). DHC count was carried out according to the method of Yeager (1945). Nucleus/nucleorlar (N/NU) ratios were also calculated for each type of cells. For biochemical analysis few larvae were collected in normal and Bacillus treated batches. The larvae were kept in refrigerator at 4-5 o C for 5-10 minutes to facilitate the free running of heamolymph. The caudal horn of larvae was punctured and the heamolymph was collected in cleaned, sterilized and pre-cooled vials. A pinch of phenylthiourea is added to prevent the oxidation of heamolymph. The vials were preserved in the refrigerator as stock and used for estimation of haemolymph protein and reducing sugar by following the standard procedures (Lowry et al., 1951 and Burton 1956). The collected data were subjected to statistical analysis of t-test (Khan and Khanum 1994) 3. Results The THC count (number per mm3) decreased significantly (P>0.010, t = , df 4, n 3) when larvae were fed orally with Bacillus. Bacterial infected silkworms showed 15.3% decrease in the number of blood cells compared to healthy worms (Figure 1). The prohaemocytes and granular haemocytes formed the bulk of the haemocytes in the present study and their population not influenced by Bacillus infection. finding in the present study is the change in the number of plasmocytes on treatment with pathogen. Infection due to Bacillus significantly increased the plasmocytes population by 40%. However, there is no much variation in oenocyte and spherule cells population on infection in haemolymph of silkworm (Table-1). Table-2 Reveals the N/NU ratio in different haemocytes of silkworm and also the changes brought out by infection with bacillus. Prohaemocytes did not show any change in N/NU on treatment, whereas notable decrease was observed in rest of the haemocytes on infection. Plasmocytes showed significantly (P>0.100) increased ratio in healthy larval batch, indicating their significant role in protein synthesis and on bacteria infection the ratio was brought down by 20%. However, significantly (P>0.100) decreased in oenocytes N/NU ratio on infection was observed when compared with other cell types. The Bacillus inoculation induced a significant (P>0.010, t , df 4, n 3) decrease in the protein content of haemolymph, and similar decrease was observed in reducing sugars. The protein contents in the healthy worms were found to be 47.9 mg/ml, which decreased to 41.7 mg/ml of haemolymph in inoculated worms. Reducing sugar was also significantly (P>0.050, t 464

3 2.2404, df 4, n 3) decreased in inoculated worms and the amount was found to be 3.0mg/ml where as in normal worms 3.26mg/ml (figure 2). 4. Discussion Flacherie, a bacterial disease infects the silkworm through injury or wounds and multiplies in the haemolymph, disrupting the normal physiological function. Haemolymph being a circulatory fluid performs several functions (Mullins, 1985) and haemolymph consists of nucleated haemocytes, which are classified into granular haemocytes, spherule cells, oenocytoids, prohaemocytes and plasmocytes (Arnold & Hinks, 1975). The most prominent haemocytes in Bombyx mori are prohaemocytes, granular haemocytes, plasmocytes, oenocytoids and spherule cells (Akai, 1976). Hence it is of interest to know the role of haemocytes and their participation in immune response. From the present experimental result, it was found that the total cell count decreased significantly when treated with Bacillus. About 15.3% of the total haemocytes were decreased when compared to normal worms. Further, the decrease in total count infected with Bacillus was due to the depletion of prohaemocytes, which accounts for most of the lowered total counts compared to normal. The DHC further helps in the investigation of cellular defense mechanisms and to know the participation of a particular type of blood cell in such function. Peake (1979) found that there was an increase in the proportion of phagocytic haemocytes in Calliphora with age. An increase with the granular haemocytes percentage was found in Bombyx mori larvae between first and fifth instar (Wago & Ishikawa, 1979). Increased numbers of phagocytic blood cells were recorded by Crossely (1968) in a study on muscle autolyses and regeneration in the larvae of Calliphora. The increase in the proportion of plasmocytes and its morphological modification was similar to lamellocyte at the end of larval development in Drosophilla (Rizki, 1957). Those findings infer that some of the haemocytes appear in the haemolymph in greater numbers at certain times and at certain physiological state. Whitter (1964) also mentioned that changes in haemocytes are likely under hormonal control. The DHC of Bacillus infected larvae of Bombyx mori in the present study showed changes in haemocytes, with a significant increase in plasmocytes and granular haemocytes. There was no significant change in proportion of oenocytes and spherule cells. Increased population of granular haemocytes and plasmocytes can be related as defense mechanisms in B.mori, since both the types of haemocytes functions as phagocytes. Haemocytes are found to undergo mitosis in insects (Wigglesworth, 1993) on the basis of studies on morphological radio isotopic and ultra-structural features. It is almost universally accepted that prohaemocytes are stem cells and divide to produce plasmocytes (Gupta & Sutherland, 1966; Lea, 1986). But it is believed that plasmocytes gives rise to granular haemocytes from which all other types developed (Gupta 1979). The formations of granular haemocytes from plasmocytes in lepidoplerans are also reported (Lai-fook, 1973; Beemam et al., 1983). Bacillus infected silkworm larvae showed that the N/NU ratio was decreased compared to healthy worms. The decreased N/NU ratio indicates decreased protein synthesis in the haemocytes on infection. In view of the above reports and the results of the experiment, it shows that the prohaemocytes undergo division in order to increase the number of granular haemocytes and plasmocytes, which take part in cellular defense mechanism. Thus, in the 465

4 inoculated larvae, we find the decreased proportion of prohaemocytes with the increased proportion of granular haemocytes and plasmocytes. In the present work the amount of protein content was found to decrease in the inoculated worms, when compared to healthy worms. Reducing sugar in the haemolymph of silkworm, Bombyx mori infected with Bacillus was also low. Perhaps, the silkworms were not able to feed at normal rate when infected by Bacillus and there could be a change in digestion and absorption of nutrient in silkworm on bacterial infection. Table 1: Effect of Bacillus Thurungiensis on different haemocyte count (%) in silkworm, Bombyx mori L. larva Different Haemocytes Prohaemocytes Granular haemocytes Plasmocytes Oenocytes Spherule cells Normal larva Inoculated larva Standard deviation t-value degrees of freedom (df) No of Replications (n) Prabability (P) Remarks Highly Table 2: Changes in Nucleus/nucleolar ratio of different haemocyte population (%) in silkworm, Bombyx mori L. inoculated with Bacillus Thurungienses. Different Haemocytes Granular Prohaemocytes haemocytes Pasmocytes Oenocytes Spherule cells Normal larva Inoculated larva Standard deviation t-value degrees of freedom (df) No of Replications (n) Prabability (P) Remarks 466

5 biochemical constituents (mg/ml) No. of haemocytes/mm3 Haematology and Haemochemistry of Silkworm, Bombyx Mori L. Infected with Bacillus Thurungenses 640 Effect of Bacillusthurungienses on total heamocyte count in silkworm Bombyx mori, L healthy infected fig-1 Fig-2 Changes in heamolymph biochemical constituents (protein and reducing sugar) in silkworm Bombyx mori, L. infected with Bacillus thurungienses protein reducing sugar healthy inoculated Figure 2: Changes in heamolymph biochemical constituents (protein and reducing sugar) in silkworm Bombyx mori, L. infected with Bacillus thurungienses 5. References 1. Akai. H., Sato, S (1976), Surface ultrastructure of the larval haemocytes of the silkworm, Bombyx mori L. International Journal of Insect Morphology and Embryology, 5(1), pp Arnold. J. W., Hinks, C. F (1976), Haemopoiesis in Lepidoptera I. The multiplication of circulating haemocytes. Cannadian Journal of Zoology, 54, pp

6 3. S. C. Beeman (1983), Structural characterization of the haemocytes of Plodia interpunctella. Journal of Morphology, 175, pp K. Burton (1956), A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemistry Journal, 62, pp A. C. Crossley (1968), The fine structure and mechanism of breakdown of larval intersegmental muscles in the blowfly, Calliphora erythrocephala. Journal of Insect Physiology, 14, pp Gupta, A. P., Sutherland, D. J (1966), In vitro transformation of the insect plasmocytes in some insects. Journal of Insect Physiology, 12, pp A. P. Gupta (1979), Haemocyte types: their structures, synonymies, interrelationships and taxonomic significance. In insect Haemocytes (Ed. A.P.Gupta), pp Irfan. A, Khan, Atiya Khanum (1994), Fundamentals of Bio-statistics, Ukaaz publications, Hydrabad, India, pp J. Lai-fook (1973), The structure of haemocytes of Calopodes ethlius (Lepidoptera). Journal of Morphology, 139, pp M. S. Lea (1986), A sericesthis iridescent virus infection of the haemocytes of the wax moth, Galleria mellanella effects on total and differential counts and haemocytogeny. Journal of Invertebrate Pathology, 48, pp O. H. Lowry (1951), Protein measurement with folin-phenol reagent. Journal of Biological Chemistry, 193, pp D. E. Mullins (1985), Chemistry and physiology of the haemolymph. In Comp. Insect. Physiol.Biochem. Pharm. Vol. 3.(Ed.,by Kerkut GA, Gilbert LF) Pergamon Press, Newyork, pp P. W. Peake (1979), Isolation and characterization of the haemocytes of Caliphora vicina on density gradients of Ficoll. Journal of Insect Physiology, 25, pp T. M. Rizki (1957), Alterations in the haemocyte population of Drosophila melanogaster. Journal of Morphology, 100, pp Raja Ram (2000), Appropriate silkworm rearing technology. In:Hari Om Agrawal, Seth MK, editors.sericulture in India,pp: :Bishen Singh Mahendra Pal Singh press, Dehradun, India. 14. M. Shapiro (1979), Changes in haemocyte populations. In Insect Haemocyte (Ed. Gupta AP) Cambridge University Press. Cambridge, pp Tauber. O. E., Yeager. J. F (1935), On total haemolymph (Blood) counts of insects-i Orthoptera, Odonata, Hemiptera and Homoptera. Annual Entamological Science of America, 28, pp

7 16. Wago, H., Ishikawa, Y (1979), Haemocytic reactions to foreign cells in the silkworms, Bombyx mori during post-embryonic development. Applied Entamological Zoology, 14, pp J. M. Whitter (1964), Haemocytes and metamorphosing tissues in Sarcophagar bullata, Drosophila melanogaster, and other Chyclorrhaphous dipter. Journal of Insect Physiology, 10, pp V. B. Wigglesworth (1993), The physiology of the cuticle and of ecdysis in Rhodinus prolixus Triatonidae (Hemiptera) with special reference to the function of the oenocytes and of the dermal glands. QJI micross. Sci., 76, pp J. F. Yeager (1935), The blood picture of the Southern army worm (prodenia eridania). Journal of agriculture Research, 71, pp