*Corresponding author email: ythangamalar4@gmail.com Madras Agric. J., 100 (7-9): 85-88, September 2013 Temporal Dynamics of Differential Haemocytes in Multivoltine Crossbreeds of Silkworm, Bombyx mori L. *A. Thangamalar 1 , S. Subramanian 2 and M. Muthuswami 3 Department of Sericulture, Tamil Nadu Agricultural University, Coimbatore - 641 003, India. The cellular immune responses of different Indian cross breeds of mulberry silkworm, Bombyx mori L. have been analysed by estimating differential haemocyte counts. The test silkworm larvae were immunized with Kanamycin resistant Escherichia coli (E. coli K R ) (10 -6 cfu/larva). Different types of haemocytes viz., prohaemocytes, granulocytes, plasmatocytes and spherulocytes were enumerated at specifed time intervals of 0h, 6h, 12h, 24h, 30h, 36h and 48h in immunized and control larvae to measure the spatial dynamics of DHC (Differential Haemocyte Count). The DHC profles of cross breed races viz., APM1 x APS8, APM2 x MVT, APM2 x APS12 and APM3 x APS12 showed that the composition of plasmatocytes, granulocytes and spherulocytes were in the range of 17-35, 69-73 and 2-8 per cent respectively. Upon immunization, there was rapid increase in granulocyte population recording as much as 122 per cent rise. Rapid rise in granulocytes was more evident in cross breeds like APM2 x APS12 and APM3 x APS12 as early as 6h after immunization. However, perceptible decrease in granulocytes and plasmatocytes was noticed in immunized larvae after 24-48h. The study brought out distinct differences in spatial and temporal dynamics of differential haemocyte counts in Indian multivoltine cross breeds of mulberry silkworm. Key words: Silkworm, Bombyx mori L., E. coli K R , Temporal dynamics and Differential haemocytes. Cellular factors in the haemolymph are participating in the protection of insects against infectious diseases. Haemocytes of different types viz., prohaemocytes, plasmatocytes, granulocytes, spherulocytes, imaginal spherulocytes and oenocytoids are the key candidates of cellular immunity. Raichoudhury and Sengupta (1959) named seven types of haemocytes in B. mori. Nittono (1960) classifed the blood cells in the silkworm, B.mori L., into six types. The chief defensive cells are the plasmatocytes and the granular cells in B. mori, which take part in phagocytosis, encapsulation and nodulation reactions in response to bacterial infection (Krishnan et al., 2000). Haemocyte counts are normally highly variable within the same species as well as among species (Jones, 1977). According to DHC (Differential Haemocyte Count) reported by Wago (1980) in B. mori , the granular cells constitute 62%, the prohaemocytes and plasmatocytes 16%, the spherule cells 19% and the oenocytoids 3%. Han et al. (1998) investigated that the haemocytic differentiation showed four types of haemocytes: prohaemocytes, plasmatocytes, granulocytes and oenocytes in the larvae of B. mori. Materials and Methods 1. Silkworm rearing Different Indian cross breed silkworm races, APM1 x APS8, APM2 x MVT, APM2 x APS12 and APM3 x APS12 were procured from the Andhra Pradesh State Sericulture Research and Development Institute, Hindupur. The silkworm hybrids were reared in rearing house at the Department of Sericulture, Tamil Nadu Agricultural University, Coimbatore, 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). 2. Immunization Escherichia coli (Kanamycin Resistant) strain was used in this study. Freshly collected mulberry leaves were dipped in 50 ppm E.coli (K R ) broth culture fltrate (10 6 cfu / ml). The leaves were shade dried and given once during frst feeding after fourth moult during ffth instar. 3. Collection of haemolymph The larvae were wiped with 70 per cent ethyl alcohol. Tip of the first proleg was snipped off, using sterilized blade / scissor. Required amount of haemolymph was collected in cleaned, sterilized and precooled eppendorf tubes. The haemolymph samples collected from control and immunized larvae were added with lepidopteran saline solution (Na Cl 2 88 mM, K Cl 2 8 mM, Ca Cl 2 9.9 mM). 4. Differential haemocyte count (DHC) DHC was taken in ffth stadium larvae of silkworm after ingestion of E. coli (K R ) to identify different types