International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol.

3, Issue 2, Jun 2013, 265-276 TJPRC Pvt. Ltd.

HAEMOCYTE POPULATION DYNAMICS IN FIFTH INSTAR SILKWORM BOMBYX MORI L INOCULATED WITH BEAUVERIA BASSIANA (BALS.) VUILL
K. RAJITHA1, G. SAVITHRI2 & P. SUJATHAMMA3
1

Research Scholar, Department of Sericulture, S. P. Mahila University, Tirupati, Andhra Pradesh, India
2

Professor, Department of Sericulture, S. P. Mahila University, Tirupati, Andhra Pradesh, India

Professor, Advanced Diploma in Sericulture (China), Department of Sericulture, S. P. Mahila University, Tirupati, Andhra Pradesh, India

ABSTRACT
Total and specific haemocyte population indicates the immunological status of the insect. In view of the significance of the haemolymph and haemocytes in the various life process and protection mechanism from foreign bodies, the present study was carried out to understand the day to day changes in Total Haemocyte Count (THC) and Differential Haemocyte Count (DHC) in 5th instar silkworm larvae inoculated with fungal pathogen Beauveria bassiana. Significant enhancement of Total Haemocyte Count (THC) was observed till third day (121.0 to 139.75 THCx103/mm3), and then gradual reduction of the haemocyte population was noticed according to the progress of the fungal pathogen (119.25 to 89.5 THCx103/mm3). In case of Differential Haemocyte Count (DHC) viz., plasmatocytes (35.75 to 45.75 DHCx103/mm3), granulocytes (39.5 to 48.75 DHCx103/mm3) and spherulocytes (16.25 to 21.0 DHCx103/mm3) number was increased up to 3rd day of the instar and from 4th day onwards the number of plasmatocytes (39.25 to 32.0 DHCx103/mm3), granulocytes (43.0 to 35.5 DHCx103/mm3) and spherulocytes (17.5 to 10.0 DHCx103/mm3) were decreased in experimental animal. Gradual reduction of oenocytes (3.75 to 1.0 DHCx103/mm3) and prohaemocytes (25.75 to 11.0 DHCx103/mm3) was noticed in the experimental silkworm.

KEYWORDS: Beauveria bassiana, Bombyx mori, Haemocytes,Silkworm INTRODUCTION

Infection in insects stimulates a complex defensive response. Insects exhibit both cellular and humoral immune responses in addition to metabolic alterations that are effective against various pathogens like bacteria, fungi, protozoa etc. Humoral reaction involves slow synthesis of anti-bacterial and anti-viral principles and requires several hours for full expression. Cellular response is direct interaction between circulatory haemocytes and invading non-self material and interaction is immediate and includes phagocytosis, nodule formation and encapsulation. The haemocytes have the ability to discriminate stranger agents, mediate phagocytosis, cytotoxicity, encapsulation, wound repair and coagulation. These defense reactions were observed against pathogens, parasites and other foreign bodies, which entered in the haemocoel. Muscardine caused by Beauveria bassiana is the most contagious disease, leads to 30-40 per cent cocoon crop loss in total loss due to diseases (Nataraju et al 2005). The progress of the pathogen in the host system is often revealed by the specific metabolic variations along with gradual changes in the infected tissue. As the fungus confines to haemolymph till the silkworm larvae approach death (Gardener 1977), it obviously influences the metabolic profiles, chemical nature, volume of haemolymph and population of haemocytes. Haemocytes in the haemolymph of insects are responsible for the defense mechanism against foreign body that enter into the haemocoel (Tepass et al 1994; Falleiros and Gregorio 1995;

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Inoue et al 2001). The degree of variations in the number of haemocytes can be used as an index for diagnosis of the disease. In view of the significance of haemocytes in defence mechanism the present study was aimed to enumerate total and differential haemocyte count during the development of fungal pathogen Beauveria bassiana in 5th instar silkworm larvae compared to control.

MATERIALS AND METHODS

Silkworm hybrid of Pure Mysore x CSR2 was selected for the study. The silkworm larvae were brushed and reared in the laboratory under optimum conditions according to Dandin et al (2003). Immediately after fourth moult i.e. on the first day of the fifth instar, the larvae were inoculated, by dipping in fungal spore suspension (2.15 x 10 6 spores/ ml @ 50 ml/100 worms for 45 sec). The larvae treated with double distilled water were used as control. After 24 hours of induction of pathogen, haemolymph was collected everyday by clipping third pair of abdominal legs and drained into prechilled eppendoff tubes. The haemolymph was used for the enumeration of total and differential haemocyte count by using phase contrast microscope. Total haemocyte count was enumerated in the haemolymph of treated and control batches following the method described by Tauber and yeager (1935) using haemocytometer. The total haemocyte count (THC) per mm3 of haemolymph was calculated using the formula suggested by Jones (1962). Haemocytes in five 1 mm2 Dilution Depth factor of chamber ___________________________________________________ No. of squares counted Differential haemocyte count (DHC) was estimated by counting different haemocytes from a haemocyte population of 200, based on the morphological features as described by Nittono (1960). Statistical analysis was performed by following t-test.

RESULTS AND DISCUSSIONS

Haemocytes are complex of several types of circulating cells and regularly found in the haemolymph of insects are responsible for the defense mechanism against foreign body that enter into the haemocoel. Nittono (1960) classified the blood cells in the silkworm, Bombyx mori L. into six types viz., prohaemocytes, plasmatocytes, granulocytes, spherulocytes, imaginal spherulocytes (observed only at the adult stage, but occasionally in pupa on the day before emergence) and oenocytoids. Akai and Sato (1973 and 1976) and Nakahara et al (2009) classified silkworm haemocytes into five types based on their morphology and function viz., granulocytes, plasmatocytes, oenocytoids, prohaemocytes and spherulocytes. The results are presented in Tables 1 & 2 and Figure 1 & 2 Present haemotological investigations envisaged no significant change in total haemocyte count on the first day (24 hours after inoculation of pathogen) of inoculation with fungal pathogen in the 5 th instar silkworm larvae, then significant enhancement of THC was recorded till 3 rd day (121.0 to 139.75 THCx103/mm3) and significant reduction of the haemocytes were noticed from 4th day to 6th day (119.25 to 89.5 THCx103/mm3) during the progress of the disease in the experimental animal compared to control. Steady enhancement of THC was noticed till 4 th day (121.75 to 146.75 THCx103/mm3) and sudden decline of the haemocyte number was noticed in the control (137.0 to 129.75 THCx103/mm3). The results are shown in Table 1 and Figure 1. The recorded data of the investigation is in conformity with the earlier studies. Once the entomophagus fungi have penetrated in the host integument and gained access nutrient rich haemocoel, they are confronted with host humoral and cellular defenses (Butt et al 1988; Butt and humber 1989; Vey and Gotz 1986). The cellular responses to infection have been carried out in many insects by earlier workers (Chain and Anderson 1982; Dunn and Drake 1983; Horohov and Dunn 1983). Nappi (1981) and Eslin and Prevost (1998) reported the increase of the haemocyte number in the haemolymph of insects as response to parasitism which indicates the activation of host defence

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mechanism. The cellular component of the capsules surrounding the parasitoids is formed by haemocytes (Ratcliffe 1993), whose concentration in the hemolymph changes in parasitized larvae (Nappi and Carton 1986; Eslin and Prvost 1998). The population of circulating haemocytes may indicate whether the host defense system was activated or not (Brehlin 1982), and any depression in the number of haemocytes contributes to the protection from the parasite (Rizki and Rizki 1980, 1992). The present study is in agreement with the earlier investigation that the number of haemocytes may increase (Balavenkatasubbaiah et al 2001) or decrease (Gillam and Shimanuki 1967) to counter foreign body when infected. CHEN Wu-guo et al (2002) observed significant enhancement of total haemocyte count (THC) in Heliothis armigera infected with Ovomermis cinensis. Pandey et al (2010) observed the alterations in haemocyte count in stress induced tasar silkworm. On the basis of above findings of the earlier scientists it is evident that the fungal pathogen Beauveria bassiana induce the defense response through fluctuation of total haemocyte count in 5 th instar of silkworm Bombyx mori. Gajendra Pal Singh et al (2011) reported the elevation of the total haemocyte count in immunized Antheraea mylitta silkworms (immunized with attenuated AmCPV) in comparison with non-immunized control indicating the positive haemocyte mediated response. He also reported that differential haemocyte count was different in immunized Antheraea mylitta silkworms from the control. Prohaemocyte, plasmatocytes and granulocytes were maximum in number whereas oenocytoids were minimum in number and the number of degenerated blood cells was increased in inoculated Antheraea mylitta silkworms. The observations made were in conformity with the present investigation. Svetlana Avulove et al (2011) reported that the progression of fungal infection in Zootermopsis angusticollis is accompanied by significant changes in the density of circulating haemocytes. Drastic reduction in the number of haemocytes during various microbial infections has also been reported by several workers. Infection by Beauveria bassiana results in a gradual suppression of the phagocytic competence of circulating haemocytes and alteration in both total and differential haemocyte counts has been reported in the case of fungal (Hung et al 1993), bacterial (Govindarajan et al 1977), viral (Narayana 1979) and parasitic infection (Narayanan and Jayaraj 1973). Hung et al (1993) noticed gradual suppression of the phagocytic competence of circulating haemocytes especially granular haemocytes and alteration in total and differential haemocyte count in Spodoptera exigua infected with Beauveria bassiana. During the progressive infection, there was a gradual decrease of prohaemocytes (25.75 to 11.0 DHCx103/mm3) and oenocytes (3.75 to 1.0 DHCx103/mm3) from the first day of inoculation to the last day compared to control. Plasmatocyte number was increased up to 3 rd day (35.75 to 45.75 DHCx103/mm3) and from 4th day (39.25 to 32.0 DHCx103/mm3) onwards the plasmatocytes count was decreased. A similar trend was also noticed in case of granulocytes and spherulocytes. Gradual increase of plasmatocytes, granulocytes and spherulocytes was noticed in the control up to fourth day then shown decreased trend (Table 2 and Figure 2). In the present investigation, it was noticed that, the number of degenerated haemocytes was increased in the experimental silkworm compared to healthy batch. Prohaemocytes are thought to be the stem cells from which all haemocytes arise (Nataraju et al 2005). Of the dividing prohaemocytes, 59.2% of the daughter cells differentiated into other types of haemocytes such as plasmatocytes, granulocytes and spherulocytes the remaining dividing prohaemocytes divided into new prohaemocytes (Yamashita and Iwabuchi 2001). The gradual decrease in prohaemocyte count may be due to the conversion of prohaemocytes to other types of haemocytes that is required for defensive mechanism during progressive infection of Beauveria bassiana. Balavenkatasubbaiah et al (2001) reported higher number of granulocytes followed by plasmatocytes and reduction of prohaemocytes and oenocytes during the progress of BmNPV infection.

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Haemocytes play an essential role in defending insects against invading parasites and pathogens. The effective physiological mechanisms of phagocytosis, encapsulation, and other related defense mechanisms were primarily due to the availability of circulatory immune cells particularly plasmatocytes and granulocytes. In Lepidoptera, most cellular defense responses involve granular cells and plasmatocytes. Elevation of the number of plasmatocytes and granulocytes up to 3rd day of the infection indicates the primary role of plasmatocytes and/or granulocytes to fight against fungal infection. Plasmatocytes and granulocytes are responsible for a number of cellular immune reactions. The plasmatocytes and granulocytes function in defense mechanism against entry of foreign body in Bombyx mori L was worked out by Sato et al (1976) and Akai and Sato (1976). Granulocytes and plasmatocytes have been reported to be deployed in combating invading bacteria in Bombyx mori, at 1 hr and 6hr post infection with bacteria (Krishnan et al 2000). Ananda kumar and Ann Sandhya Michael (2011) reported significant increase of plasmatocytes and granulocytes in Bombyx mori infected with Bacillus thuringiensis and there was no significant change in proportion of oenocytes and spherulocytes. Increased population of granulocytes and plasmatocytes can be related as defense mechanism in silkworm Bombyx mori, since both the types of haemocytes functions as phagocytes. The response of haemocytes during progressive infection especially THC and DHC with regard to granulocytes, plasmotocytes and spherulocytes in silkworm indicate the correlation between THC and DHC and susceptibility to pathogenic infection (Salt 1970; Ratcliffee and Rowley 1975). Hung and Boucias (1992); Gillespie et al (1997); Vilcinskas and Gotz (1999) reported that once fungal invasion progresses, termites start exhibiting mycosis-related symptoms, plasmatocyte number is significantly reduced. In other insect species, plasmatocytes exhibit decreased density and phagocytic activity during advanced fungal infection. Increased numbers of phagocytic blood cells were recorded by Crossley (1968) in a study on muscle autolyses and regeneration in the larvae of Calliphora. The increase in the proportion of plasmotocytes and its morphological modifications was observed at the end of larval development in Drosophilla (Rizki 1957a). Those findings infer that some of the haemocytes appear in the haemolymph in great numbers at certain times and at certain physiological state. Whitter (1964) mentioned that changes in haemocytes are likely under hormonal control. Bora and Handique (2008) reported significant variation in THC and DHC in the fifth instar Antheraea assama larvae, exposed to sub lethal dose of leaf extract of Catharanthus roseus and noticed the clumping behavior, phagacytosis, change of shape, granulocytes breakdown etc. following Catharanthus treatment. The same authors reported the fluctuations occurring in plasmatocytes and prohaemocytes and suggested that it may be due to detoxification action of plasmatocytes against Catharanthus, prohaemocytes number decreased and because of transformation of prohaemocytes to plasmatocytes was probably more. Granulocytes were found to decrease at 6 h and 24 h which may be due to their involvement in phagocytosis of non-self material. Granulocytes are reported to be involved in phagocytosis in cell mediated defence of different insects (Wago 1982; Pathak 1990). In the present investigation spherulocytes number was enhanced up to 3 rd day (16.25 to 21.0 DHCx103/mm3) and then significant reduction of the cells was recorded (17.5 to 10.0 DHCx103/mm3) from 4th day to 6th day of inoculated silkworm larvae. Bora and Handique (2008) observed initial increase and then degeneration of spherulocytes following Catharanthus treatment. Begum et al (1998) recorded similar trend of spherulocyte count and she suggested that it may be due to effective utilization of fat reserves during the period decreased respiratory metabolism and also to produce extra energy under stress condition. Carlos Ribeiro et al (1996) reported that the functions of spherule cells are unknown. For Nittono (1960) spherule cells of Bombyx mori could be related to silk synthesis. Gradual reduction of oenocytes was observed from 1 st day to 6th day (3.75 to 1.0 DHCx103/mm3) of the inoculation during the progress of fungal pathogen. Bora and Handique (2008) also recorded decreased trend of oenocytes

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in muga silkworm following Catharanthus treatment. Oenocytes of muga silkworm contain small crystal like structures and electron dense spherules. It has been reported that such crystals in oenocytes contain tyrosine and involved in synthesis of phenoloxidase (Rizki 1957b). The involvement of oenocytoids in synthesis of phenoloxidase enzyme cascade (Soderhall and Smith 1986) may be responsible for decrease in their level after Catharanthus treatment. Ashida and Brey (1998) suggested that oenocytoids produce prophenoloxidase, and the active form of prophenoloxidase causes melanization to entrap and kill the invading pathogen. These studies are in conformity with the present investigation. Variations in the susceptibility of insect species to fungal invasion may result from several factors, including differences in the structure and composition of the exoskeleton, the presence and activity of antifungal proteins in haemolymph, as well as the efficiency of cellular and humoral defense reactions (Vilcinskas and Go tz 1999). The effective physiological mechanisms of phagocytosis, encapsulation, and other related defense mechanisms were primarily due to the availability of circulatory immune cells particularly plasmatocytes and granulocytes. As haemocytes respond very instantly against adversaries, it is expected that by using haemocyte catalogue as indicator, impact of several biotic and abiotic factors can be evaluated. Present investigation tried to analyze cellular immunopotency in Bombyx mori larvae during the progress of fungal pathogen i.e. Beauveria bassiana by quantifying the density of total and differential haemocytes. The research obviously provides evidence for the differential impact of different categories of haemocytes in silkworm Bombyx mori affected with Beauveria bassiana.

Figure 1: Variations in Total Haemocyte Count in Silkworm, Bombyx Mori L. Inoculated with Beauveria Bassiana

Figure 2: Variations in Differential Haemocyte Count in Silkworm, Bombyx Mori L. Inoculated with Beauveria Bassiana

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Table 1: Total Haemocyte Count in Haemolymph of Silkworm Bombyx Mori Inoculated with Fungal Pathogen Beauveria Bassiana Vuill Compare to Control Total Haemocyte Count (THC) V Instar Control Inoculation Day 1 121.750.96 1210.82 NS Day 2 125.51.29 130.52.65 *** Day 3 133.51.73 139.752.22 *** Day 4 146.750.96 119.250.96 **** Day 5 1372.16 992.16 **** Day 6 129.751.71 89.51.29 **** Table 2: Differetial Haemocyte Count in the Haemolymph of Silkworm Bombyx Mori Inoculated with Fungal Pathogen Beauveria Bassiana Vuill Compare to Control Differential Haemocyte Count (DHC) I day II day III day IV day 23.252.50 24.51.29 26.252.22 29.752.22 25.751.71 22.752.22 21.752.22 17.252.22 NS NS ** **** 35.52.08 36.251.71 37.751.71 41.52.65 35.751.71 41.252.50 45.752.50 39.252.63 NS *** **** NS 40.252.22 411.83 42.52.08 452.58 39.52.38 43.51.29 48.752.50 432.58 NS NS *** NS 18.251.71 190.82 19.52.08 221.83 16.251.71 20.251.71 211.83 17.51.29 NS NS NS *** 4.51.29 4.752.22 7.51.29 8.52.65 3.751.71 2.751.71 2.51.29 2.251.71 NS NS **** ***

Haemocytes C Prohaemocte I C Plasmatocytes I C Granulocytes I C Spherulocytes I C Oenocytes I

V day 27.251.71 131.83 **** 392.16 351.83 * 432.16 382.16 *** 21.51.29 120.82 **** 6.250.96 10.82 ****

VI day 25.502.65 112.16 **** 37.254.27 324.24 NS 41.753.50 35.53.11 * 20.251.71 101.83 **** 51.41 11.15 ***

ACKNOWLEDGEMENTS
This work was supported by University Grants Commission (UGC), New Delhi, INDIA. I thank UGC for the financial support to carry out the work.

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APPENTICES
Microscpic Observation of Haemocytes in Beauveria Bassiana Infected Silkworm Bombyx Mori Compared to Control Day 1 Control Inoculated

Day 2 Control Inoculated

Day 3 Control Inoculated

Haemocyte Population Dynamics in Fifth Instar Silkworm Bombyx mori L Inoculated with Beauveria bassiana (Bals.) Vuill

275

Day 4 Control Inoculated

Day 5 Control Inoculated

Day 6 Control Inoculated