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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

MORIL INOCULATED WITH BEAUVERIA BASSIANA (BALS.) VUILL

K. RAJITHA1, G. SAVITHRI

2& P. SUJATHAMMA

3

1Research Scholar, Department of Sericulture, S. P. Mahila University, Tirupati, Andhra Pradesh, India

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

3Professor, 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/mm

3), and then

gradual reduction of the haemocyte population was noticed according to the progress of the fungal pathogen (119.25 to

89.5 THCx103/mm

3). In case of Differential Haemocyte Count (DHC) viz., plasmatocytes (35.75 to 45.75 DHCx10

3/mm

3),

granulocytes (39.5 to 48.75 DHCx103/mm

3) and spherulocytes (16.25 to 21.0 DHCx10

3/mm

3) 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/mm

3), granulocytes

(43.0 to 35.5 DHCx103/mm

3) and spherulocytes (17.5 to 10.0 DHCx10

3/mm

3) were decreased in experimental animal.

Gradual reduction of oenocytes (3.75 to 1.0 DHCx103/mm

3)

and prohaemocytes (25.75 to 11.0 DHCx10

3/mm

3) 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 al2005). 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 al1994; Falleiros and Gregorio 1995;


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266 K. Rajitha, G. Savithri & P. SujathammaInoue 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 106

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 pre-

chilled 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

mm3of 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 (1973and 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 5th

instar silkworm larvae, then

significant enhancement of THC was recorded till 3rd

day (121.0 to 139.75 THCx103/mm

3) and significant reduction of the

haemocytes were noticed from 4th

day to 6th

day (119.25 to 89.5 THCx103/mm

3) during the progress of the disease in the

experimental animal compared to control. Steady enhancement of THC was noticed till 4th

day (121.75 to 146.75

THCx103/mm

3) and sudden decline of the haemocyte number was noticed in the control (137.0 to 129.75 THCx10

3/mm

3).

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 al1988; 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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Haemocyte Population Dynamics in Fifth Instar Silkworm Bombyx moriL Inoculated with Beauveria bassiana(Bals.) Vuill 267

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 Rizki1980, 1992). The present study isin agreement with the earlier investigation that the number of haemocytes may increase

(Balavenkatasubbaiah et al2001) 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 5th

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 al1993), bacterial (Govindarajan et al1977), 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/mm

3)

and oenocytes (3.75 to 1.0 DHCx103/mm

3) from the first day of inoculation to the last day compared to control.

Plasmatocyte number was increased up to 3rd

day (35.75 to 45.75 DHCx103/mm

3) and from 4

thday (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.
http://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamashita%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamashita%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamashita%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamashita%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamashita%20M%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Yamashita%20M%22%5BAuthor%5D


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268 K. Rajitha, G. Savithri & P. SujathammaHaemocytes 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 3

rd

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 al2000). 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 typesof 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 haemocytesappear 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 3rd

day (16.25 to 21.0 DHCx103/mm

3) and

then significant reduction of the cells was recorded (17.5 to 10.0 DHCx103/mm

3) from 4

thday to 6

thday 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 countand 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 ofBombyx moricould be related to silk synthesis.

Gradual reduction of oenocytes was observed from 1st

day to 6th

day (3.75 to 1.0 DHCx103/mm

3) 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 MoriL. Inoculated with Beauver ia Bassiana

Figure 2: Variations in Differential Haemocyte Count in Silkworm,

Bombyx MoriL. Inoculated with Beauver ia Bassiana


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270 K. Rajitha, G. Savithri & P. SujathammaTable 1: Total Haemocyte Count in Haemolymph of Silkworm Bombyx MoriInoculated

with Fungal Pathogen Beauveri a BassianaVuill Compare to Control

Total Haemocyte Count (THC)

V Instar Control Inoculation

Day 1 121.750.96 1210.82 NSDay 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 ****

Table2: Differetial Haemocyte Count in the Haemolymph of Silkworm Bombyx MoriInoculated

with Fungal Pathogen Beauveri a BassianaVuill Compare to Control

Differential Haemocyte Count (DHC)

Haemocytes I day II day III day IV day V day VI day

ProhaemocteC 23.252.50 24.51.29 26.252.22 29.752.22 27.251.71 25.502.65I

25.751.71

NS

22.752.22

NS

21.752.22

**

17.252.22

****

131.83

****

112.16

****

Plasmatocytes

C 35.52.08 36.251.71 37.751.71 41.52.65 392.16 37.254.27

I35.751.71

NS

41.252.50

***

45.752.50

****

39.252.63

NS

351.83

*

324.24

NS

Granulocytes

C 40.252.22 411.83 42.52.08 452.58 432.16 41.753.50

I39.52.38

NS

43.51.29

NS

48.752.50

***

432.58

NS

382.16

***

35.53.11

*

Spherulocytes

C 18.251.71 190.82 19.52.08 221.83 21.51.29 20.251.71

I16.251.71

NS20.251.71

NS211.83

NS17.51.29

***120.82

****101.83

****

Oenocytes

C 4.51.29 4.752.22 7.51.29 8.52.65 6.250.96 51.41

I 3.751.71NS

2.751.71NS

2.51.29****

2.251.71***

10.82****

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 Beauveri a BassianaInfected

Silkworm Bombyx MoriCompared to Control

Day1

Control Inoculated

Day2

Control Inoculated

Day3

Control Inoculated


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Day4

Control Inoculated

Day

5Control Inoculated

Day6

Control Inoculated


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27.haemocyte population.full

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