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ISSN 2278 –1358 Original Article IMPACT OF BOTANICAL FORMULATION ON CONTAINMENT OF VIROSIS DISEASE AND CELLULAR AND HUMORAL RESPONSE IN TASAR SILKWORM ANTHERAEA MYLITTA D. G.P. Singh, K.P. Kiran Kumar, K.N. Madhusudan and Alok Sahay Silkworm Pathology Laboratory, Central Tasar Research and Training institute, Nagri, Ranchi – 835303, India Received 11 June 2015; accepted 24 June 2015 Abstract Among diseases in tasar silkworm, Antheraea mylitta D. virosis caused by Cytoplasmic Polyhedrosis Virus (CPV) is highly contagious and more prevalent. A botanical formulation prepared from the plants known for their antiviral properties was tested against Antheraea mylitta Cytoplasmic Polyhedrosis Virus (AmCPV) infection in tasar silkworm in laboratory. The influence of botanical formulation on haematological and biochemical changes in silkworm larvae infected with AmCPV was also observed. The aqueous extract of botanical formulation at its 2.5% concentration reduced the larval mortality to 28.14 and 25.99% respectively in 1st and 2nd crop rearing than 88.36 and 91.58% in inoculated control. The Pooled analysis of data of two crops revealed that the highest reduction in AmCPV infection in tasar silkworm than inoculated control was 69.88% with the treatment of 2.5% aqueous extract of botanical formulation. It was observed that total haemocyte count increased in haemolymph up to 6th days post inoculation in the silkworm treated with botanical formulation. While in the inoculated control the increase was within 3 days indicating the positive haemocyte mediated response in silkworm treated with botanical formulation. After two days in the inoculated control, as the multiplication of AmCPV increased, defense capacity of the silkworm decreased. Similarly, differential haemocyte count was different from the inoculated control. The prohaemocyte plasmatocytes and granulocytes were maximum in number whereas oenocytoids were minimum in number. The number of degenerated blood cells was increased in inoculated control up to 8 th days of post inoculation. It is thus observed that haemocytes involve in defense mechanism against AmCPV and botanical formulation prevents the multiplication of virus by enhancing the haemocyte mediated defense response. The hemolymph protein in the silkworm treated with botanical formulation was significantly higher than treated control. The gradual increase 1st day to 8th day was observed in botanical formulation treated silkworm. In inoculated control, the total hemolymph proteins have shown increasing trend from 1st to 5th day and decreasing from 6th day onwards. © 2015 Universal Research Publications. All rights reserved Key words: Botanical formulation, Virosis, Tasar silkworm. INTRODUCTION Tasar silkworm, Antheraea mylitta D., an economically important insect reared on food plants in outdoor conditions. Silkworm larvae often suffer from various diseases causing heavy losses to the economy of the silk industry. Among diseases in silkworm, cytoplasmic polyhedrosis commonly known as virosis caused by Cytoplasmic Polyhedrosis Virus (CPV), a reovirus is highly contagious and prevails all through the year in tasar culture regions, which accounts considerable loss of 25-30% to cocoon production (Sahay et al., 2000).

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The chemical disinfectants and antibiotics practiced as preventive measures involve high cost have limitations to be effective in open field and hazardous to the users and environment. In contrast to the use of chemicals and antibiotics, we have tried biodegradable materials like plant extracts, which are now a days on the top priority for protection of plants and animals (Jesper and Ward, 1993; Kumar et al., 1999). Phytoextracts have been reported to control of grasserie disease (caused by Nuclear Polyhedrosis Virus) in silkworm, Bombyx mori L. (Kumar et al., 1998; Manimegalai et al., 2000) and virosis (caused

International Journal of Research in Zoology 2015; 5(1): 13-20

by CPV) in tasar silkworm, A. mylitta D. (Singh et al., 2004, 2006, 2008, 2010). Insects exhibits defense response through cellular and humoral components (Kimbrell, 1991). Humoral reactions involve slow synthesis of antibacterial and antiviral principles and require several hours for full expression. Cellular responses are direct interactions between circulatory haemocytes and invading non self-materials. The interaction is immediate and includes phagocytosis, nodulation and encapsulation (Gupta, 1986). In insects, several types of haemocytes are observed in the haemolymph (Butt and Shields, 1996). Studies on biochemical changes in tasar silkworm related to stress have been carried out by Pandey et al, 2010 and Kumar et al, 2011. Singh et al, (2011) have noticed six types of blood cells in his studies on cellular and biochemical changes in immunized tasar silkworm. A variety of functions like mechanization and immobilization of invading organism by encapsulation and for phagocytosis, wound repair, coagulation have been reported to haemocytes (Pech et al., 1995). Effect of various pathogens have been reported in the insects (Lea, 1986), To date reports on influence of botanicals on cellular and humoral defense in the insects infected with Cytoplasmic Polyhedrosis Virus (CPV) are scanty. In the present investigation, the influence of botanical formulation on suppression of virosis and the cellular and humoral responses is reported through reduction in mortality and estimation of haemocyte count and total proteins in the haemolymph in the tasar silkworm infected with Antheraea mylitta Cytoplasmic Polyhedrosis Virus (AmCPV). MATERIALS AND METHODS Botanical formulation was developed from the medicinal plant (Andrographis paniculata, Phyllanthus niruri and Aloe vera) known for their antibiotic property in silkworm pathology laboratory, Central Tasar Research and Training Institute, Ranchi for the control of virosis in tasar silkworm, Antheraea mylitta. The aqueous extract (0.5, 1.0, 1.5, 2.0 and 2.5% W/V) was used to test their efficacy against virosis and its influence on cellular and humoral response. The required quantity of botanical formulation (0.5, 1.0, 1.5, 2.0 and 2.5g ) was soaked in 100 ml. clean waster for 10-12 hours, filtered through muslin clothe and squeezed completely for preparation of 0.5, 1.0, 1.5, 2.0 and 2.5% aqueous extract respectively. Cytoplasmic Polyhedrosis Virus (AmCPV) inoculum Fresh cytoplasmic polyhedrosis virus inoculum’s was prepared from diseased silkworm. Completely whitened mid-gut obtained from cytoplasmic polyhedrosised silkworm at an advanced stage of infection were homogenized in sterile distilled water. The polyhedral suspension was filtered through a cheese - cloth and the filtrate was centrifuged at 3000 rpm for 15 min. and the polyhedra were purified following Aizawa, (1971) by repeated and differential centrifugation. The resultant pellet suspended in distilled water was examined by light microscope for purity. The polyhedral suspension in sterile

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distilled water was prepared to contain 1 x 105 polyhedra/ml. Inoculation of Polyhedra Occlusion Bodies (POBs) of AmCPV and treatment of larvae with botanical formulation Silkworm larvae of Daba eco-race were orally inoculated (by feeding food plant leaf smeared with polyhedra suspension) with 200 l of AmCPV polyhedra (1 x 105 polyhedra/ml) inoculum 24 hrs after 2nd moult. After 6 hrs of virus inoculation three subsequent feedings and once in 3rd, 4th and 5th instar larvae were allowed to feed on leaves of its primary food plant Terminalia tomentosa (asan) treated with 0.5, 1.0, 1.5 and 2.0% aqueous extract of botanical formulation. Three replications with 100 silkworm larvae were maintained for each treatment for observations on effect of botanical formulation on virus infection. For the observations on influence of botanical formulation on cellular and humoral defense, a separate batch of silkworms was maintained on fresh leaves of food plant up to 4th moult. The larvae after 4th moult in three replications (100 larvae each) were orally inoculated with POBs of AmCPV (1 x 105) and following three feedings were given with the leaves treated with effective concentration of aqueous extract of botanical formulation. Both treated and control batches were reared on leaf of its primary food plant, Terminalia tomentosa (Asan) in two rearing seasons (July – August and September – October 2011) in indoor under normal rearing conditions up to spinning. Estimation of haemocytes count Every day total haemocyte counts (THC) estimation in the haemolymph of all treated and control batches was determined following the method described by Tauber and Yeager (1934, 1935) using haemocytometer. The THC per mm3 of haemolymph was estimated according to the formula suggested by Jonesh (1962). Every day from 0 to 8th day 6 larvae were collected from each replication, the haemolymph from all the 6 larvae was collected in to three eppendoff tubes (2 larvae haemolymph/tube) on ice and stored at 4°C. A total of 6 tubes represented 3 replication collections. The differential haemocyte count (DHC) was estimated by counting different haemocytes from a haemocyte population of 200. Different haemocytes were identified based on the morphological features as described by Nittono (1960). The observations were made on THC and DHC counts Estimation of total proteins The total protein content in hemolymph was estimated by the method of Lowry et al. (1995). To 100 µl of hemolymph, 100 µl of 20% trichloro acetic acid was added and kept for 30 min. the contents were centrifuged at 3,000 rpm for 5 min and the pellet was washed twice with 10% trichloroacetic acid. Finally the palate was dissolved in 0.1 N sodium hydroxide. To the 10 µl of pellet sample 5 ml of alkaline copper sulphate reagent (reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide; reagent B: copper sulphate in 0.1 % potassium sodium tartrate in 1:1 ratio) was added. To make alkaline copper sulphate reagent, reagents A and B were mixed well in the ratio of 50:1.


After 10 min, 0.5 ml of folin-phenol reagent (the commercial solution was diluted once with distilled water) was added and shaken well. After 30 min, the colour intensity was read at 660 nm in a Jasco V – 530 Spectrophotometer. The blank sample contained 10 µl of distilled water, 5 ml of alkaline copper sulphate reagent and 0.5 ml of folin-phenol reagent. The protein content was recorded from the standard curve prepared for bovine serum albumin (10 - 100 µg). The protein content in the samples were expressed as mg/ml of hemolymph. Data recorded for mortality due to concerned pathogen,

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THC and DHC counts and total protein content were statistically analyzed using Completely Randomized Design (Snedecor and Cockron, 1971). RESULTS AND DISCUSSION Effect of botanical formulation on virosis disease in tasar siikworm The observations with regard to larva mortality due to virosis and reduction in virus infection in tasar silkworm, after POBs of AmCPV (1 x 105) inoculation and treatment with aqueous extracts of botanical formulation are depicted in Figs. 1and 2.


Table 1: Effect of botanical formulation on the total haemocyte count in AmCPV infected tasar silkworm, Antheraea mylitta D. Days post inoculation Treatment 1 2 3 4 5 6 7 8 Botanical 12550 14460 14810 15116 15207 15312 14303 15120 formulation Inoculated 12760 14380 14350 12471 9810 6200 4809 3715 control Normal control

12340

12430

12902

13415

13870

13672

12290

13751

S.E. ± C. D. at 5%

122.16 127.27

136.24 142.26

116.17 169.21

101.38 118.23

148.72 251.21

175.28 301.25

187.26 279.92

193.13 325.46

Table 2. Differential haemocyte counts after AmCPV infection in silkworm, A. mylitta and treatment with botanical formulation

Treatment

Haemocyte

Botanical formulation

PR PL GR SP OE VER DEG

Inoculated control

Normal control

PR PL GR SP OE VER DEG PR PL GR SP OE VER DEG

1 24 39 42 25 8 1 12 1 28 33 35 24 13 1 22 22 32 35 17 5 10

2 23 41 47 25 7 16 2 25 39 45 20 11 24 25 33 36 18 6 12

3 21 44 48 24 6 15 3 24 44 48 18 10 25 27 35 38 20 8 13

Days post inoculation 4 5 19 24 41 39 48 43 19 20 8 9 17 12 4 5 23 18 42 38 45 44 17 15 8 8 26 30 29 30 37 39 39 42 22 25 10 10 4 14 16

6 24 36 40 22 8 12 6 17 35 41 14 5 37 33 41 40 26 12 1 9

7 26 35 39 22 9 10 7 15 32 32 12 4 39 34 37 38 27 13 7

8 27 37 41 25 10 9 8 12 25 27 9 3 42 35 38 38 28 15 8

PR = Prohaemocyte PL = Plasmatocyte GR = Granulocyte SP = Spherulocyte OE = Oenocytoid VER = Vermiform cell DEG = Degenerated cell

Results revealed that the larva mortality due to virus infection was highest in the larvae inoculated with POBs of AmCPV (Treated control). The larva mortality was considerably reduced in the larvae treated with the aqueous extracts of botanical formulation. The larva mortality ranged 28.14 to 48.76% and 25.99 to 45.28% in 1 st and 2nd crop rearing respectively when the larvae were treated with different concentrations of aqueous extracts of botanical formulation. The lowest mortality 28.14 and 25.99% in 1 st and 2nd crop respectively was recorded with treatment of 2.5% concentration of botanical formulation. In treated

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control larva mortality was 88.36 and 91.58% during 1st and 2nd crop respectively. Pooled analysis of data revealed that the reduction in virus infection than treated control was 47.68 to 69.88% with the treatment of different concentrations of botanical extract. In the present study the botanical formulation found effective in reducing virus infection significantly (P < 0.05) at the lower concentrations of 0.5 to 2.5%. Reduction in virus infection was comparatively higher at the higher concentration of 2.5%. However, aqueous extract of botanical formulation (2.5%) was much effective and reduced the virus infection in


silkworm to the maximum level, may be due to its antiviral action as reported by Singh et al. (1999). The observations on antiviral action of plant extracts have been made by Samuel Manohraj (1994) and Mallika (1997) who reported 72.46 and 79.50% reduction in grassarie disease (caused by Nuclear Polyhedrosis Virus) in mulberry silkworm, Bombyx mori L. by application of leaf extract of Psoralia coryleifolia respectively. Similarly Sivaprakasham (1994) reported that the aqueous extract of P. coryleifolia was better than gentamycin and calcium hydroxide in reducing the larval mortality due to grassarie. Manimagalai et al (2000) observed 63% reduction in grassarie disease in B. mori with the application of turmeric and chalk powder. The extracts of leaf, root and flower of A. zeylanica have been reported for the treatment of bacterial and viral diseases in human being (Singh et al, 1999). However, the plant extracts tested in the present investigation have not been reported earlier against the virus diseases in tasar silkworm. Effect of Antheraea mylitta Cytoplasmic Polyhedrosis Virus (AmCPV) and botanical formulation treatment on Total Haemocyte Counts (THC) The results on the effect of botanical formulation on total haemocyte count in AmCPV infected tasar silkworms are presented in table 1. In the treatment and normal control, the total haemocyte counts increased from 1st day to 6th days and decreased on 7th day after the next moult. In botanical formulation treated batch, the total haemocyte counts was 12550/mm3 of haemolymph and increased to 15312/mm3 by 6th day. On 7th day total haemocyte counts were 14303/mm3. On 8th day the count was increased up to 15120/mm3. The similar trend was observed in normal control. While in inoculated control the counts were increased after inoculation with AmCPV up to 2nd day of infection. Then there was a decrease for a period ranging from 3-8 days. In normal control the total haemocyte count was significantly low as compared to the treatment because the increase may represent the defense response of silkworm, A. mylitta against the invading pathogen. The observed data agreed with the earlier workers as they investigated that once entomophagus fungi have penetrated in the host integument and gained access nutrient-rich haemocoel, they are confronted with host humoral and or cellular defenses (Butt et al., 1988, Butt and Humber, 1989, Vey & Gotz, 1986). As humoral response, the phenoloxidase system will be activated to induce the phagocytic process and melanization which works as toxin to invading microorganism (Tanada and Kaya, 1993). The cellular responses to infection have been worked out in many insect by earlier workers (Chain and Anderson, 1982; Dunn and Drake, 1983; Horohove and Dunn, 1983). Similar observations have also been made Mallikarjuna et al (2002) in his study on the effect of systematic fungicide on the total haemocyte count in Beauveria bassiana infected silkworm, Bombyx mori. Salt (1970) reported that Haemocytes are exteremely efficient in removing pathogens by accomplishing a series of reactions designated as phagocytosis, nodule formation or encapsulation. The present observations are in agreement

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with the earlier investigation that the number of haemocytes may increase (Balavenkatasubaiah et al., 2001) and decrease (Gillium and Shimanuki, 1967) to counter foreign body when infected. On the basis of the above findings of the earlier workers it is evident that CPV induce the defense response through multiplication of haemocytes as is indicated by the increase in total haemocyte counts of the haemolymph of the worms. Effect of Antheraea mylitta Cytoplasmic Polyhedrosis Virus (AmCPV) and botanical formulation treatment on Differential Haemocyte Counts (DHC) The observation with regard to DHC in tasar silkworm, Antheraea mylitta D. after POB of AmCPV (1 x 105) inoculation and treatment with botanical formulation is depicted in table 2. The prohaemocyte and spherulocytes decreased up to 4th day and increasing trend was observed from 5 th day onwards when worms were first inoculated with AmCPV(1 x105) and then treated with botanical formulation. While during progressive infection the gradual decrease in number of prohemocyte, spherulocytes and oenocytoid viz., 28-12, 24-9 and 13-3 respectively was noticed in inoculated control where worm were inoculated with AmCPV (1 x105). In the normal control (free from pathogen and treatment) all the recorded haemocytes showed an increasing trend during developmental period and number of degenerated cells was comparatively less than the botanical formulation treatments and inoculated control. The number of plasmatocytes and granulocytes increased up to 4th day and then decreased in the treatment. However, more or less similar observations have been recorded in inoculated control and in normal control. On 8 th day there is slight increase in number of the haemocyte observed in the treatment. The increasing trend in number of these heamocyte was not high in treatments as compared to the inoculated control because of the possibility of interaction of both pathogen and treatment. The number of prohemocyte decreased due to the conversion of prohemocyte to other types of haemocyte during course of infection and number of plasmatocytes and granulocytes increased as both are involved in defense mechanism against entry of pathogens. The palsmatocyte and granulocytes function in defense against largest foreign body in Bombyx mori L. was worked out by Satto et al (1976) and Akai and Sato (1976). The number of oenocytoid was less in the treatment including inoculated and normal control and ranged from 3 to 15. Few vermiform cells, synonyms of plasmatocyte (Lee and Gilbert, 1966; Devauchelle, 1971) were recorded in both the treatments and control. Humoral changes in AmCPV infected and botanical formulation treated tasar silkworm, A. mylitta D. Total protein contents The data on the effect of immunization on hemolymph total protein contents in AmCPV infected silkworm are presented in fig. 3. The hemolymth protein in immunized silkworms increased gradually from 32.18 mg/ml on 1st day to 57.13 mg/ml on day 8th. Similar trend was observed in non - immunized silkworms but the total hemolymph


proteins were significantly low. In inoculated control, the total hemolymph proteins have shown increasing trend from 1st to 5th day and decreasing trend from 6th day onwards. The results indicated that changes occur in the hemolymph protein, during the course of AmCPV infection. The difference in hemolymph protein between immunized, non-immunized silkworm and inoculated control becomes more pronounced as the diseases progresses. This would probably indicate that during infection the synthesis and release of proteins from fat bodies are greatly increased. There are reports of production of antimicrobial substances such as lectin, defensin and attacin with the entry of foreign bodies (Wago, 1995). Pombo et al., (1998) reported that several viral induced proteins were also produced during infection of baculovirus and a sharp decrease in overal protein synthesis was observed. Sinha et al. (1985) studied the changes in protein content in haemolymph of tasar silkworm, Antheraea mylitta, and reported that protein content in it increases enormously during larval development. A great reduction in all the protein fractions was reported in the haemolymph of heavily diseased larvae. The effect of infection by cytoplasmic polyhedrosis virus on the midgut protein metabolism in silkworms was studied by Watanabe (1971) and reported that the active synthesis of midgut proteins, as well as polyhedron proteins, is induced by infection and continues until later stages. REFERENCES 1. Aizawa K. (1971) Structure of polyhedra and virus particles of cytoplasmic polyhedrosis, In the cytoplasmic polyhedrosis of the silkworm (Eds: Aruga, H. and Tanada, Y.) Univ. Tokyo press, Tokyo, pp. 2336. 2. Akai H. & Sato S. (1976) Surface ultastructure of larval haemocytes of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Int. J. Insect Morphol. Embryol., 5: 17-21.

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Source of support: Nil; Conflict of interest: None declared

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