EFFECT OF NEW MICROSPORIDIAN INFECTION ON THE RELEVANT ECONOMIC CHARACTERS OF SILKWORM AND ITS MANAGEMENT

Transcription

1 EFFECT OF NEW MICROSPORIDIAN INFECTION ON THE RELEVANT ECONOMIC CHARACTERS OF SILKWORM AND ITS MANAGEMENT

2 Chapter III Effect of new microsporidian infection on the relevant economic characters of silkworm and its management T he Microsporidia have been reported to cause a wide range of damage and disease in silkworm Bombyx mori L. They can infect virtually any organ system and causes devastating loss in silkworm crop. Microbiological studies of silkworm microsporidians started after the pandemic of pebrine happened in Southern France in 1845, which attracted the interest of scientists in Europe (Steinhaus, 1949, 1956). Ever since Louis Pasteur (1870) established that pebrine disease (Corpuscles disease or Microsporidiosis) of the silkworm (Bombyx mori L.), is caused by Nosema bombycis, a parasite producing the spores. The disease transmit both vertically (trans-ovarial transmission) and horizontally (trans-ovam transmission) and spread in the colony and to their progeny. Silkworm diseases are the main constraint for successful harvest of quality cocoon in India. The estimated cocoon crop loss due to different silkworm diseases was 27-35% and cocoon yield loss was from kg / 100 dfls during different seasons (Selvakumar et al., 2002). Among the different diseases, pebrine is the only disease which comes both by primary and secondary infection and occurs during all seasons. Several authors reported that the spores of different microsporidian sp. infect different tissues and spore formation occurs in mid-gut epithelium, malphigian tubules, silk glands, fat bodies adipose tissue, gonads and trachea (Fujiwara, 1980, 1985; Watanabe, 1988; Ishiwata et al., 1990; Kawarabata, 2003). Pebrine disease causing agent Nosema bombycis is a unique pathogen transmitted by way of egg i.e., through transovarial transmission and by the ingestion of contaminated mulberry leaf by silkworm. The disease is also transmitted by transovum transmission through the contamination of egg s surface (Masera, 1938). Louis Pasteur established the control of pebrine disease by individual mother moth examination. Of late Fujiwara (1980), who worked on microsporidians of silkworm suggested group mother moth examination. Once the pathogen enters and germinates in the mid-gut cells of silkworm larvae and spreads to other tissues.

3 In the silkworm seed production process, maximum precaution is taken to avoid transovarially transmitted infection. The silkworms hatched out of eggs laid by healthy moths are only used for silkworm rearing and seed production thus eliminating chances of disease occurrence by transovarial transmission. Transovum transmission is avoided by surface disinfection of eggs. However, the secondary infection in the larval stage can pose serious problem. Disinfection of silkworm rearing environment and adaptatiing hygienic practices during the rearing is followed to avoid secondary infection. There had been several attempts to cure the silkworm microsporidiosis resulting from secondary as well as primary infection. Chemotherapy is an effective way to control the diseases in insects. In the past, chemotherapy was considered as non-realistic way to control silkworm diseases, however in recent years, some of the therapeutic drugs / chemicals have proved to be effective. Chemotherapy though did not cure the insects microsporidiosis but found promising in suppressing the incidence. Chinese scientists are reported to have identified antimicosporidian drug, which cure silkworm of microsporidiosis resulting from transovarial infection. Analogues of benzimidazole and Benlate (Liu Shi Xian, 1979) Bavistin, Derosal (Baig, 1994), Fumidil-B or fumagillin (Lewis and Lynch, 1970; Hayasaka, 1991; Schmahl and Benini, 1998; Frankenhuyzen et al., 2004), methylthiophanate and ethyl thiophanate (Liu, 1987; Hayasaka, 1991) anisomycin (Hayasaka, 1991) to be effective against different microsporidians. Buquinolate is reported to control microsporidiaosis in Blue crab (Overstreet, 1975). Griyaghey (1976) and Alok Sahay et al., (2005) studied the effect of chemotherapeutic agents on Pebrine in Tassar silkwom, Antheraea mylitta and found an effective way to control the microsporidiosis. More recently, two groups of therapeutic agents have been identified as being useful in the treatment of human microsporidiosis (Costa and Weiss, 2000). The benzimidazoleles, which bind tubulin, include albendizole, the benzimidazole of choice for treatment of microsporidiosis due to the Encephalozoonidae. The second group of compounds derived from fungi has activity against several groups of parasites and they have been demonstrated to bind irreversibly to a common bi functional protein identified by mass spectrometry as methionine amino peptidase type 2 (MetAP2) in other microsporidia excluding Nosema bombycis (Griffith et al., 1997). A review of literature

4 indicated that it is possible to control Microsporidiosis with the use of benzimidazoles such as albendazole and its derivatives to control human microsporidiosis in vitro and in animal models of microsporidiosis (Hong Zhang et al., 2005). Most of the chemicals/ drugs/ antibiotics used for suppression of the pebrine disease are cost prohibitive and not eco-friendly. In the recent past, there is a shift towards using botanical based products for the suppression of diseases in silkworm. Several medicinal plants have been screened to control insect disease (Sujatha et al., 2005; Singh et al., 2005, Jaiswal and Deka, 2005). Girijadevi (2006) tested aqueous extracts of different botanicals against N. bombycis. Thymol, an active ingredient of Ammi copticum (Ajowan) of family Umbelliferae have found effective in controlling Nosema disease in honey bees (Rice, 2001). Chemotherapeutic agents are fed to silkworm either through artificial diet or through mulberry sprayed with the antimicrosporidian agent (Iwano and Ishihara, 1981; Hayasaka, 1991; Brooks et al., 1978; Frankenhuyzen et al., 2004). Several preventive measures against microsporidiosis are in practice in sericulture. The inspection of mother moth and destruction of eggs laid by moths infected by the microsporidia during the silkworm seed production is the most meticulously followed method to prevent the outbreak of pebrine disease in sericultural areas. The other preventive measures may emphasis on elimination of secondary sources of infection. The major sources of secondary infection are the diseased and dead larvae, faces and the gut juice vomited by the diseased silkworm. Alternate hosts also pose problem in management of silkworm microsporidiosis. The disinfection of silkworm rearing house, silkworm rearing appliances and the silkworm seed production centers is followed to eliminate the spores of microsporidians which cause the secondary infection. It is coupled with good hygiene practices during silkworm rearing and egg production. Several physical and chemical disinfectants have been screened for their efficacy against the microsporidian spores. Microsporidia are diverse group of organisms and with the exception to the direct effects of sunlight, which quickly kill/inactivate most organisms, the different group of microsporidians respond differently to different environmental stresses (Maddox, 1977; Kava, 1977; Brooks, 1988). Very little information is available on the effect of sunlight or ultraviolet (uv)

5 radiation on the survival of the microsporidian spores (Maddox, 1973). However, it is known that microsporidian spores are susceptible to ultraviolet (uv) radiation. Baribeau and Burkhardt (1970) reported the unpublished work of I. L. Revell where it is observed that Nosema apis spores are inactivated in 5 hr by radiation. Subsequently, Wilson (1974) demonstrated that N. fumiferanae spores on cherry leaves and artificial diet were inactivated by a 30-W germicidal lamp within 4 and 5hr.The effect of temperature on microsporidian spores is reviewed by Maddox (1973). According to Maddox (1973)the inactivation of microsporidian spores occurs at fairly high temperature, but is dependent on condition and length of exposure. Various disinfectants viz., Formalin (Kagawa, 1980), Asiphore (Venkata Reddy et al., 1990), Chlorinated lime and hydrochloric acid (Miyajima, 1979), Chlorine dioxide (Nataraju, 1995; Balavenkatasubbaih et al., 1999), Kao haiter (Balavenkatasubbaih et al., 2003), Serichlor (Balavenkatasubbaih et al., 2006), Calcium chloride (Iwashita and Zhou, 1988; Patil, 1991) have been screened against silkworm pathogens including N. bombycis. Chemical disinfectants such as Hilite, Sodium hypochlorite, Bleaching powder and Formalin are used as surface disinfectants of silkworm egg against N. bombycis (Baig et al., 1989). Chemical germicides play a crucial role in infection control and combat with wide range of pathogens. However, there is a concomitant heightening of concerns on the human and environmental safety of many germicidal chemicals. This, in turn is focusing greater attention on the search for formulations, which are not only good broadspectrum germicides, but are also safer for humans and benign for the environment. While the objective of finding highly effective and fast-acting germicidal chemicals which are, at the same time, totally harmless for humans and the environment may be unattainable at the moment. In this objective it was to test the sporicidal, fungicidal, bactericidal and virucidal activities to determine its potential as a broad-spectrum disinfectant. Based on these objectives different drugs/chemicals/botanicals were identified for the management of microsporidiosis. Hence in the present study, efforts will be made to screen NIK-5hm microsporidia for its response to different Drugs, chemical and botanicals to develop convenient and efficient preventive/control measures to contain the microsporidiosis disease caused by NIK-5hm microsporidia in CSR2 breed. Attempt has also been made to develop drug/chemical/ botanical formulations

6 against NIK-5hm infection. MATERIALS AND METHODS Effect of NIK-5hm micrsporidian infection in CSR2 and PM breeds of silkworm: The Effect of NIK-5hm microsporidia on the economic characters of CSR2 and PM breeds of silkworm was determined by per os inoculation of the microsporidian spores in conc. of sporesml to 3 rd instar silkworm. The eggs of identified bivoltine and multivolotine breeds viz., CSR2 and PM were received from the Germplasm bank of CSRTI, Mysore for this study. The larvae of the selected breeds were reared under hygienic condition till the beginning of 3 rd instar and were inoculated per os with the purified spores of NIK-5hm microsporidia. Each breed formed a treatment. The inoculum was prepared from purified spores of the microsporidia and quantified by standard method using haemocytometer. The silkworm larvae were inoculated by feeding mulberry leaf smeared with one ml inoculum of spores/ml to 100 larvae. The larvae were allowed to feed on the treated leaves for 24 h, to ensure complete consumption of treated leaves. After 24 h, the larvae were continued to be reared on uncontaminated mulberry leaves till cocooning. Two controls for each of the breed were maintained for comparative purpose. The first control larvae were treated with mulberry contaminated with 1 ml of spores / ml of N. bombycis to 100 larvae and the second control larvae were treated with mulberry leaves smeared with sterilized distilled water and reared on uncontaminated mulberry. The controls and treatments had three replications of 100 larvae each. The larvae were observed for growth, behavior and other economic characters such as larva, cocoon and shell weight, silk ratio and mortality due to microsporidiosis during the rearing and pupal stage, etc. recorded and analyzed. The reason for mortality was confirmed by microscopic examination of the dead larvae and pupa collected during the rearing and cocooning for the microsporidian spores. Viability response of NIK-5hm spores to chemical disinfectants Disinfection ability of Chemical disinfectants such as Chlorine dioxide (20,000ppm ClO 2 ) from M/s Sericare, Divn. of Ashchem Agrotech (Pvt Ltd. Bangalore), Formaldehyde (37% HCHO from M/s Hindustan Organic Chemicals Ltd. India), Bleaching powder (30% DCM Sriram consolidated Ltd (India) and Asthra (M/s Seri Gro, Pvt. Ltd., Bangalore)) were determined by suspending the spores in the chemical disinfectants. The purified spores were suspended in different concentrations of the chemical disinfectants for a period of 5 to 30, min and diluted, pelleted by centrifugation

7 and washed by repeated centrifugation to remove the traces of chemical disinfectant. The final pellet was suspended in distilled water and examined for viability and infectivity. The different concentrations of different disinfectants forming the treatment are listed in Table 3.1. The following method was adopted for preparation of different concentration of different disinfectants. Chlorine dioxide of 50, 100, 200, 300, 400 and 500 ppm in water was prepared from commercially available stabilized chlorine dioxide (Sanitech) containing 20,000 ppm. The stabilized chlorine dioxide was activated by adding 0.25g of activator in 2.5ml of Sanitech solution. To the activated Sanitech solution, 97.5 ml of water was added to obtain 100 ml of 500 ppm Chlorine dioxide solution. The 500ppm solution was serially diluted with distilled water to get the required chlorine dioxide solution of concentration 400, 300, 200, 100 and 50 ppm. Formalin of 0.5, 1, 1.5 and 2.0% concentrations were prepared from commercial formaldehyde (37% formaldehyde) by using the formula N1V1/ N2V2 where N1 is the original concentration, V1 is the original volume, N2 is the required concentration and V2 is the required volume. Bleaching powder of 0.5, 1.0, 1.5 and 2.0% in water was prepared from Bleaching powder containing 30% Chlorine by mixing 0.5, 1, 1.5 and 2 g of Bleaching powder in 100 ml sterilized distilled water respectively. Asthra solution of 0.01, 0.03 and 0.05% prepared by dissolving 0.01, 0.03 and 0.05 g of Asthra powder in 100 ml sterilized distilled water respectively. In vivo tests: The treated spores were subjected to bioassay to determine the infectivity of the spore exposed to different chemical disinfectants. One ml of the inoculum containing treated spores / ml were smeared on mulberry leaves and fed to the 100 silkworm immediately after 2 nd moult. A control batch of silkworm fed with spores without exposure to chemical disinfectants was also maintained. Each treatment had three replications of 100 larvae. The larvae were reared till cocooning and pupae were allowed to emerge and moths were examined for infection. The dead larvae, pupae and moth were also examined for infection by the spores exposed to different chemical disinfectants. The observations were recorded and analyzed.

8 Screening of different drugs, chemicals and botanicals. Drug based: Based on the literature available several anti - protozoan drugs such as benzimidazole derivatives viz., Albendazole, (M/s Cipla Ltd. by Meditab Specialties Pvt. Ltd., Goa, India), Mebendazole, (M/s Cipla Ltd. by Meditab Specialties Pvt. Ltd., Goa, India), Quine derivatives viz., Sapriquine (M/s Fulford India, limited, Pathampur, Distt. Dhar, M.P.), Chloroquine phosphate (M/s Rhone-Poulenc Pvt. Ltd. Aurangabad, India), were selected for the screening of anti-microsporidian activity against NIK-5hm spores. The drugs were screened in-vivo for their toxicity and the non-toxic drugs were screened for efficacy by in vivo and bioassay method for anti-microsporidian activity. In vivo toxicity studies: The drugs were screened for their toxicity to silkworm by feeding the drug at 1% concentration in water. The drug at 1% concentration was smeared on to mulberry and fed to silkworms immediately after 2 nd moult once on day 1, 2 and 3. The larvae were observed for symptoms and mortality due to the drugs for 7 days, recorded and the non toxic drugs, to the silkworm were short listed. The non toxic drugs were selected for further study and screened in vivo for anti- microsporidians activity. In vivo screening: Second instar disease free silkworm of CSR2 breed inoculated with NIK-5hm microsporidia were used for in vivo screening of drugs against the NIK-5hm microsporidia. 100 silkworms of these breeds were inoculated by per os with NIK-5hm spores of spores/ml immediately after 2 nd moult. The breeds were then fed on mulberry leaf treated with the drugs in 0.25, 0.50 and 1.00 % concentrations. The drugs in identified concentrations were smeared on the surface of mulberry leaf (1 ml / 50 sq. cms) and fed to rd instar silkworm on alternate days up to spinning. A control group of larvae inoculated with NIK-5hm spores of concentration spores/ml were reared on normal leaves for comparative purpose. Observations were recorded for mortality due to microsporidiosis and its impact on economic characters. Botanical based: To develop eco and user friendly approach in management of microsporidiosis in CSR2 breed, four botanicals, identified based on the literature (Thomas, 1998), were screened for anti-microsporidian activity. The identified botanicals were the sap of Ficus bengalensis, Carica papaya, Jetropha gossypifolia and Ruta chalepensis.

9 In vivo toxicity studies: The aqueous content (sap) of botanicals were screened for toxicity to silkworm. The extracts of 2% concentration was smeared on to mulberry leaf and fed to silkworm after 2 nd moult once on day 1, 2 and 3. The larvae were observed for symptoms and mortality due to the toxicity for 7 days. The non-toxic botanicals were selected for further study and screened in vivo and bioassay for anti- microsporidians activity. In vivo screening: Second instar silkworm of CSR2 breed inoculated with NIK-5hm microsporidia were used for in vivo screening of botanical extract (sap) against the NIK- 5hm microsporidia. 100 silkworms of these breeds were inoculated per os with NIK-5hm spores of spores/ml concentration immediately after 2nd moult. The breeds were then fed with botanical extract of 0.5, 1, 1.5 and 2 %concentrations. The extract was smeared on the surface of mulberry leaf (1 ml / 50 sq. cms) and fed to 100 third instar silkworm larvae on alternate days up to spinning. A control group of larvae inoculated with NIK-5hm spores of spores/ml were reared on normal leaves for comparative purpose. The dead larvae were collected and examined microscopically to determine the cause of death. Observations were recorded for mortality due to microsporidiosis and its impact on economic characters. The data were statistically analyzed. Chemical based: Based on the literature available several anti - protozoan chemicals such as Hydrogen peroxide, Glutaraldehyde, Aldol and Accelerated Hydrogen peroxide (AHP) were selected for the screening for anti-microsporidian activity against NIK- 5hm spores. The chemicals were screened in-vivo for their toxicity and the nontoxic chemicals were screened for efficacy by in vivo and bioassay method for antimicrosporidian activity. In vivo toxicity studies: The chemicals were screened for their toxicity to silkworm by feeding the chemicals at 2% concentration in water. The chemicals at 2% concentration was smeared on to mulberry leaf and fed to silkworms immediately after 2 nd moult once on day 1, 2 and 3. The larvae were observed for symptoms and mortality due to the chemicals for 7 days, recorded and the non toxic chemicals, to the silkworm were short listed. The non toxic chemicals were selected for further study and screened in vivo and bio-assay method for anti- microsporidians activity.

10 In vivo screening: Second instar disease free silkworm of CSR2 breed inoculated with NIK-5hm microsporidia were used for in vivo screening of chemicals against the NIK- 5hm microsporidia. 100 silkworms of these breeds were inoculated by per os with NIK- 5hm spores of spores/ml immediately after 2 nd moult. The breeds were then fed on mulberry leaf treated with the chemicals in 0.50, 1.00, 1.50 and 2.00 % concentrations. The chemicals in identified concentrations were smeared on the surface of mulberry leaf (1 ml / 50 sq. cms) and fed to 100 third instar silkworm on alternate days up to spinning. A control group of larvae inoculated with NIK-5hm spores of concentration spores/ml were reared on normal leaves for comparative purpose. Observations were recorded for mortality due to microsporidiosis and its impact on economic characters. RESULTS Effect of NIK-5hm micrsporidian infection in CSR2 and PM breeds of silkworm: The results of the studies on the effect of NIK-5hm microsporidia and N. bombycis in silkworm on CSR2 and PM breed of silkworm are presented in Table 3.2. It is observed that the infection by NIK-5hm microsporidia resulted in significant impact on the economic parameters both in CSR2 and Pure Mysore (PM). A comparison with the respective healthy control indicate that the infection with NIK-5hm microsporidia shows high rate of reduction in all economic characters viz., percent survival (ERR %), larval weight, single cocoon weight, shell weight and percent silk content. The survival percent after NIK-5hm infection was lowest in CSR2 (23.00%) compared PM (41.00 %). Similar trend was in N. bombycis inoculated batches where survival percent after N.bombycis infection was lowest in CSR2 (24.33%) and in PM (42.67%) The single cocoon weight was significantly lower in CSR2 and PM breeds silkworm infected with NIK-5hm microsporidian when compared to control batches. The cocoon weight in infected batches was 1.32g and 0.86g respectively, while the healthy cocoon weight was 1.62g in CSR2 and 1.07g in PM. Similar trend was noticed in N.bombycis infected batches also. The cocoon weight in infected batches was 1.34g and 0.89g in CSR2 and PM respectively. The single shell weight was significantly lower in CSR2 and PM breeds silkworm infected with NIK-5hm microsporidian when compared to control batches. The shell weight in infected batches 0.27 g and 0.10 g respectively, where the shell weight

11 of control batches were 0.38 g and 0.14 g in CSR2 and PM respectively. Similar trends were noticed in N. bombycis infected batches also. The shell weight in infected batches was 0.29g and 0.11g in CSR2 and PM respectively The silk content was significantly lower in CSR2 and PM breeds silkworm infected with NIK-5hm microsporidia when compared to control batches. The silk content in infected batches was 20.40% and 11.96% respectively, where the silk content of control batches were and 13.44% in CSR2 and PM breeds respectively. Similar trend was noticed in N.bombycis infected batches also. The silk content in infected batches was and 12.41% in CSR2 and PM breeds respectively. A comparative study of NIK-5hm microsporidia and N. bombycis infections in CSR2 and PM breeds indicate that the NIK-5hm is more virulent than N. bombycis microsporidia. The survival percent of infected batches with NIK-5hm in CSR2 and PM breed was 23.00% and 41.00% respectively and it is less than the N. bombycis inoculated batches. Chemical disinfectants The response of microsporidian strain, NIK-5hm to chemical disinfectants are presented in Table 3.3. All the chemical disinfectants were sporicidal to spores of NIK-5hm. NIK-5hm spores suspended for 20 min in Chlorine dioxide solution of 400 ppm and higher concentration resulted in killing of all the spores suspended. At lower concentration and shorter suspension period, all the spores were not killed. The per os inoculation of spores exposed to 400 ppm for 20 and 30min and 500 ppm for 5 to 30min., Chlorine dioxde did not cause infection in silkworm. At lower concentration and the exposure period of 5 to 30 min the microsporidia caused an infection in CSR2 breed ranging from 4.33 to 63.33%. Bleaching powder of 30% chlorine content at 0.5 % concentration with exposure period of 5 to 30 min. was not sporicidal in CSR2 breed. At higher concentration of 1.5%, from 10 minute and above duration and 2.00%, bleaching powder suspended for 5 to 30 minutes was sporicidal and killed all the spores. Per os inoculation of spores suspended in 0.5% and 1% bleaching powder for 5 to 30 minutes exposure caused infection in silkworm ranging from 4.67 to

12 Formalin 1.5% was sporocidal to NIK-5hm spores when exposed for 10 min or longer period. 0.5% Formalin with exposure period of 5 to 30 min., 1% Formalin with exposure period of 5 to 10 min. did not cause 100 % killing of spores. At higher concentration of 1.5% for 10 to 30 min, and 2% for 5 to 30 min. exposure, period formalin was sporicidal and killed all the spores exposed. In such cases, 100 % mortality of spores was achieved. Per os inoculation of spores exposed to formalin of 1.5% for 10 to 30 min. and 2% for 5 to 30 min did not cause infection in silkworm while spores exposed to lower concentration of 0.5% for 5 to 30min and 1% for 5 to 30 min. and 1.5% for 5 minute lead to the infection ranging from 1.67 to 38.00% in silkworm. Asthra of 0.03% concentration with exposure period of 5 to 30 min was sporicidal in CSR2 breed. At higher concentration of and 0.05% from 5 minute and above duration was sporicidal and killed all the spores. Per os inoculation of spores suspended in 0.01% for 5 to 30 minutes exposure caused infection in silkworm ranging from 5.00 to 26.25%. Chemotherapy Drugs based: The observations on the efficacy of drugs on the microsporidiosis caused by NIK-5hm microsporidia in CSR2 breed of silkworm is presented in Table Among the drugs screened, four drugs were found non-toxic to the silkworm and were considered for screening for their anti-microsporidian activity viz., Albendazole, Mebendazole, Sapraquine and Chloroquine. The result of in vivo screening of drugs for anti-microsporidia activity in CSR2 indicated that among the drugs Sapraquine (1.00%) is effective in reduction of larval mortality due to microsporidiosis to an extent of 97.42%. The other drugs at concentrations ranging from 0.25 to 1.00% reduced the mortality due to microsporidiosis caused by NIK-5hm to the extent ranging from 87.75%-96.08% in CSR2 breed. The percent of infected moths was significantly low in all treatments. In the inoculated control it was 54.00%. The economic characters such as larval weight, single cocoon weight, shell weight and silk ration are significantly higher in the treatment batches with these drugs derivative in CSR2 breed.

13 Chemical based: The results of screening of chemicals for anti microsporidian activity against NIK-5hm microsporidian are presented in Table The chemicals screened, viz., Hydrogen peroxide, Glutaraldehyde, aldol and Accelerated Hydrogen Peroxide (AHP) did not show any toxic effect in silkworm at 0.5 to 2.0% concentration and were considered and for screened as non toxic chemicals to silkworm. The result of in vivo screening of chemicals for anti-microsporidia activity in CSR2 indicated that among the chemicals AHP (2.00%) is effective in reduction of larval mortality due to microsporidiosis to an extent of 100%. The other chemicals at concentrations ranging from 0.5% to 2.00% reduced the mortality due to microsporidiosis caused by NIK-5hm to the extent ranging from 89.92% % in CSR2 breed. The percent of infected moths was significantly low in all treatments over the inoculated control it was %. The economic characters such as larval weight, single cocoon weight, shell weight and silk content were significantly increased in the treatment batches with all chemicals in CSR2 breed, However in case of AHP all the % of ERR (89.33%), larval weight (41.67 g), single cocoon weight (1.623), single shell weight (0.380) and silk content (23.410) which were significantly higher than the control batches. Botanical based: The observations on the efficacy of botanicals on the microsporidiosis caused by NIK-5hm microsporidia in CSR2 breed of silkworm is presented in Table Among the botanicals screened, four botanicals were found non-toxic to the silkworm and were considered for screening for their anti-microsporidian activity viz., Ficus bengalensis, Carica papaya, Jetropha gossypolia and Ruta chalepensis. The result of in vivo screening of botanicals for anti-microsporidia activity in CSR2 indicated that among the botanicals, Carica papaya (2.00%) is effective in reduction of larval mortality due to microsporidiosis to an extent of 97.95%. The other botanicals at concentrations ranging from 0.5 to 2.00% reduced the mortality due to microsporidiosis caused by NIK-5hm to the extent ranging from 72.66% to in CSR2 breed. The percent of infected moths was significantly low in all treatments. In the inoculated control it was 53.00%. The economic characters such as larval weight, single cocoon weight, shell weight and silk content were significantly increased in the treatment batches with all botanicals in CSR2 breed, However in case of Carica

14 papaya % of ERR (89.33%), Larval weight (41.67 g), single cocoon weight (1.623), single shell weight (0.380) and silk content (23.410) which were significantly higher than the control batches. DISCUSSION A comparison of infectivity and transmission of NIK-5hm microsporidia with that of Nosema bombycis indicate that NIK-5hm microsporidia have high rate of infectivity, transmission and mortality in the larval and pupal stage. The NIK-5hm microsporidia infects different breeds of silkworm and its infectivity and transmission is higher in CSR2 breed compare to PM. Its impact on the economic characters is significant. The survival percentage of larvae is less, the cocoon weight, shell weight and silk content is reduced. While the CSR2 breed is of great economic value in terms of quality and quantity of silk produced, the ability of the breed to survive infection is of great interest. The low percent of survival of CSR2 breed to NIK-5hm microsporidia confirms that the breed is comparatively more susceptible to microsporidian infection. In view of this, the understanding and management of the disease in CSR2 breed is of practical importance in the process of introduction and improvement of mulberry sericulture in the area. The understanding will also be useful in management of microsporidiosis in general. The characterization of NIK-5hm spores for viability response to physical and chemical agents is important in management of the disease. Among the chemical disinfectants use in sericulture, 500 ppm Chlorine dioxide, 2% bleaching powder (30% chlorine) and 2% formalin are effective sproricides against NIK-5hm in CSR2 breed. Asthra was also found ineffective even at low concentration of 0.05% in water. This suggests the existing practice of use of Chlorine dioxide, bleaching powder and asthra as disinfectant in sericulture is good enough for the disinfection of silkworm rearing house and appliances. The management of microsporidiosis in silkworm through Chemotheraptic approach has not given good success till date. However, it is an effective way to control the diseases in insects. Analogues of benzimidazole (Colbourn, et al., 1994; Schmahl and Benini, 1998), Benlate, Bavistin, Derosal (Baig, 1994), Fumidil - B or Fumagillin (Lewis and Lynch, 1971; Hayasaka, 1991; Frankenhuyzen et al., 2004),

15 methylthiophanate and ethyl thiophanate (Liu, 1987; Hayasaka, 1991) and Anisomycin (Hayasaka, 1991) have been found to be effective against different microsporidians. Buquinolate is reported to control microsporidiaosis in Blue crab (Overstreet, 1975). Griyaghey (1976) and Alok Sahay et al., (2005) studied the effect of chemotherapy on Pebrine in Tassar silkworm, Antheraea mylitta D. and found an effective way to control the microsporidiosis. In the present study benzimidazole derivatives viz., Albendazole and Mebendazole were found effective anti-sporozoan drugs. At % concentration the drugs could inhibit the development of the disease to an extent of 100%. Different benzimidazole derivitaves were tested in vivo against Glugae anomala parasiting the connective tissues of sticks Gasterosteus aculeatus (Schimaha and Benini 1998). In N. bombycis clumping of chromatin in the nuclei, inhibition of spindle formation and also malformation of spores after exposure to albendazole has been shown by transmission electron microscopy (Haque et al., 1993). In addition, an enlargement of their nuclei and disruption of the nuclear membrane was also observed. An antimicrosporidial activity has also been demonstrated for another benzimidazole compounds, benomyl (Hsiao and Hsiao, 1973). The authors states that the mode of action of albendazole and the related benzimidazole derivitives, is to a large extent the prevention of microtubule assembly which in the case of susceptible microsporidian species will inhibit the formation of intranuclear spindle, the only known case of microtubule formation in microsporidians. The distorted and leached cytoplasm observed in the merogonic and sporogonic stages of Glugea anomala after medication was also reported for Encephalitozoon cuniculi as an effect following albendazole treatment (Colbourn et al., 1994). The authors argue that this effect in E. cunicuculi, and also the paucity of ribosomes, is likely to result from the loss of cytoplasm from disrupted merogonic and sporogonic stages rather than prevention of ribosomal synthesis, since ribosomes were abundant in other samples which had other damage. All the three quinine derivatives viz., Chloroquine, Primiquine, and Sapraquine were also found effective in reduction of mortality to an extent of 96.46%. In view of the high cost of drugs/chemicals and their hazardous consequenceces, now a days use of biodegradable materials like fresh plant extracts have been on the top priority for the control of diseases in plants (Jesper and Ward, 1993) and animals (Kumar et al., 1999). Use of botanical for the control / suppression of microsporidiosis disease in mulberry silkworm, B. mori L. are scanty. Nathan et al., (2005) reported that feeding Azidirachta exctracts along with leaves of food plants to

16 Spodoptrera litura resulted in reduction of microsporidiosis disease as well as reduction in the ingestion and digestion of food. Kalaivani et al., (2003) reported that neem at a concentration of 2, 4 and 6% reduced the virulence of pebrine spores during 4 th and 5 th instar. He also reported that higher doses of neem affects the endocrine system and kills the pebrine spores. Girijadevi (2006) reported that aqueous extracts of som botanical like neem, turmeric, tulsi and garlic kills 10 26% spores of N. bombycis. In present study it is also observed that the seeds of A.copticum of family Umbelliferae, C. paradisi of family Rutaceae are effective in reduction of mortality due to CSR2 microsporidia to an extent of 100%. Thymol the active ingredient of seeds of A. copticum is reported to be effective in control of microsporidians disease in Apis mellifera, the Honey bee (Rice, 2001). Thymol, (3-Hydroxy-p-cymcnc), a phenol compound is a constituent of essential oil derived from Thymc, Sage and many other plant species. Thymol, is also made synthetically and in pure form, thymol is colorless crystal with a pleasant, yet strong odour. Thymol is reported to be an anti-microbial agent and acts directly on the spore, penetrate the spore coat and disrupt the plasma membrane. This action prevents the germination of spore and subsequently the disease (Rice, 2001). Grape fruit seeds of C. paradisi are known to contain numerous polyphenolic compounds such as Aquercetin, Hesperidin, Rutin, Apigenin and Campherol. It is rich in vitamin C and potassium, folate, iron, calcium, and other minerals. It is also high in fiber and low in calories, and contains bioflavonoids and other plant chemicals that are known to protect against cancer and heart disease. Grapefruit seeds are well known as an anti-fungal agent in that their consumption kills many different types of parasites and assists the body in producing beneficial bacteria. A biologically active natural ingredient found in the seeds kills Streptococcus, Staphylococcus, Salmonella, Esturia coli, Candida, Herpes, Influenza parasites and fungi. It is also used to control traveler's diarrhoea and is used commonly as an antibiotic, anti-fungal., anti-protozoan, antiviral, antiseptic and disinfectant. The indication in the present study that it is effective against microsporidiosis caused by CSR2 microsporidia and it is most encouraging not only in the management of microsporidiosis but also in the management of several other diseases in silkworm. It is expected to function against different bacteria, fungus and viruses infecting silkworm.

17 The investigations on the management of microsporidiosis caused by CSR2 microsporidia have given vital information which could go long way in management of microsporidiosis in silkworm caused by NIK-5hm and N. bombycis. Drugs such as Saproquine and Mebendazole and botanicals viz., Carica papaya and Ruta chalepensis and chemicals viz., AHP are potent in control of microsporidiosis. Apart from the general management practices with the use of disinfectants such as bleaching powder and chlorine dioxide, Asthra chemicals and botanicals such as Carica papaya and chemical AHP could be used as an important component in integrated management of microsporidiosis in silkworm.

18 Table 3.1: Viability response of NIK-5hm microsporidian spore to different chemical disinfectants. Treatments Chlorine dioxide Bleaching powder Formalin Asthra + Ineffective; - Effective Concentration (%/ppm) Sporocidal activity in different treatments durations (min) ppm % % % % % % % % % % %

19 Table 3.2: Impact of NIK-5hm microsporidian infection on the economic characters of different bivoltine and multivoltine silkworm breed Silkworm Breed CSR2 Treatment Larva % Mortality pupa Larval wt. (g) Larval duration (D:H) % ERR Single cocoon wt. (g) Single shell wt. (g) NIK-5hm 40.33± ± ± ± ± ± ± ±0.77 N.bombycis 39.33± ± ± ± ± ± ± ±0.61 Control 0.00± ± ± ± ± ± ± ±0.26 SR% CD@5% NIK-5hm 33.67± ± ± ± ± ± ± ±0.47 PM N.bombycis 32.33± ± ± ± ± ± ± ±1.12 Control 0.00± ± ± ± ± ± ± ±0.74 CD@5%

20 Table 3.3: Percent mortality after inoculation of NIK-5hm spores treated with different chemical disinfectants. Treatment Chlorine dioxide Bleaching powder Formalin Asthra Concentration (%/ppm) % mortality in different treatment duration caused by treated spores (min) ppm ppm ppm ppm ppm ppm Control SE CD@5% % % % % Control SE CD@5% % % % % Control SE CD@5% % % % Control SE CD@5%

21 Table 3.4: Screening of different benzimidazole and quine derivatives for antimicrosporidian action against NIK-5hm microsporidian spores. Sl.No. Name of the drug Chemical formula Concentrations (%) Effective Albendazole C 12 H 15 N 3 O 2 S Mebendazole C 16 H 13 N 3 O Sapraquine NA Chloroquine C 18 H 29 C 1 N 3 -H 3 PO Control Ineffective; - Effective

22 Table 3.5: Efficacy of different Drugs in disease control/suppression of NIK-5hm microsporidian in the larvae of CSR2 breed Treatment % mortality and disease reduction due to treatment of different concentrations 0.25% 0.50% 1.00% Control Treatment Albendazole 10.83± ± ± ±1.94 Mebendazole 9.00± ± ± ±1.80 Sapraquine 8.33± ± ± ± ±1.69 Chloroquine 12.25± ± ± ±1.41 Concentrations 18.88± ± ± ±2.34 Source of Variations Df Sum of Squares Mean Squares F Ratio Probability η 2 ηр 2 ω 2 Treatment *** Concentration *** Treat Conc *** Error (B) Total Comparison Std. Error S.E. Difference t value 5% C.Difference Treatment Conc Treat. Conc Significant at 5% level; ** Significant at 1% level; *** Significant at 0.1% level;

23 Table 3.6: Efficacy of different Drugs in disease suppression of NIK-5hm microsporidian in moth stage of CSR2 breed. Treatment % mortality and disease reduction due to treatment of different concentrations 0.25% 0.50% 1.00% Control Treatment Albendazole 23.25± ± ± ±3.85 Mebendazole 14.58± ± ± ±3.33 Sapraquine 16.50± ± ± ± ±3.84 Chloroquine 14.08± ± ± ±3.44 Concentrations 19.08± ± ± ±3.61 Source of Variations df Sum of Squares Mean Squares F Ratio Probability η 2 ηр 2 ω 2 Treatment *** Concentration *** Treat. Conc *** Error (B) Total Comparison Std. Error S.E. Difference t value 5% C.Difference Treatment Concentration Treat. Conc Significant at 5% level; ** Significant at 1% level; *** Significant at 0.1% level;

24 Table 3.7: Efficacy of different Drugs on the economic characters of CSR2 breed Treatment Conc. (%) ERR (%) Larval wt. (g) (10 larvae) SCW (g) SSW (g) SR% ± ± ± ± ±0.42 Albendazole ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.31 Mebendazole ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.33 Sapraquine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.28 Chloroquine ± ± ± ± ± ± ± ± ± ±0.25 Control 55.67± ± ± ± ± Conc *: Significant at 5%; **Significant at 1%; ***Significant at 0.1%

25 Table 3.8: Screening of different chemicals for antimicrosporidian action against NIK- 5hm microsporidian spores. 2 Glutaraldehyde C 16 H 13 N 3 O 3 Sl.No. Name of the drug Chemical formula Concentrations (%) Effective Hydogen peroxide C 12 H 15 N 3 O 2 S Aldol NA AHP C 18 H 29 C 1 N 3 - H 3 PO Control Ineffective; - Effective

26 Table 3.9: Efficacy of different chemicals in disease control/suppression of NIK-5hm microsporidian in the larvae of CSR2 breed. Treatment % infection and reduction in infection in due to treatment of different concentrations 0.50% 1.00% 1.50% 2.00% Control Treatment Hydogen peroxide 14.25± ± ± ± ±4.40 Glutaraldehyde 15.92± ± ± ± ± ±1.782 Aldol 9.83± ± ± ± ±3.88 AHP 8.42± ± ± ±3.62 Concentration 20.68± ± ± ±20.70 Source of Variations df Sum of Squares Mean Squares F Ratio Probability η 2 ηр 2 ω 2 Treatment *** Concentration *** Treat. Conc *** Error (B) Total Comparison Std. Error S.E. Difference t value 5% C. Difference Treatment Control Treat. Conc Significant at 5% level; ** Significant at 1% level; *** Significant at 0.1% level;

27 Table 3.10: Efficacy of different chemicals in disease suppression of NIK-5hm microsporidian in moth stage of CSR2 breed Treatment % infection and reduction in infection in due to treatment of different concentrations 0.50% 1.00% 1.50% 2.00% Control Treatment Hydogen peroxide 25.75± ± ± ± ±7.42 Glutaraldehyde 25.75± ± ± ± ± ±3.057 Aldol 16.17± ± ± ± ±6.82 AHP 14.42± ± ± ± ±6.22 Concentrations 23.22± ± ± ±12.36 Source of Variations df Sum of Squares Mean Squares F Ratio Probability η 2 ηр 2 ω 2 Treatment *** Concentration *** Treat. Conc *** Error (B) Total Comparison Std. Error S.E. Difference t value 5% C. Difference Treatment Concentration Treat. Conc Significant at 5% level; ** Significant at 1% level; *** Significant at 0.1% level;

28 Table 3.11: Efficacy of different Chemicals on the economic characters of CSR2 breed Treatment Conc. (%) ERR (%) Larval wt. (g) (10 larvae) SCW (g) SSW (g) SR% ± ± ± ± ±0.501 Hydogen peroxide ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.483 Glutaraldehyde ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.555 Aldol ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.484 AHP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.087 Control 55.67± ± ± ± ± Conc

29 Table 3.12: Screening of different botanicals for antimicrosporidian action against NIK- 5hm microsporidian spores. Sl.No. Name of the botanical Common botanical 1 Ficus Ficus bengalensis 2 Papaya Carica papaya 3 Jetropha Jetropha gossypifolia 4 Nagadali Ruta chelepensis Concentrations (%) Effective Control Ineffective; - Effective

30 Table 3.13: Efficacy of different botanicals in disease control/suppression of NIK-5hm microsporidian in the larvae of CSR2 breed. % infection and reduction in infection in due to treatment of different concentrations Treatment 0.50% 1.00% 1.50% 2.00% Control Treatment Ficus bengalensis 21.00± ± ± ± ±8.10 Carica papaya 18.17± ± ± ± ±7.12 Jetropha 22.42± ± ± ± ± ±3.13 gossypifolia Ruta chelepensis 16.42± ± ± ± ±6.75 Concentrations 26.20± ± ± ± ±7.68 Source of Variations df Sum of Squares Mean Squares F Ratio Probability η 2 ηр 2 ω 2 Treatment *** Concentration *** Treat. Conc *** Error (B) Total 179 Comparison Std. Error S.E. Difference t value 5% C.Difference Treatment Concentration Treat. Conc Significant at 5% level; ** Significant at 1% level; *** Significant at 0.1% level;