EFFECT OF NEW MICROSPORIDIAN INFECTION ON THE...

EFFECT OF NEW MICROSPORIDIAN INFECTION ON THE RELEVANT ECONOMIC CHARACTERS OF

SILKWORM AND ITS MANAGEMENT

Chapter III

Effect of new microsporidian infection on the relevant economic characters of silkworm and its management

The 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 11-15 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.

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

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)

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 broad-

spectrum 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

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 1×106 sporesml to 3rd 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 3rd 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 1×106 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 1×106 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 ClO2) 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

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 1 ×106 treated spores / ml were smeared on mulberry leaves and fed to the

100 silkworm immediately after 2nd 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.

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 2nd 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 1 ×106 spores/ml immediately after 2nd 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 100 3rd instar silkworm on alternate days up to spinning. A control group

of larvae inoculated with NIK-5hm spores of concentration 1 ×106 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.

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 2nd 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 1×106 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 1 × 106 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 non-

toxic chemicals were screened for efficacy by in vivo and bioassay method for anti-

microsporidian 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 2nd 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.

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 1 ×106 spores/ml immediately after 2nd 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 1

×106spores/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

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 23.30 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 21.59 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 47.33.

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 3.4-

3.7. 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.

Chemical based: The results of screening of chemicals for anti microsporidian activity

against NIK-5hm microsporidian are presented in Table 3.8-3.11. 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%- 99.67 % in

CSR2 breed. The percent of infected moths was significantly low in all treatments over

the inoculated control it was 34.00 %. The economic characters such as larval weight,

single cocoon weight, shell weight and silk content were significantly increased in the

treatment batches with all 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

3.12-3.15. 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 96.33 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

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),

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 0.25-1% 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

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 4th and 5th

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.

http://www.viable-herbal.com/singles/herbs/s914.htm




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.

Table 3.1: Viability response of NIK-5hm microsporidian spore to different chemical disinfectants.

Treatments Concentration (%/ppm)

Sporocidal activity in different treatments durations (min)

5 10 20 30

Chlorine dioxide

50ppm + + + +100 + + + +200 + + + +300 + + + +400 + + - -500 + + + -

Bleaching powder

0.5% + + + +1% + - - -

1.5% - - - -2% - - - -

Formalin

0.5% + + + +1% - - - -

1.5% - - - -2% - - - -

Asthra0.01% + + + +0.03% + + + -0.05% + + - -

+ Ineffective; - Effective

Table 3.2: Impact of NIK-5hm microsporidian infection on the economic characters of different bivoltine and multivoltine silkworm breed

Silkworm Breed Treatment

% MortalityLarval wt. (g)

Larval duration (D:H)

% ERRSingle

cocoon wt. (g)

Single shell wt. (g) SR%Larva pupa

CSR2

NIK-5hm 40.33±058 36.67±0.58 34.33±0.58 25.00±0.58 23.00±1.00 1.32±0.01 0.27±0.01 20.40±0.77

N.bombycis 39.33±0.58 36.33±0.58 36.00±3.00 25.00±0.58 24.33±0.58 1.34±0.02 0.29±0.01 21.59±0.61

Control 0.00±0.00 0.00±0.00 41.33±0.00 24.00±0.00 95.33±0.58 1.62±0.021 0.38±0.89 23.30±0.26

CD@5% 0.641 0.873 1.369 0.342 1.210 0.015 0.008 0.733

PM

NIK-5hm 33.67±1.16 27.67±1.16 20.67±0.58 26.33±0.58 41.00±1.73 0.86±0.02 0.10±0.01 11.96±0.47

N.bombycis 32.33±0.58 26.33±1.53 21.67±0.58 26.33±0.58 42.67±1.53 0.89±0.01 0.11±0.01 12.41±1.12

Control 0.00±0.00 0.00±0.00 22.67±0.58 26.00±0.00 96.33±1.16 1.07±0.15 0.14±0.01 13.44±0.74

CD@5% 0.641 1.067 1.677 0.419 1.482 0.018 0.010 0.733

Table 3.3: Percent mortality after inoculation of NIK-5hm spores treated with different chemical disinfectants.

Treatment Concentration(%/ppm)

% mortality in different treatment duration caused by treated spores (min)

5 10 20 30

Chlorine dioxide

50ppm 63.33 34.33 23.33 10.67100ppm 35.67 23.67 14.00 8.67200ppm 22.67 13.67 8.67 5.67300ppm 20.00 11.33 7.33 4.33400ppm 4.67 1.67 0.00 0.00500ppm 0.00 0.00 0.00 0.00Control 96.67

SE 7.26 7.08 7.22 7.37CD@5% 3.52 4.66 2.49 2.15

Bleaching powder

0.50% 47.33 32.67 12.33 8.001.00% 21.33 12.33 8.00 4.671.50% 0.33 0.00 0.00 0.002.00% 0.00 0.00 0.00 0.00Control 98.67

SE 9.87 9.88 10.08 10.24CD@5% 4.09 2.94 1.56 1.05

Formalin

0.50% 38.00 20.33 12.33 4.671.00% 25.00 9.00 4.67 1.671.50% 3.33 0.00 0.00 0.002.00% 0.00 0.00 0.00 0.00Control 98.67

SE 9.61 10.04 10.24 10.46CD@5% 4.04 3.52 1.82 1.41

Asthra

30 60 90 1200.01% 26.25 17.67 13.33 5.670.03% 5.67 5.00 0.00 0.000.05% 4.92 2.00 0.00 0.00Control 98.67

SE 11.66 11.99 12.46 12.70CD@5% 4.59 3.69 2.18 1.44

Table 3.4: Screening of different benzimidazole and quine derivatives for anti-microsporidian action against NIK-5hm microsporidian spores.

Sl.No. Name of the drug Chemical formula Concentrations (%) Effective

1 Albendazole C12H15N3O2S0.25 +0.50 +1.00 -

2 Mebendazole C16H13N3O3

0.25 +0.50 +1.00 -

3 Sapraquine NA0.25 +0.50 +1.00 +

4 Chloroquine C18H29C1N3-H3PO4

0.25 +0.50 +1.00 -

Control - ++ Ineffective; - Effective

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 Treatment0.25% 0.50% 1.00% ControlAlbendazole 10.83±2.29 9.16±1.95 7.75±1.82

54.00±2.734

11.556±1.94Mebendazole 9.00±2.05 4.83±1.27 3.08±0.67 10.00±1.80Sapraquine 8.33±1.72 3.92±1.08 2.58±0.79 8.11±1.69Chloroquine 12.25±2.73 10.17±1.80 8.58±1.505 7.33±1.41Concentrations 18.88±17.9 16.42±19.15 15.20±19.75 9.250±2.34Source of Variations Df Sum of Squares Mean Squares F Ratio Probability η2 ηр2 ω2

Treatment 4 62920.055556 15730.013889 16557.91 0.0000 *** 0985 0.998 0.496Concentration 2 422.633333 211.316667 222.44 0.0000 *** 0.007 0.788 0.007Treat × Conc. 8 154.811111 19.351389 20.37 0.0000 *** 0.002 0.576 0.002Error (B) 120 114.000000 0.950000 0.73Total 63611.5000 15961.63195 16801.45Comparison Std. Error S.E. Difference t value 5% C.DifferenceTreatment 0.16245 0.22973 1.97994 0.45486Conc. 0.12583 0.17795 1.97994 0.35233Treat. × Conc. 0.28137 0.39791 1.97994 0.787844

Significant at 5% level; ** Significant at 1% level; *** Significant at 0.1% level;

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 Treatment0.25% 0.50% 1.00% ControlAlbendazole 23.25±3.44 19.00±2.86 17.42±2.71

27.00±4.36

19.89±3.85Mebendazole 14.58±4.27 13.17±2.79 12.50±2.61 13.42±3.33Sapraquine 16.50±4.89 13.83±2.79 13.08±2.84 14.47±3.84

Chloroquine 14.08±4.36 12.58±2.10 11.83±2.59 12.83±3.44Concentrations 19.08±6.36 17.12±6.02 16.37±6.17 15.15±3.61

Source of Variations df Sum of Squares Mean Squares F Ratio Probability η2 ηр2 ω2

Treatment 4 5168.633333 1292.158333 1314.06 0.0000 *** 0.738 0.978 0.424Concentration 2 236.211111 118.105556 120.11 0.0000 *** 0.034 0.667 0.032Treat. × Conc. 8 118.233333 14.779167 15.03 0.0000 *** 0.017 0.500 0.015

Error (B) 120 118.000022 0.983334Total 134 5641.0778 1426.02639 1449.2

Comparison Std. Error S.E. Difference t value 5% C.DifferenceTreatment 0.16527 0.23373 1.97994 0.46277

Concentration 0.12802 0.18105 1.97994 0.35846Treat. × Conc. 0.28626 0.40483 1.97994 0.80154


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%

Albendazole0.25 81.67±0.58 39.33±0.577 1.51±0.02 0.35±0.010 21.92±0.42

0.50 83.67±0.58 39.33±0.58 1.60±0.01 0.36±0.006 22.71±0.251.00 85.67±0.58 40.33±0.58 1.61±0.01 0.37±0.00 23.03±0.09

Mebendazole0.25 83.33±0.58 40.33±0.58 1.61±0.01 0.36±0.006 22.20±0.310.50 84.67±0.58 39.67±0.58 1.60±0.01 0.37±0.006 22.87±0.201.00 87.67±0.58 40.67±0.58 1.61±0.00 0.38±0.006 23.39±0.36

Sapraquine0.25 83.33±0.58 40.00±1.33 1.51±0.01 0.35±0.006 22.13±0.330.50 85.67±0.58 40.33±1.16 1.61±0.01 0.37±0.006 23.24±0.321.00 88.33±0.58 41.33±0.58 1.62±0.01 0.38±0.006 23.30±0.28

Chloroquine0.25 82.33±0.58 39.67±1.16 1.59±0.01 0.35±0.006 21.85±0.280.50 83.67±0.58 40.33±0.58 1.60±0.01 0.37±0.006 22.92±0.251.00 86.67±1.18 40.67±0.58 1.61±0.01 0.37±0006 23.19±0.25

Control 55.67±0.58 33.67±0.58 1.32±0.01 0.27±0.010 20.40±0.77CD@5%Treatment 0.60888 0.688 0.00919 0.00673 0.41479

CD@5%Concentration 0.47164 0.53314 0.00712 0.00521 0.32129

CD@5%Treat. × Conc. 1.05462 1.19213 0.01592 0.01166 0.71843*: Significant at 5%; **Significant at 1%; ***Significant at 0.1%

Table 3.8: Screening of different chemicals for antimicrosporidian action against NIK-5hm microsporidian spores.

Sl.No. Name of the drug Chemical formula Concentrations (%) Effective

1 Hydogen peroxide C12H15N3O2S

0.50 +1.00 +1.50 -2.00 -

2 Glutaraldehyde C16H13N3O3

0.50 +1.00 +1.50 -2.00 -

3 Aldol NA

0.50 +1.00 +1.50 +2.00 -

4 AHP C18H29C1N3-H3PO4

0.50 +1.00 +1.50 -2.00 -


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 Treatment0.50% 1.00% 1.50% 2.00% ControlHydogen peroxide

14.25±5.85 12.33±4.45 10.58±2.68 10.08±3.11

55.00±1.782

11.81±4.40

Glutaraldehyde 15.92±6.85 13.50±5.00 12.25±3.72 10.00±2.86 12.92±5.15Aldol 9.83±3.01 5.33±1.23 3.58±1.38 0.33±0.65 4.77±3.88AHP 8.42±1.78 4.58±1.24 0.50±0.67 0.00 3.38±3.62Concentration 20.68±1.80 18.15±1.91 16.38±0.07 15.08±20.70

Source of Variations

df Sum of Squares Mean Squares F Ratio Probability η2 ηр2 ω2

Treatment 4 87413.941667 21853.485417 20250.52 0.0000*** 0.962 0.998 0.490Concentration 3 1057.250000 352.416667 326.57 0.0000*** 0.012 0.860 0.011Treat. × Conc. 12 413.791667 34.482639 31.95 0.0000*** 0.005 0.706 0.004Error (B) 160 172.665104 1.079157Total 179 89057.64844 22241.46386Comparison Std. Error S.E. Difference t value 5% C. DifferenceTreatment 0.14994 0.21205 1.97490 0.41878Control 0.13411 0.18966 1.97490 0.37457Treat. × Conc. 0.29988 0.42410 1.97490 0.83755


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 Treatment0.50% 1.00% 1.50% 2.00% ControlHydogen peroxide

25.75±6.92 18.58±6.11 15.75±5.26 13.50±5.44

34.00±3.057

18.37±7.42

Glutaraldehyde 25.75±6.92 18.58±6.11 15.75±5.26 14.25±3.98 18.58±7.08Aldol 16.17±5.62 8.83±3.54 2.75±2.53 1.67±1.67 7.35±6.82AHP 14.42±5.05 7.75±3.79 2.00±1.60 1.50±1.78 6.42±6.22Concentrations 23.22±8.98 17.55±10.46 14.05±12.23 12.98±12.36



Treatment 4 23927.60833 5981.902083 3643.82 0.0000*** 0.728 0.989 0.421Concentration 3 3826.53333 1275.511111 776.97 0.0000*** 0.116 0.936 0.104Treat. × Conc. 12 1046.758333 87.229861 53.14 0.0000*** 0.032 0.799 0.030Error (B) 160 262.665104 1.641657Total 179 29063.5651 7346.284712Comparison Std. Error S.E. Difference t value 5% C. DifferenceTreatment 0.18494 0.26154 1.97490 0.51651Concentration 0.16541 0.23393 1.97490 0.46198Treat. × Conc. 0.36987 0.52308 1.97490 1.03303


Table 3.11: Efficacy of different Chemicals on the economic characters of CSR2 breed


Hydogen peroxide

0.50 71.67±2.08 36.33±0.58 1.520±0.010 0.327±0.006 21.493±0.5011.00 72.00±1.00 37.33±0.58 1.537±0.015 0.333±0.006 21.693±0.3951.50 73.67±1.16 37.67±0.58 1.553±0.015 0.343±0.012 22.103±0.7382.00 74.33±2.52 37.33±0.58 1.573±0.015 0.353±0.006 22.457±0.176

Glutaraldehyde

0.50 67.67±1.53 35.00±1.00 1.513±0.012 0.317±0.006 20.923±0.4831.00 67.33±0.58 35.33±0.58 1.523±0.015 0.327±0.006 21.447±0.5821.50 68.67±2.08 35.67±0.58 1.527±0.006 0.340±0.010 22.273±0.6602.00 71.00±2.00 37.00±1.00 1.560±0.010 0.350±0.006 22.437±0.145

Aldol

0.50 81.00±2.65 37.33±0.58 1.553±0.015 0.343±0.006 22.110±0.5551.00 84.00±1.00 38.00±0.58 1.567±0.015 0.347±0.006 22.127±0.1761.50 86.33±0.58 39.00±1.00 1.590±0.010 0.357±0.006 22.430±0.2522.00 87.67±0.58 40.67±0.58 1.610±0.010 0.373±0.006 23.190±0.384

AHP

0.50 86.67±0.58 38.67±0.58 1.590±0.010 0.357±0.006 22.433±0.4841.00 87.33±0.58 39.67±0.58 1.607±0.006 0.370±0.000 23.030±0.0871.50 88.33±0.58 41.33±0.58 1.623±0.006 0.377±0.006 23.207±0.4392.00 89.33±1.33 41.67±0.58 1.623±0.006 0.380±0.000 23.410±0.087



CD@5%Treat. × Conc. 2.21399 1.06520 0.01917 0.01167 0.85598

Table 3.12: Screening of different botanicals for antimicrosporidian action against NIK-5hm microsporidian spores.

Sl.No. Name of the botanical

Common botanical

Concentrations (%) Effective

1 Ficus Ficus bengalensis

0.50 +1.00 +1.50 -2.00 -

2 Papaya Carica papaya

0.50 +1.00 +1.50 -2.00 -

3 Jetropha Jetropha gossypifolia

0.50 +1.00 +1.50 +2.00 -

4 Nagadali Ruta chelepensis

0.50 +1.00 +1.50 -2.00 -


Table 3.13: Efficacy of different botanicals 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 Treatment0.50% 1.00% 1.50% 2.00% Control

Ficus bengalensis 21.00±4.90 10.50±5.37 6.50±4.25 2.83±2.86

53.00±3.13

10.21±8.10Carica papaya 18.17±5.02 9.25±3.93 6.17±4.41 2.17±2.25 8.94±7.12Jetropha gossypifolia

22.42±6.27 12.08±5.85 7.33±4.66 3.17±2.73 11.25±8.76

Ruta chelepensis 16.42±4.19 8.00±3.59 5.50±4.08 0.333±0.65 7.56±6.75Concentrations 26.20±14.38 18.57±17.90 15.750±19.19 12.300±20.64 9.49±7.68



Treatment 4 73063.641667 18265.910417 10077.87 0.0000*** 0.867 0.996 0.464Concentration 3 6311.650000 2103.883333 1160.78 0.0000*** 0.075 0.956 0.070Treat. × Conc. 12 1664.225000 138.685417 76.52 0.0000*** 0.020 0.852 0.019Error (B) 160 289.996354 1.812477Total 179Comparison Std. Error S.E. Difference t value 5% C.DifferenceTreatment 0.19432 0.27481 1.97490 0.54272Concentration 0.17380 0.24580 1.97490 0.48542Treat. × Conc. 0.38864 0.54962 1.97490 1.08544


Table 3.14: Efficacy of different botanicals 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 Treatment0.50% 1.00% 1.50% 2.00% Control

Ficus bengalensis 21.50±9.18 13.50±4.38 8.25±4.20 3.67±2.31

36.00±5.46

11.73±8.63Carica papaya 19.75±8.17 12.17±3.66 6.33±3.28 2.25±1.27 10.13±8.12Jetropha gossypifolia

24.42±12.49 17.66±9.91 8.92±4.56 5.33±2.87 14.08±11.10

Ruta chelepensis 27.33±10.66 15.75±4.48 9.83±4.15 4.42±2.54 14.33±10.53Concentrations 25.80±10.57 19.02±10.25 13.87±11.76 10.33±13.14 12.57±9.59



Treatment 4 21664.433333 5416.108333 1714.86 0.0000*** 0.552 0.977 0.355Concentration 3 8130.645833 2710.215278 858.12 0.0000*** 0.207 0.941 0.171Treat. × Conc. 12 2206.000000 183.833333 58.21 0.0000*** 0.056 0.814 0.052Error (B) 160 505.333594 3.158335Total 179 32506.41276 8313.315279Comparison Std. Error S.E. Difference t value 5% C.DifferenceTreatment 0.25651 0.36276 1.97490 0.71642Concentration 0.22943 0.32447 1.97490 0.64079Treat. × Conc. 0.51302 0.72553 1.97490 1.43285


Table 3.15: Efficacy of different Botanicals on the economic characters of CSR2 breed


Ficus bengalensis

0.50 74.67±2.08 32.00±0.00 1.503±0.012 0.317±0.006 21.063±0.2191.50 79.33±1.00 34.67±0.58 1.563±0.015 0.350±0.010 22.383±0.5231.50 91.33±0.58 35.33±0.58 1.600±0.010 0.363±0.006 22.707±0.2472.00 94.00±0.00 40.67±1.16 1.613±0.006 0.373±0.006 23.140±0.277

Carica papaya

0.50 73.67±0.58 33.33±1.16 1.533±0.021 0.323±0.006 21.087±0.2701.50 85.67±1.16 34.67±1.53 1.547±0.006 0.337±0.006 21.770±0.4221.50 71.33±36.57 35.67±0.58 1.563±0.023 0.353±0.006 22.600±0.352.00 95.33±0.58 39.00±1.00 1.590±0.006 0.367±0.006 23.060±0.364

Jetropha gossypifolia

0.50 72.33±1.16 31.33±0.58 1.500±0.010 0.307±0.006 20.450±0.5071.50 76.33±0.58 33.33±0.58 1.553±0.023 0.333±0.006 21.467±0.5961.50 88.33±0.58 35.00±0.58 1.593±0.006 0.350±0.010 21.967±0.6962.00 94.33±0.58 40.67±1.16 1.613±0.006 0.370±0.000 21.933±0.081

Relepensis

0.50 87.33±0.58 35.00±1.00 1.537±0.006 0.330±0.010 21.477±0.5811.50 90.67±0.58 37.00±1.00 1.543±0.006 0.337±0.006 21.817±0.3421.50 92.33±0.58 37.00±1.00 1.550±0.010 0.357±0.015 23.013±0.9452.00 97.00±1.0080 41.00±1.00 1.607±0.012 0.377±0.006 23.447±0.310



CD@5%Treat. × Conc. 13.59629 1.39700 0.02032 0.01330 0.886340

EFFECT OF NEW MICROSPORIDIAN INFECTION ON THE...

Documents

Transcript of EFFECT OF NEW MICROSPORIDIAN INFECTION ON THE...