Chapter 2 STUDIES ON THE PATHOGENICITY OF...

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Chapter 2

STUDIES ON THE PATHOGENICITY OF MICROSPORIDIA ISOLATED

FROM INSECT PESTS OF MULBERRY AND SOME OTHER

AGRICULTURAL CROPS TO SILKWORM AND SUSCEPTIBILITY OF

DIFFERENT BREEDS TO PATHOGENIC MICROSPORIDIA

icrosporidiosis of silkworm is caused by a highly virulent parasitic

microsporidian, Nosema bombycis. Interestingly, the first

microsporidian to be named was Nosema bombycis Naegeli in 1857. It was

discovered as the etiological agent of the “Pebrine” epidemic of silkworm that

occurred in Europe in the mid nineteenth century, commanding the attention of many

eminent scientists. Later, by the end of the 19th century, the disease spread to all

sericultural countries of Europe resulting in a drastic decline of silk production. The

noted French microbiologist, Louis Pasteur (1870) was the first who made a detailed

study on the biology of the pathogen. Later, in addition to Nosema bombycis, the

microsporidia belonging to the genera Nosema, Pleistophora and Thelohania were

isolated from silkworm (Fujiwara, 1984a, b) and tentatively designated as M11, M12

and M14 (Nosema sp.), M24, M25 and M27 (Pleistophora sp.) and M32 (Thelohania

sp.). Three microsporidia designated as NIK-2r (Nosema sp., Mysore), NIK-3h

(Nosema sp. M11, Hassan) and NIK-4m (Nosema sp. M12, Mysore) have been

isolated from Karnataka and were found to be different from Nosema bombycis

(Ananthalakshmi et al., 1994). Kishore et al. (1994) have reported that certain species

of Catopsilia which are frequent visitors / inhabitants of the fields around mulberry

gardens carry microsporidian spores which after testing were found to be infective to

silkworm. Samson et al. (1999a, b) also reported certain butterflies containing

microsporidia infective to silkworm.

Microsporidia invade insects through three natural portals of entry viz., oral,

cuticular and ovarial pathways (Kramer, 1976). Entrance by the oral and cuticular

portals results in horizontal transmission and by the ovarial portal in vertical

transmission. These types of transmissions are believed to occur commonly in the

same host generation, but in the European corn borer, Ostrinia nubilalis, the

microsporidium Nosema pyrausta is transmitted primarily by transovarial (vertical)

`

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infection in the first generation and by both vertical and horizontal transmissions in

the second generation (Siegel et al., 1988).

Microsporidian spores are unique in the way they invade a new host. The spores

sometimes leave the host with the faeces but are usually released in large numbers

after the host dies. The infection cycle commences after another host ingests the

environmental spores. By per os means, the microsporidia infect silkworm and the

process of infection initiates with the germination of environmental spores in the

larval midgut. A stimulus due to high pH in the gut environment sets off a series of

events initiating the process of germination. The spores germinate in the larval midgut

and the sporoplasm, extruded from spores, invade the host cells within the reach of

the polar tube and initiate the developmental cycle. The sporoplasms of microsporidia

with long polar tubes penetrate through the midgut and arrive directly at the body

cavity (Ishihara, 1985). Normally, the per oral infection of silkworm larvae with

N.bombycis NIS-001, Nosema sp. NIS-M11, Vairimorpha sp. NIS-M12, Nosema sp.

NIS-M14 and Microsporidium sp. NIS-M25 in silkworm larvae is systemic and

severe. They form environmental spores in all tissues and organs viz., midgut

epithelium, muscles, fat body, silk gland, malpighian tubules etc. as presented in the

Table given below.

Site of infection of different microsporidia in silkworm

Host tissues Microsporidia Gut

epithelium Malpighian

tubules Muscle Fat body

Silk gland

Gonad

N.bombycis NIS-001 + + + + + +

Nosema sp. NIS-M11 - + + + + -

Vairimorpha sp. NIS-M12 - + + + + -

Nosema sp. NIS-M14 - + + + + -

Microsporidium NIS-M25 - + + + + -

Pleistophora sp. NIS-M27 + - - - - -

Thelohania sp. NIS-M32 - + - - - -

Nosema sp. NIK-1s + + + + + +

Nosema sp. NIK-2r + + + + + +

Nosema sp. NIK-3h - + + - + -

Vairimorpha sp. NIK-4m + - - - - - Source: Nataraju et al., 2005

Nosema bombycis NIS-001 is highly gonadotropic while Nosema sp. NIS-M11

is moderately gonadotropic (Han and Watanabe, 1988). Pleistophora sp. exhibits no

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evident production of primary spores and produces only uninucleate environmental

spores (Becnel and Andreadis, 1999). Pleistophora sp. NIS-M27 infects merely the

midgut epithelium of silkworm larvae (Tanaka et al., 1972), Thelohania sp. produces

environmental sporogony alone (Becnel and Andreadis, 1999) and Thelohania sp.

NIS-M32 produces uninucleate octospores only in larval muscles of the silkworm

(Fujiwara, 1984a). In a recent study, two new microsporidia isolated from Bihar Hairy

Caterpillar, Spilosoma obliqua have been reported to form spores in all the tissues of

silkworm larvae (Singh et al., 2008).

Multiplication and spore yield of microsporidia in various host systems have

been studied by Lai and Canning (1983), the influence of temperature has been

studied by Fowler and Reeves (1975b), Becnel and Undeen (1992), Madan Mohanan

et al. (2006) and the type of tissue has been studied by Sasidharan et al. (1994) and

Shabir Ahmad Bhat and Nataraju (2007a). Microsporidia penetrate into the

haemocoel and exist extracellularly in the haemolymph or intracellularly within the

cells of various tissues and organs (Tanada and Kaya, 1993). A microsporidian,

Nosema carpocapsae isolated from the larvae of the codling moth, Carpocapsa

(Paillot, 1939) which is an agricultural pest has been reported to parasitize most of the

cells of the host, but the most frequently affected are those of the silk glands,

malpighian tubules, adipose tissue, muscles and the oenocytes. The spores are also

found in the pericardial cells, the epidermal cells, the cells at the base of the hairs, the

genital capsule and the epithelial cells of the posterior midgut (Steinhaus, 1949).

Microsporidian spore formation has been recently observed inside the haemocytes of

silkworm (Selvakumar et al., 2005). As the intensity of infection by microsporidia

varies from one tissue to the other (Sasidharan et al., 1994; Shabir Ahmad Bhat and

Nataraju, 2007a), testing of individual tissues where the intensity of infection is

generally high can be more accurate in the diagnosis of microsporidian infection than

the whole larval crushing. The site of infection and the intensity of infection are

important parameters in characterization of microsporidia infecting silkworm.

As defined by Lacey (1997), the intrinsic capability of a microorganism to

penetrate the host defenses is known as Pathogenicity. Fuxa and Tanada (1987)

described pathogenicity as the disease producing power of the pathogen and the

ability to invade and injure the tissues of the host. The infectivity and pathogenicity of

silkworm microsporidians are determined on the basis of infection rate and mortality

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of silkworm larvae, respectively after inoculating orally with environmental spores.

Nosema bombycis NIS-001, Vairimorpha sp. NIS-M12 and Pleistophora sp. NIS-

M27 show the highest infectivity to silkworm larvae. Nosema sp. NIS-M11, NIS-

M14 and Microsporidium sp. NIS-M25 show moderate infectivity to silkworm. On

the other hand, Thelohania sp. NIS-M32 is low in infectivity. With regard to

pathogenicity to silkworm larvae, N.bombycis NIS-001 and Vairimorpha sp. NIS-

M12 are highly pathogenic and usually cause the death of infected larvae before

pupation (Tanaka et al., 1972; Fujiwara, 1980, 1984a, b).

Microsporidia infect silkworm by per os means and the process of infection

initiates with germination of environmental spores in larval midgut. The mode of

transmission of the microsporidian infection from infected host to uninfected host has

been categorized into two types-1) Vertical transmission wherein there is direct

transmission of the pathogen from the parent organism to its progeny (Fine, 1975). 2)

Horizontal transmission wherein transmission of the pathogen is from individual to

individual (Canning, 1982). The extrusion of spores by infected worms through gut

juice and faecal matter leads to the spread of the disease within a healthy population

(Ichikawa, 1935; Ishihara and Fujiwara, 1965; Baig et al., 1988). Horizontal

transmission is a common mechanism for the spread of disease in forest lepidopteran

population that can result in second-order density dependent regulation in the number

of individuals (Dwyer, 1994). Successful horizontal transmission has two components

1) encounter of pathogen propagules in the environment 2) initiation of infection after

encounter (Knell et al., 1998). The probability of healthy individuals encountering

pathogen propagules depends on the host’s life history, behaviour and susceptibility to

the pathogen (Knell et al., 1998; Hajek, 2001), the density of uninfected larvae

(Dwyer, 1991) and the density of propagules in the environment (Onstad and

Maddox, 1989; Reeson et al., 2000). The later depends to a large extent on the density

of infected hosts (Knell et al., 1996; D’Amico et al., 1996; Dwyer et al., 1997; Siegel

et al., 1988; Fenton et al., 2002). Factors to be considered when examining horizontal

transmission include the density of microsporidian spores egested into the

environment by infected individuals that are available for ingestion by other larvae,

and the number of the microsporidian spores ingested to give rise to new infection.

The younger the larvae, the fewer the spores are needed to establish infection

(Wilson, 1974). The silkworm, Bombyx mori L is reared indoor in wooden or iron

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racks which are called rearing bed and the contamination takes place through the

faecal matter voided by infected larvae, contaminated trays, seat paper, dust from the

contaminated room and the larvae died due to the disease. The spores, thus, liberated

settle along with the dust on the mulberry leaf forming the source of secondary

contamination in the rearing bed (Singh et al., 2007).

All silkworm breeds are susceptible to microsporidiosis and no breed has been

found to be resistant to infection. Different silkworm breeds differ in their

susceptibility to the microbial infection. Such differences are genetically determined

and have been studied extensively involving silkworm viruses (Tanada and Kaya,

1993). In silkworm, large differences exist among various breeds in their

susceptibility to infection by Nosema bombycis (Chinnaswamy and Devaiah, 1984).

Chinese breeds are more resistant to pebrine pathogen than the Japanese breeds and

the European breeds are the least resistant (Govindan et al., 1998; Singh and

Saratchandra, 2003; Nataraju et al., 2005). Patil and Geethabai (1989) reported that

multivoltine breeds are relatively more resistant than bivoltine breeds. Among

bivoltines, NB7 is most susceptible followed by NB4D2 and KA (Patil and Geethabai,

1989). Silkworm races such as Pure Mysore, Nistari and C.Nichi have high survival

ability than other silkworm races (Devaiah and Krishnaswamy, 1975; Devaiah, 1973;

Patil and Geethabai, 1989). The high survival of Pure Mysore breed is attributed to

the high regenerative capacity of their midgut enabling it to recover fast from the

damages caused by infection (Fujiwara, 1993). However, Liu (1984) reported that a

silkworm race Baipidan is resistant to Nosema bombycis. The wild silkworm,

Antheraea pernyi and Platysamia cecropia are comparatively more resistant to

microsporidiosis than others (Weiser, 1969). In honeybee also, resistance to infection

by Nosema apis is attributed to heterosis and polygenic system (Sidorov et al., 1975).

The studies on the mode of infection, site of infection, pathogenicity, rate of

spread and susceptibility of different silkworm breeds to the five different

microsporidia isolated from lepidopteran insect pests of mulberry and other

agricultural crops constitute the subject matter of this chapter. The results of the study

on the above aspects have been reported and discussed in light of works of other

researchers in the field.

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MATERIALS AND METHODS

Mode of infection of the isolated microsporidia in the silkworm, Bombyx mori L.:

To determine the mode of infection of the isolated microsporidia, the layings of CSR2

breed were received from germplasm bank of Central Sericultural Research and

Training Institute, Mysore. The layings were surface sterilized and incubated

following the standard procedure at 25±1ºC and 80±5% RH. The hatched larvae were

reared as per standard methods till the beginning of 3rd instar. The 3rd instar silkworm

larvae were fed with purified spores of the isolated microsporidia. Also one set of

larvae was fed with spores of Nosema bombycis. The inoculum was prepared from

purified spores of the microsporidia by proper quantification as per standard method

(Cantwell, 1970). One ml of the inoculum (of each microsporidia separately)

containing 1×107 spores/ml was smeared on the mulberry leaf disc (100 cm2 surface

area) and fed to 100 silkworm larvae immediately after second moult (T1). In another

treatment (T2), the surface of eggs of the CSR2 breed at the blue egg stage was

smeared with 1×107 spores/ml of each microsporidia separately. The eggs were

incubated till the onset of hatching and the larvae hatched from treated layings were

reared following the standard procedure (Datta, 1992). Another set of healthy larvae,

immediately after second moult were topically smeared with inoculums of each

microsporidia separately containing 1×107 spores/ml (T3) and kept on sterile surface

for 24 h. After 24 h, the topically treated larvae were dusted with Vijetha. The larvae

were then transferred to disinfected rearing trays and fed with mulberry leaf. In yet

another treatment (T4), the third instar larvae were topically smeared with the

microsporidian spores at a concentration of 1×107 spores/ml, kept on sterile surface

for 24 h. The larvae were then transferred to disinfected rearing trays and fed with

mulberry leaf. In this treatment, dusting of Vijetha was not carried out. Yet another

set of larvae (T5) were reared without any inoculation and it served as control for

comparison purpose. Each treatment consisted of three replications with 100

larvae/replication. The treated and control larvae were reared till cocooning. The dead

larvae and pupae were homogenized and examined for the microsporidian infection

under phase contrast microscope, Nikon (Type-104). The moths were allowed to lay

eggs. All the moths were subjected to microscopic examination. The eggs were

treated with HCl of specific gravity of 1.075 at 46.1oC for 5 minutes to break the

diapause. The layings were incubated at 25±1oC and 80±5% RH for 10 days in an

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incubator. Fecundity and Hatching percent were recorded. The dead eggs were

subjected to microscopic infection. The progeny larvae from each treatment were

homogenized individually and examined for microsporidian infection. The

observations were recorded, tabulated and analyzed.

Site of infection of the isolated microsporidia in the silkworm, Bombyx mori L.:

To get different infected tissues, an inoculum dosage of 1×107 spores/ml was prepared

by using Neubar haemocytometer (Cantwell, 1970). One ml of the inoculum (1×107

spores/ml) of each microsporidian strain (NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So

and NIK-1Dp) was smeared separately onto a mulberry leaf disc (100 cm2 surface

area) and fed separately to the silkworm larvae of CSR2 breed (100 larvae/treatment)

immediately after 3rd moult. Also one group of 100 larvae was inoculated per orally

with Nosema bombycis spores (1×107 spores/ml) to serve as control for comparison of

results. The second normal feeding was provided to each inoculated batch after 24

hours of microsporidian inoculation and the rearing was continued as per standard

methods till the onset of spinning.

Tissue preparation and microscopic examination:

From second day of post inoculation till the onset of spinning, five larvae were

collected every day at random from each treated batch and different tissues viz.,

midgut, fat bodies, malpighian tubules, trachea, silk gland and gonads were dissected

out in insect saline (0.85% NaCl). The tissues were washed in sterilized distilled

water. Also, the haemolymph was collected by puncturing the proleg of the larvae

with a fine disposable needle in a pre chilled eppendorf tube containing a pinch of

phenyl thiourea and examined under the microscope for spore stage. To examine the

other host tissues, each tissue was weighed and 1 mg of each tissue was homogenized

in 1 ml of distilled water. The microsporidian spores in the homogenate of each tissue

and also in haemolymph were quantified using a Neubar haemocytometer and the

intensity of infection in different tissues during progressive infection was determined

by the standard formula (Cantwell, 1970). The observations were recorded on the

intensity of infection which was shown as (-) for no infection, (+) for low-level

infection, (++) for high-level infection and (+++) for very high infection. The terms

nil, low, high and very high for severity of infection are quantitative indicating no

presence of spores, 0.25 to 7.5×106, 7.75 to 15×106 and 15.25 to 22.5×106 spores/mg

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tissue respectively. The same in case of haemolymph was expressed as spores/ml. To

take the microphotographs of the infected tissues, a sample of each tissue was placed

on a clean micro slide. A drop of water was added and another slide was placed over

the tissue, pressed and dragged gently. Mechanical and osmotic pressure cause the

cells to lyse and release the spores from the host tissues. Each tissue smear was then

observed and photographed under phase contrast microscope (Nikon, Type-104 with

camera attachment).

Pathogenicity of the isolated microsporidia to the silkworm, Bombyx mori L.: To

determine the pathogenicity of the microsporidia isolated from lepidopteran insect

pests of mulberry and agricultural crops, a popular bivoltine silkworm breed viz.,

CSR2 was selected. The eggs of this breed were received from germplasm bank of

Central Sericultural Research and Training Institute, Mysore and were incubated after

surface disinfection at 25±1ºC and 80±5% RH. The hatched larvae were reared till the

beginning of third instar following standard silkworm rearing practices under hygienic

conditions. The third instar silkworm larvae were inoculated separately with different

concentrations of the isolated microsporidian spores. Also, one set of larvae inoculated

with different concentrations of Nosema bombycis spores was maintained separately

for comparison. Six concentrations of the pathogen inoculum i.e., 1×103, 1×104,

1×105, 1×106, 1×107 and 1×108 spores/ml were tested for pathogenicity determination

of each microsporidia. These different concentrations were prepared from purified

spores of each isolated microsporidia by serial dilution of the quantified stock

inoculum. The quantification was done following the standard method using Neubar

haemocytometer (Cantwell, 1970). Each inoculum concentration formed a treatment.

For each treatment five replications of 100 larvae were maintained. Different sets of

larvae were inoculated separately with different concentrations of each of the

microsporidian spores isolated from insect pests of mulberry and some other

agricultural crops. One ml of specific concentration of specific microsporidian spores

was smeared on the ventral surface of mulberry leaf and fed to the silkworm larvae

just out of 2nd moult.

Observations on the mortality due to the concerned microsporidia were

recorded daily. The dead larvae were homogenized and examined for the specific

microsporidian infection under phase contrast microscope. The LC50 values for each

microsporidia were determined in terms of cumulative larval mortality for 12 and 15

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days PI and in terms of cumulative mortality up to pupal stage for 21 days PI

following Probit analysis (Finney, 1971). The pathogenicity of the isolated

microsporidia was compared with that of N. bombycis and the results were discussed.

Rate of spread of microsporidian infection in silkworm population: To determine

the rate of spread of infection in a fixed population of silkworm, a specific number of

carrier larvae with specific microsporidian infection were introduced into a fixed

healthy population of silkworm larvae. A popular bivoltine silkworm breed CSR2 was

selected. CSR2 larvae were brushed and reared under optimum conditions as

recommended. Immediately after I moult, 600 newly ecdysed second instar larvae

were taken and divided into six groups, each group containing 100 larvae. To ensure

the availability of infected carrier larvae for introduction into a healthy colony of

silkworm, the newly ecdysed second instar larvae were inoculated separately with

NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp microsporidia at infective

spore dosages of 1×108, 1.5×108, 1×107, 1×107 and 1.5×108 spores/ml/100 larvae

respectively to ensure infection in the carrier larvae. Also, one group of 100 larvae

inoculated with the spores of Nosema bombycis (1×106 spores/ml) was kept for

obtaining carrier larvae to be introduced in control batch for comparison. The rearing

of healthy larvae and inoculated larvae was continued till second moult. Before

resumption of feeding after second moult, the microscopical examination of faeces of

the larvae from each inoculated group was made to confirm the presence of spores of

the concerned microsporidia. On zero day of third instar, the infected carrier larvae

were introduced into the healthy colonies in the combinations of 1 in 99 healthy

worms, 3 in 97 healthy worms, 5 in 95 healthy worms and 7 in 93 healthy worms.

Three replications for each treatment were maintained and the rearing was continued

following the standard procedure till spinning and moth emergence. Dead larvae were

microscopically examined for the presence of microsporidian spores to ascertain the

cause of mortality. The mortality due to respective microsporidia was recorded daily.

Data was also collected in respect of larval weight, larval duration, pupal mortality

and number of moths emerged. The moths emerged were tested individually to

determine the percentage of infection at moth stage. Total spread of infection by the

five different microsporidia by way of secondary contamination was calculated. The

data was analyzed statistically to arrive at conclusion.

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Susceptibility of different silkworm breeds to the isolated microsporidia: To

determine the susceptibility of different silkworm breeds to the microsporidia isolated

from insect pests of mulberry and agricultural crops, ten popular silkworm

breeds/hybrids viz., Pure Mysore, Nistari, ND7, NP1 (Multivoltine breeds), CSR2,

CSR4, CSR18, CSR19 (Bivoltine breeds) and Pure Mysore×CSR2 and CSR2×CSR4

(hybrids) were selected. The said breeds/hybrids were selected based on the

characteristics presented in the following Table.

Characteristics of the breeds/hybrids selected for the studies on their susceptibility to the isolated microsporidia

Silkworm breed/hybrid Characteristic (s)

MULTIVOLTINE BREEDS Pure Mysore Most popular and local breed of South India.

Nistari Most popular and local breed of Eastern India. ND7 Newly developed, highly productive multivoltine

breed and component of Jayalaxmi hybrid; under National level race authorization test.

NP1 Newly developed robust multivoltine breed which is in pipeline and subjected to race authorization.

BIVOLTINE BREEDS CSR2 Popular and productive bivoltine breed and an

authorized component of popular hybrid CSR2×CSR4.

CSR4 Popular and productive bivoltine breed and an authorized component of popular hybrid CSR2×CSR4.

CSR18 Authorised robust bivoltine breed, high temperature tolerant and component of CSR18×CSR19.

CSR19 Authorised robust bivoltine breed, high temperature tolerant and component of CSR18×CSR19.

HYBRIDS (CROSS BREEDS) PM×CSR2 Popular multi×bi hybrid.

CSR2×CSR4 Popular bi×bi hybrid.

The layings of the selected breeds were received from the silkworm

germplasm of CSR&TI, Mysore and incubated at 25±1ºC and 80±5% RH. The

hatched larvae were reared following standard rearing method till the beginning of 3rd

instar. The 3rd instar larvae (100 larvae/replication) immediately after 2nd moult were

inoculated separately with the inoculum dosage of 1×107 spores/ml of the isolated

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microsporidia and also N. bombycis and were reared till cocooning. The inoculum of

each microsporidia was prepared from the purified stock. Three replications of 100

larvae each were maintained. The observations were recorded on the mortality due to

the microsporidiosis caused by specific microsporidia. For the purpose, the dead

larvae and pupae were homogenized and microscopically examined. Mortality due to

the specific microsporidian infection was recorded. The live moths were examined for

the infection. Data with respect to single cocoon weight, single shell weight and silk

ratio also was recorded and the data was statistically analyzed. The susceptibility of

these breeds to the isolated microsporidia was compared with the susceptibility of

these breeds to N. bombycis.

RESULTS

Mode of infection of the isolated microsporidia in the silkworm, Bombyx mori L.:

NIK-1Pr: The results with regard to the mode of infection of NIK-1Pr microsporidian

are presented in Table 2.1. The data indicates that the inoculation of the said

microsporidian to CSR2 larvae by mulberry leaf contamination (T1) resulted in

infection of silkworm leading to a larval and pupal mortality of 42.0 and 9.7%

respectively. The infection at moth stage was recorded as 44.7%. Fecundity and

Hatching percent of the layings from infected parents were reduced significantly (384

and 80.3% respectively) due to infection. The microscopic examination of dead eggs

showed that 86.9% of the dead eggs were infected with the said microsporidian. The

progeny larvae showed an infection of 97.4%. The egg surface contamination with the

microsporidia (T2) also resulted in infection leading to a larval and pupal mortality of

54.3 and 20.0% respectively. 67.0% of the moths emerged were found to be infected.

Fecundity and Hatching percent were significantly reduced (354 and 74.3%

respectively). Infection in dead eggs was 90.1% and the progeny larvae showed

98.5% infection. The set of larvae smeared with the spores of NIK-1Pr on the

integument (T3) followed by Vijetha dusting did not develop infection. Microscopic

examination of the homogenate of larvae, pupae, moths, dead eggs and the progeny

larvae did not show the presence of microsporidian spores and also, there was no

significant reduction in Fecundity and Hatching percent (498 and 95.5% respectively).

However, the larvae smeared with NIK-1Pr microsporidian spores on the integument

and not dusted with Vijetha (T4) developed infection and a mortality of 30.7 and

8.3% was recorded at larval and pupal stage respectively. The infection at moth stage

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was recorded as 40.0%. In this treatment also, Fecundity (413) and Hatching percent

(84.5%) were reduced significantly. In the dead eggs and progeny larvae, infection of

83.9 and 91.6% respectively was recorded. The larvae, pupae, moths, dead eggs and

progeny larvae from control batches (T5) did not show the presence of microsporidian

spores. The Fecundity and Hatching percent of the control batches were recorded as

505 and 97.0% respectively.

NIK-1Cc: The data as presented in Table 2.2 indicates that the inoculation of NIK-

1Cc microsporidian to CSR2 larvae by mulberry leaf contamination (T1) resulted in

infection of silkworm leading to a larval and pupal mortality of 32.0 and 7.7%

respectively. The infection at moth stage was recorded as 29.3%. Fecundity and

Hatching percent of the layings from infected parents were reduced significantly (417

and 85.8% respectively). However, the microscopic examination of dead eggs and the

progeny larvae did not show the presence of microsporidian spores indicating that

though the said microsporidian infects the silkworm, Bombyx mori L. but the infection

is not passed to progeny from the infected parents. The egg surface contamination

(T2) also resulted in infection leading to a larval and pupal mortality of 39.7 and

18.0% respectively. 38.0% of the emerged moths were found to be infected.

Fecundity and Hatching percent were significantly reduced (406 and 81.4%

respectively). In this treatment also, the dead eggs and progeny larvae were found to

be free from microsporidian infection. The set of larvae smeared with the spores of

NIK-1Cc on the integument (T3) followed by Vijetha dusting did not develop

infection. Microscopic examination of the homogenate of larvae, pupae, moths, dead

eggs and the progeny larvae did not show the presence of microsporidian spores and

also, there was no significant reduction in Fecundity and Hatching percent (499 and

96.4% respectively). However, in the treatment wherein the larvae were smeared with

NIK-1Cc microsporidian spores on the integument and not followed by Vijetha

dusting (T4) developed infection and a mortality of 21.3 and 7.0 % was recorded at

larval and pupal stage respectively. The infection at moth stage was recorded as

23.7%. In this treatment also, Fecundity (429) and Hatching percent (88.3%) were

reduced significantly. The dead eggs and progeny larvae did not show the presence of

microsporidian spores. The larvae, pupae, moths, dead eggs and progeny larvae from

control batches (T5) did not show the presence of microsporidian spores. The

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Fecundity and Hatching percent of the control batches were recorded as 509 and

97.4% respectively.

NIK-1Cpy: The results with regard to the mode of infection of NIK-1Cpy

microsporidian are presented in Table 2.3. The data indicates that the inoculation of

the said microsporidian to CSR2 larvae by mulberry leaf contamination (T1) resulted

in infection of silkworm leading to a larval and pupal mortality of 70.0 and 10.7%

respectively. The infection at moth stage was recorded as 100%. Fecundity and

Hatching percent of the layings from infected parents got significantly reduced (362

and 77.5% respectively). The microscopic examination of dead eggs as well as the

progeny larvae showed an infection of 100%. The egg surface contamination (T2)

also resulted in infection leading to a larval and pupal mortality of 76.0 and 13.3%

respectively and the infection at moth stage was recorded as 100%. Fecundity and

Hatching percent were significantly reduced (343 and 72.7% respectively). Infection

in dead eggs and the progeny larvae was recorded as 100%. The set of larvae smeared

with the spores of the said microsporidian on the integument (T3) followed by Vijetha

dusting did not develop infection and also, there was no significant reduction in

Fecundity and Hatching percent (498 and 95.3% respectively). However, the larvae

smeared with NIK-1Cpy microsporidian spores on the integument and not dusted with

Vijetha (T4) developed infection and a mortality of 41.7 and 9.3% was recorded at



reduced significantly. In the dead eggs and progeny larvae, infection of 90.3 and

94.0% respectively was recorded. The larvae, pupae, moths, dead eggs and progeny

larvae from control batches (T5) did not show the microsporidian infection and the

Fecundity and Hatching percent were recorded as 504 and 97.0% respectively.

NIK-1So: The data as presented in Table 2.4 indicates that the inoculation of NIK-

1So microsporidian by mulberry leaf contamination (T1) resulted in infection of

silkworm leading to a mortality of 85.7 and 11.3% at larval and pupal stage

respectively. The infection at moth stage was recorded as 100%. Fecundity and

Hatching percent were reduced significantly (359 and 76.5% respectively). The dead

eggs as well as the progeny larvae showed an infection of 100%. The egg surface

contamination (T2) also resulted in infection and a larval and pupal mortality of 90.3

and 3.7% respectively was recorded. The infection at moth stage was 100%.

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72

Fecundity and Hatching percent were significantly reduced (340 and 71.6 %

respectively). 100% infection was recorded in the dead eggs and the progeny larvae.

The set of larvae smeared with the spores of the said microsporidian on the

integument (T3) followed by Vijetha dusting did not develop infection and also, there

was no significant reduction in Fecundity and Hatching percent (498 and 95.7%

respectively). However, the larvae smeared with NIK- 1So microsporidian spores on

the integument and not followed by Vijetha dusting (T4) developed infection and a

mortality of 45.7 and 10.7% was recorded at larval and pupal stage respectively. The

infection at moth stage was recorded as 95.0%. Fecundity (385) and Hatching percent

(79.5%) were reduced significantly. In the dead eggs and progeny larvae, infection of

94.0 and 96.3% respectively was recorded. The larvae, pupae, moths, dead eggs and

progeny larvae from control batches (T5) did not show the microsporidian infection

and the Fecundity and Hatching percent were recorded as 503 and 97.0% respectively.

NIK-1Dp: The results with regard to the mode of infection of NIK-1Dp

microsporidian are presented in Table 2.5. The data indicates that the inoculation of

the said microsporidian to CSR2 larvae by mulberry leaf contamination (T1) resulted

in infection of silkworm and a larval mortality of 33.0% was recorded. Similarly,

pupal mortality of 8.3% was recorded. The infection at moth stage was recorded as

32.7%. Fecundity and Hatching percent of the layings from infected parents were

reduced significantly (407 and 84.0% respectively). The microscopic examination of

dead eggs showed that 85.3% of the dead eggs were infected with the said

microsporidian. The progeny larvae showed an infection of 84.1%.The egg surface

contamination (T2) also resulted in infection leading to a larval and pupal mortality of

44.7 and 18.7% respectively. 40.7% of the moths emerged were found to be infected.

Also, Fecundity and Hatching percent were significantly reduced (395 and 79.7%

respectively). Infection in dead eggs was 88.0% and the progeny larvae showed

86.5% infection. The set of larvae smeared with the spores of the said microsporidian

on the integument (T3) followed by Vijetha dusting did not develop infection.

Microscopic examination of the homogenate of larvae, pupae, moths, dead eggs and

the progeny larvae did not show the presence of microsporidian spores and also, there

was no significant reduction in Fecundity and Hatching percent (499 and 96.3%

respectively). However, in the treatment wherein the larvae were smeared with NIK-

1Dp microsporidian spores on the integument and not dusted with Vijetha (T4),

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microsporidian infection developed and a mortality of 24.0 and 8.0% was recorded at



reduced significantly. In the dead eggs and progeny larvae, infection of 81.8 and

78.5% respectively was recorded. The larvae, pupae, moths, dead eggs and progeny

larvae from control batches (T5) did not show the presence of microsporidian spores

and the Fecundity and Hatching percent were recorded as 509 and 97.4% respectively.

Nosema bombycis: The data regarding the mode of infection of the standard

microsporidian strain, Nosema bombycis as presented in Table 2.6 indicates that the

inoculation of the said microsporidian by mulberry leaf contamination (T1) resulted in

high level of infection in silkworm leading to 100% mortality within the larval stage.

The egg surface contamination treatment (T2) also resulted in higher level of infection

and a mortality of 100% was recorded at larval stage. The set of larvae smeared with

the spores of N.bombycis on the integument (T3) followed by Vijetha dusting did not

develop infection and also, there was no significant reduction in the Fecundity and

Hatching percent (499 and 95.7% respectively). However, the larvae smeared with

Nosema bombycis spores on the integument and not followed by Vijetha dusting (T4)

developed infection and a mortality of 52.0 and 17.0% was recorded at larval and

pupal stage respectively. At moth stage 100% infection was recorded. Fecundity (360)

and Hatching percent (72.1%) were reduced significantly. In the dead eggs and

progeny larvae, infection of 100 % was recorded. The larvae, pupae, moths, dead eggs

and progeny larvae from control batches (T5) did not show infection and the

Fecundity and Hatching percent were recorded as 501 and 97.0% respectively.

Site of infection of the isolated microsporidia in the silkworm, Bombyx mori L.:

The results of microscopic examination of different tissues of silkworm infected

separately with five different microsporidia are presented in Tables 2.7 to 2.12 and

Figures 2.1 to 2.6. In batches inoculated with NIK-1Pr (Table 2.7 and Fig. 2.1), the

microsporidian was first noticed in midgut on 8th day Post inoculation (PI) with low

intensity of infection. From 9th day PI onwards, the intensity of infection was high.

The microsporidial presence of low intensity in the fat bodies was first noticed on 9th

day PI. The infection intensity in fat bodies became high on 11th day PI onwards. Low

intensity infection was first sighted on 11th day PI in malpighian tubules, trachea and

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haemolymph, whereas in silk glands and gonads, the presence of NIK-1Pr spores was

first noticed only on 12th day PI.

Infection in different tissues of silkworm by microsporidian NIK-1Cc (Table 2.8

and Fig. 2.2) indicates that the microsporidian was first sighted in midgut with low

intensity of infection on 10th day PI. The intensity became high from 12th day PI

onwards. In fat bodies, the microsporidian was first noticed on 11th day PI, where as

in malpighian tubules, trachea and haemolymph, the microsporidian spores were first

noticed on 12th day PI. In silk gland, the microsporidian was first noticed on 13th day

PI. The intensity of infection in tissues other than midgut and fat bodies was low

throughout the observation period of 13 days. In gonads, no microsporidian infection

was noticed till the end of the observation period.

In case of microsporidian NIK-1Cpy, the microsporidian presence of low

intensity was first observed in midgut on 7th day PI (Table 2.9 and Fig. 2.3). The

intensity of infection became high from 8th day PI onwards. In fat bodies,

microsporidian first appeared with low intensity on 8th day PI, which subsequently

became high from 9th day PI onwards. In malpighian tubules, trachea and silk gland,

the microsporidian was first observed on 9th day PI, whereas in gonads and

haemolymph, the microsporidial presence was first noticed on 10th day PI. The

intensity of infection was low till 11th day PI in malpighian tubules, whereas in

trachea, intensity of infection was low till 12th day PI. In silk gland, gonads and

haemolymph, the intensity of infection was low throughout the observation period.

The intensity of infection in different tissues of silkworm by microsporidian

NIK-1So (Table 2.10 and Fig. 2.4) shows its presence with low intensity first in the

midgut on 8th day PI. The infection intensity in the midgut increased from 10th day PI

onwards. In fat bodies, malpighian tubules, trachea, silk gland and gonads, the low

intensity infection was first noticed on 9th day PI, whereas in haemolymph, the

microsporidial presence was first noticed on 10th day PI. In fat bodies, the intensity of

infection increased on 12th day PI whereas in malpighian tubules, the increase in

intensity was noticed on 13th day PI. The intensity of infection remained low in

trachea, silk gland, gonads and haemolymph throughout the observation period.

In the batches inoculated with the microsporidian NIK-1Dp, the low intensity

infection was first noticed in midgut on 9th day PI, in fat bodies and trachea on 10th

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day PI, malpighian tubules and silk gland on 11th day PI and gonads and haemolymph

on 12th day PI. The intensity of infection was high on 12th day of PI in midgut and fat

bodies whereas in malpighian tubules, high intensity infection was noticed on 13th day

PI. In remaining tissues viz., trachea, silk gland, gonads and haemolymph, the

intensity of infection was low throughout the observation period of 13 days (Table

2.11 and Fig. 2.5).

The intensity of infection in different tissues of silkworm inoculated with

Nosema bombycis, the standard strain of microsporidia causing pebrine disease in

silkworm, which served as control is presented in Table 2.12 and Fig. 2.6. It is clear

from the data that the first infection of low intensity was noticed as early as on 6th day

PI in midgut, fat bodies and malpighian tubules whereas in trachea, silk gland, gonads

and haemolymph, the N. bombycis spores were first noticed on 7th day PI. In midgut,

the intensity of infection was high on 7th day PI and very high on 9th day PI onwards.

In fat bodies, the intensity of infection was very high on 10th day PI whereas the same

in case of malpighian tubules, trachea and silk gland was observed on 11th day PI. In

other tissues viz., gonads and haemolymph, the intensity of infection was very high on

12th and 13th day PI respectively. For comparison of the infected tissues with that of

the normal tissues, microphotographs of the normal tissues also were taken and are

presented in Fig. 2.7.

Pathogenicity of the isolated microsporidia to the silkworm, Bombyx mori L.: The

results with regard to the pathogenicity of microsporidian spores isolated from insect

pests of mulberry and other agricultural crops are presented in Table 2.13. It is

observed that the microsporidia viz., NIK-1Pr, NIK-1Cpy and NIK-1So did not cause

any mortality in larvae and pupae at an inoculum dose of 1×103 spores/ml whereas,

the microsporidian NIK-1Cc did not elicit any mortality in larvae and pupae at a

concentration of up to 1×105 spores/ml. In the batches infected with microsporidian

NIK-1Dp, no mortality due to microsporidiosis was recorded in the larval stage up to

an innoculum dose of 1×105 spores/ml. However, at this dose, mortality was observed

in pupal stage. The mortality in larvae and pupae due to microsporidiosis was first

recorded in NIK-1Pr, NIK-1Cpy and NIK-1So at a minimum spore dose of 1×104

spore/ml. In batches inoculated with NIK-1Cc, larval and pupal mortality was

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observed at a spore concentration of 1×106 spores/ml and above. The microsporidian

NIK-1Dp was found to elicit larval mortality at a spore dose of 1×106 spore/ml.

Data presented in Table 2.13 shows that percent larval mortality due to infection

with different microsporidia ranged from 1.0 (1×104 spores/ml) to 66.0 (1×108

spores/ml); 19.7 (1×106 spores/ml) to 42.3 (1×108 spores/ml); 6.7 (1×104 spores/ml) to

81.7 (1×108 spores/ml); 9.7 (1×104 spores/ml) to 97.0 (1×108 spores/ml) and 22.7

(1×106 spores/ml) to 45.7% (1×108 spores/ml) in NIK-1Pr, NIK-1Cc, NIK-1Cpy,

NIK-1So and NIK-1Dp treated batches respectively. Likewise, the percent mortality

at pupal stage ranged from 1.7 (1×104 spores/ml) to 20.3 (1×108 spores/ml); 9.0

(1×106 spores/ml) to 14.0 (1×108 spores/ml); 3.7 (1×104 spores/ml) to 12.0 (1×108

spores/ml); 3.0 (1×108 spores/ml) to 10.3 (1×107 spores/ml) and 1.7 (1×105 spores/ml)

to 17.3% (1×108 spores/ml) in NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-

1Dp treated batches respectively.

It was also observed that adults could successfully emerge in all the batches

treated separately with the isolated microsporidia at different spore concentrations

except in NIK-1So treated batches wherein the adult emergence was nil at the highest

concentration (1×108 spores/ml). A decreasing trend was observed in the percentage

of adult emergence with an increase in the spore concentration of the inoculum in all

the microsporidia tested. At different inoculum doses (1×103,1×104 ,1×105, 1×106,

1×107 and 1×108), the total infection percentage in NIK-1Pr infected batches ranged

from 29.7 to 99.0; in NIK-1Cc, 14.3 to 71.7; in NIK-1Cpy, 31.7 to 100; in NIK-1So,

36.3 to 100 and in NIK-1Dp, 17.7 to 77.3% from the lowest concentration (1×103

spores/ml) to the highest concentration (1×108 spores/ml).

In comparison to the microsporidia isolated from different lepidopteran pests of

mulberry and agricultural crops, in the standard strain, Nosema bombycis, the

mortality at larval and pupal stages was observed at the lowest inoculum dosage

tested (1×103 spores/ml) which increased with the increase in the inoculum dose. The

mortality recorded at larval stage ranged from 3.7 (1×103 spores/ml) to 100% (1×108

spores/ml) and at pupal stage, from 8.3 (1×103 spores/ml) to 18.0% (1×106

spores/ml). At the inoculum dosage of 1×107 and 1×108 spores/ml, all the larvae died

prior to adult eclosion. The total infection recorded was 87.3, 89.7, 92.3, 100, 100 and

100% in the batches treated with the spore concentration of 1×103, 1×104, 1×105,

1×106, 1×107 and 1×108 spores/ml respectively.

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The median lethal concentration (LC50) and fiducial limits for the isolated

microsporidia viz., NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp and also

the standard strain N. bombycis were calculated for cumulative larval mortality up to

12 and 15 days PI and for cumulative pupal mortality up to 21 days PI in CSR2 breed

of the silkworm, Bombyx mori L. following the Probit method and the results are

presented in Tables 2.14, 2.15 and 2.16 and graphically represented in Figures 2.8, 2.9

and 2.10 respectively. The LC50 values for 12th day PI (Table 2.14 and Figure 2.8) for

NIK-1Pr microsporidian was 1×108.1 spores/ml with 7.817 and 8.403 as lower and

upper fiducial limits respectively, for NIK-1Cc, 1×1014.6 spores/ml with 9.621 and

19.607 as lower and upper fiducial limits; for NIK-1Cpy, the LC50 value was 1×107.5

spores/ml with 7.317 and 7.870 as lower and upper fiducial limits; for NIK-1So, it

was 1×107.2 spores/ml with 7.028 and 7.506 as lower and upper fiducial limits and for

NIK-1Dp, the same was 1×1011.6 spores/ml with 9.430 and 13.903 as lower and upper

fiducial limits respectively. However, when compared to the isolated microsporidia,

the LC50 value for N.bombycis for 12 days PI was as low as 1×106.9 spores/ml with

6.759 and 7.206 as lower and upper fiducial limits respectively.

The LC50 values estimated for 15th day PI are presented in Table 2.15 and Figure

2.9. The LC50 value for 15 days PI for NIK-1Pr was 1×107.5 spores/ml with 7.275 and

7.781 as lower and upper fiducial limits; for NIK-1Cc, 1×109.5 spores/ml with 8.459

and 10.553 as lower and upper fiducial limits; for NIK-1Cpy, 1×106.7 spores/ml with

6.529 and 6.932 as lower and upper fiducial limits; for NIK-1So 1×106.1 spores/ml

with 5.998 and 6.359 as lower and upper fiducial limits and for NIK-1Dp, 1×108.9

spores/ml with 8.125 and 9.711 as lower and upper fiducial limits respectively. In

comparison to the isolated microsporidia, the estimated LC50 value for 15 days PI for

Nosema bombycis was as low as 1×105.6 spores/ml with 5.526 and 5.856 as lower and

upper fiducial limits respectively.

The estimated values for median lethal concentration (LC50) for 21st day PI for

the isolated microsporidia and also Nosema bombycis are presented in Table 2.16 and

Figure 2.10. For NIK-1Pr microsporidian, the LC50 value for 21 days PI was 1×106.6

spores/ml with 6.443 and 6.811 as lower and upper fiducial limits, for NIK-1Cc, it

was 1×107.5spores/ml with 7.081 and 7.974 as lower and upper fiducial limits, for

NIK-1Cpy, it was 1×105.9 spores/ml with 5.767 and 6.122 as lower and upper fiducial

limits, for NIK-1So, it was 1×105.4 spores/ml with 5.312 and 5.616 as lower and upper

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fiducial limits and for NIK-1Dp, the same was as 1×107.2 spores/ml with 7.032 and

7.436 as lower and upper fiducial limits. The LC50 value for 21 days PI for Nosema

bombycis was 1×105.3 spores/ml with 5.150 and 5.579 as lower and upper fiducial

limits respectively.

From the above results, it is observed that among five microsporidia isolated

from lepidopteran pests of mulberry and other agricultural crops, the microsporidian

NIK-1Cc is least virulent with the highest LC50 values of 1×1014.6, 1×109.5 and 1×107.5

spores/ml followed by NIK-1Dp with LC50 values of 1×1011.6, 1×108.9 and 1×107.2

spores/ml; NIK-1Pr with the same as 1×108.1, 1×107.5 and 1×106.6 spores/ml and NIK-

1Cpy with LC50 values of 1×107.5, 1×106.7 and 1×105.9 spores/ml for 12, 15 and 21

days PI respectively. Also, the results reveal that the microsporidian NIK-1So is most

virulent with the lowest LC50 values of 1×107.2, 1×106.1 and 1×105.4 spores/ml for 12,

15 and 21 days respectively. However, based on LC50 values, these microsporidia

were less virulent compared to the standard strain Nosema bombycis wherein the LC50

values were only 1×106.9, 1×105.6 and 1×105.3 spores/ml for 12, 15 and 21 days post

inoculation respectively.

Rate of spread of microsporidian infection in a fixed population of silkworm:

The data on the spread of infection in the healthy colony of silkworm after

introducing carrier larvae (larvae infected with respective microsporidia) and the

effect of this spreading infection with respective microsporidia on larval duration and

larval weight is presented in Table 2.17.

Larval duration: Introduction of microsporidian carrier larvae into healthy colonies

significantly prolonged the larval duration. The larval duration recorded in batches

introduced separately with carrier larvae infected with the five isolated microsporidia

was 24 days. In the batches introduced with Nosema bombycis infected carrier larvae,

the larval duration was recorded as 24 days and 12 hours as against a larval duration

of 23 days in healthy colonies.

Larval weight: The V instar, 6th day larval weight did not differ significantly among

batches introduced separately with carrier larvae (1, 3, 5 and 7) infected with five

isolated microsporidia and ranged between 33.4 and 34.1 g in NIK-1Pr, 33.9 and 34.7

g in NIK-1Cc, 33.4 and 34.8 g in NIK-1Cpy, 34.2 and 34.5 g in NIK-1So and 34.1

and 34.6 g in NIK-1Dp batches. In the standard strain, Nosema bombycis infected

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carrier larvae introduced batches, the larval weight recorded was between 33.8 and

34.3 g as against a larval weight of 36.0 g in healthy colonies.

Spread of infection: The results as presented in Table 2.17 show that the larval

mortality due to microsporidian infection in batches introduced with 1, 3, 5 and 7

carrier larvae infected separately with the five isolated microsporidia ranged between

1.0-7.0% in NIK-1Pr, 1.0-5.7% in NIK-1Cc, 3.7-9.0% in NIK-1Cpy, 3.3-7.7% in

NIK-1So and 1.7-7.0% in NIK-1Dp. However, this larval mortality was marginally

high (4.3-10.0%) in the batches introduced with 1, 3, 5 and 7 carrier larvae infected

with the standard strain, Nosema bombycis.

The mortality at pupal stage ranged between 1.0-2.7%, 0.3-1.7%, 0.7-3.7%,

0.7-3.3% and 0.0-1.0% in the batches introduced with 1, 3, 5 and 7 carrier larvae

infected separately with the isolated microsporidia viz., NIK-1Pr, NIK-1Cc, NIK-

1Cpy, NIK-1So and NIK-1Dp respectively. The pupal mortality was slightly more

(2.7-5.0%) in the batches introduced with 1, 3, 5 and 7 carrier larvae infected with

Nosema bombycis.

The percent infection at moth stage ranged between 19.7-25.3% in NIK-1Pr,

9.0-17.7% in NIK-1Cc, 30.7-37.7% in NIK-1Cpy, 23.0-34.7% in NIK-1So and 11.7-

19.3% in NIK-1Dp which was significantly lower than that of Nosema bombycis

infected carrier larvae (1, 3, 5 and 7) introduced batches where it ranged between

40.0-59.0%.

The lowest total spread of infection after introducing the carrier larvae

infected separately with the five isolated microsporidia was recorded in the batches

introduced with 1, 3, 5 and 7 carrier larvae infected with NIK-1Cc which ranged

between 10.3-25.0% followed by NIK-1Dp (13.3-27.3%), NIK-1Pr (21.7-35.0%),

NIK-1So (27.0-45.7%) and NIK-1Cpy (35.0-50.3%) which in turn was significantly

lower compared to the batches introduced with 1, 3, 5 and 7 carrier larvae infected

with the standard strain Nosema bombycis and ranged between 47.0-74.0% (Figures

2.11 to 2.16).

Susceptibility of different silkworm breeds to the isolated microsporidia:

The results of screening of ten silkworm breeds viz., four multivoltine breeds,

four bivoltine breeds and two cross breeds are presented in Tables 2.18 to 2.23.

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Multivoltine breeds: The results regarding the susceptibility of multivoltine breeds to

the isolated microsporidia in terms of larval and pupal mortality, infection at moth

stage, total percentage of infection and effect on larval and cocoon characters are

presented in Tables 2.18 and 2.19 respectively.

Larval mortality: The data as presented in Table 2.18 shows that among the

multivoltine breeds screened, ND7 is the most susceptible to all the five isolated

microsporidia with 21.3, 18.3, 27.0, 28.7 and 20.7% larval mortality due to NIK-1Pr,

NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp microsporidia respectively followed by

NP1 with a larval mortality percent of 18.3, 14.7, 22.3, 25.3 and 18.1 which in turn

was followed by Nistari with a larval mortality percent of 15.3, 12.0, 20.3, 22.3 and

14.0 due to NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp microsporidia

respectively. Pure Mysore was found to be least susceptible to the isolated

microsporidia with a larval mortality % of 12.3, 11.3, 17.7, 19.7 and 12.0 due to NIK-

1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp respectively. ND7 was also found

to be most susceptible to Nosema bombycis with a larval mortality of 54.3% followed

by NP1 (50.3%) and Nistari (48.0%). Pure Mysore exhibited lowest susceptibility to

Nosema bombycis also with a larval mortality of 40.7%.

Among the five microsporidia viz., NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So

and NIK-1Dp, lowest larval mortality was recorded in Pure Mysore (11.3%), Nistari

(12.0%,), ND7 (18.3%) and NP1 (14.7%) respectively due to NIK-1Cc closely

followed by the batches infected with NIK-1Dp with a larval mortality of 12.0, 14.0,

20.7 and 18.1% respectively. Highest larval mortality of 19.7, 22.3, 28.7 and 25.3% in

Pure Mysore, Nistari, ND7 and NP1 respectively was observed in the batches

inoculated with the microsporidian NIK-1So. The other microsporidia viz., NIK-1Pr

and NIK-1Cpy resulted in intermediate level of larval mortality (12.3, 15.3, 21.3 and

18.3% due to NIK-1Pr microsporidian and 17.7, 20.3, 27.0 and 22.3% due to NIK-

1Cpy microsporidian in Pure Mysore, Nistari, ND7 and NP1 batches respectively).

When the mortality caused by the five microsporidia tested is compared with

that due to the standard strain Nosema bombycis, it is observed that infection due to

Nosema bombycis caused significantly higher level of larval mortality in the

multivoltine breeds (40.7, 48.0, 54.3 and 50.3% in Pure Mysore, Nistari, ND7 and

NP1 batches respectively).

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Pupal mortality: The highest mortality percent at pupal stage due to the isolated

microsporidia was recorded in ND7 (18.0, 13.7, 17.0, 18.7 and 17.3) followed by NP1

(14.7, 10.3, 13.0, 15.0 and 13.3) which was closely followed by Nistari (12.0, 8.7,

10.3, 11.3 and 9.7) due to infection with the isolated microsporidia viz., NIK-1Pr,

NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp respectively. The lowest mortality at

pupal stage was recorded in Pure Mysore (9.7, 6.7, 7.7, 8.7 and 8.7%) in the batches

treated with NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK- 1Dp respectively.

The percent pupal mortality due to the standard strain Nosema bombycis also showed

a similar trend and was recorded to be highest in ND7 (21.7%) followed by NP1

(17.7%), Nistari (13.3%) and Pure Mysore (11.3%).

Percent infection at moth stage: The percent infection in moths in the

microsporidian-inoculated batches ranged from 22.3 to 70.7% in Pure Mysore, 22.7 to

67.3% in Nistari, 22.3 to 56.0% in ND7 and 22.7 to 63.7% in NP1.

Total percentage of infection: The total infection recorded in the batches inoculated

with NIK-1Cpy and NIK-1So was 96.0% and above in all the four multivoltine breeds

tested. The lowest total infection was recorded in NIK-1Cc infected batches (40.7,

43.7, 56.7, and 48.7% in Pure Mysore, Nistari, ND7 and NP1 respectively). In NIK-

1Pr and NIK-1Dp infected batches, total infection of intermediate level was recorded

(56.7, 60.7, 71.3 and 65.3% due to NIK-1Pr and 43.0, 46.4, 60.3 and 54.2% due to

NIK- 1Dp in Pure Mysore, Nistari, ND7 and NP1 batches respectively). In Nosema

bombycis inoculated batches, which served as control for comparison, the total

infection recorded was 100% in all the four multivoltine breeds tested (Table 2.18 and

Figures 2.17 to 2.22).

The data with regard to the effect of infection with the isolated microsporidia

on the quantitative characters such as larval and cocoon characters of the survivors is

presented in Table 2.19.

Larval weight: The V instar 6th day larval weight of the microsporidian inoculated

batches got significantly reduced and ranged from 16.18 to 20.18 g in Pure Mysore,

18.11 to 21.31 g in Nistari, 33.12 to 37.48 g in ND7 and 31.49 to 34.03 g in NP1

compared to the normal control batches wherein the V instar 6th day larval weight was

recorded as 22.02 g in Pure Mysore, 24.41 g in Nistari, 38.92 g in ND7 and 36.62 g in

NP1 batches.

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Larval duration: The larval duration was increased from 26 to 28 days in Pure

Mysore, 23 to 25 days in Nistari, 25 to 26 days in ND7 and 24 to 25 days in NP1

batches infected separately with the five isolated microsporidia when compared with

the normal control. A similar trend with respect to larval duration was observed in the

batches inoculated with the standard strain Nosema bombycis except in Pure Mysore

wherein larval duration was increased from 26 to 29 days.

Single cocoon weight: Infection with the isolated microsporidia and N.bombycis

significantly lowered the cocoon weight and the cocoon weight of the treated batches

ranged from 0.730 to 0.788 g in Pure Mysore, 0.825 to 0.875 g in Nistari, 1.170 to

1.197 g in ND7 and 1.173 to 1.202 g in NP1 compared to the normal control batches

wherein the cocoon weight of 0.805 g in Pure Mysore, 0.911 g in Nistari, 1.220 g in

ND7 and 1.221 g in NP1 batches was recorded.

Single shell weight: Results presented in Table 2.19 show that the single shell weight

in the batches treated separately with the isolated microsporidia and Nosema

bombycis got reduced due to infection and ranged from 0.085 to 0.109 g in Pure

Mysore, 0.105 to 0.122 g in Nistari, 0.207 to 0.222 g in ND7 and 0.193 to 0.216 g in

NP1 compared to the normal control batches where the same was recorded as 0.115 g

in Pure Mysore, 0.133 g in Nistari, 0.246 g in ND7 and 0.227 g in NP1 batches.

Shell percentage (SR%): Infection with the isolated microsporidia significantly

reduced the shell percentage of the multivoltine breeds studied. The highest reduction

in the shell percentage was recorded in NIK-1So inoculated batches (13.32% in Pure

Mysore, 13.56% in Nistari, 18.17% in ND7 and 17.39% in NP1) followed by NIK-

1Cpy inoculated batches (13.38% in Pure Mysore, 13.64% in Nistari, 18.25% in ND7

and 17.43% in NP1), NIK-1Pr inoculated batches (13.54% in Pure Mysore, 13.75% in

Nistari, 18.36% in ND7 and 17.55% in NP1), NIK-1Dp inoculated batches (13.79%

in Pure Mysore, 13.91% in Nistari, 18.46% in ND7 and 17.71% in NP1) which in turn

was followed by NIK-1Cc inoculated batches (13.87% in Pure Mysore, 13.97% in

Nistari, 18.57% in ND7 and 17.94% in NP1).

Compared to the reduction in SR% by the isolated microsporidia, the reduction

in SR% was significantly higher in the batches inoculated with the standard

microsporidian strain, N.bombycis and was recorded as 11.64, 12.72, 17.69 and

16.45% in Pure Mysore, Nistari, ND7 and NP1 respectively. The results, therefore,

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clearly indicate that the shell percentage in the microsporidia inoculated batches was

significantly lower compared to that of normal control batches wherein shell

percentage of 14.24, 14.64, 20.13 and 18.55% was recorded in Pure Mysore, Nistari,

ND7 and NP1 respectively. The percentage reduction in the SR% of the multivoltine

breeds inoculated separately with the isolated microsporidia viz., NIK-1Pr, NIK-1Cc,

NIK-1Cpy, NIK-1So, NIK-1Dp and the standard strain N.bombycis compared to that

of the non inoculated control batches is graphically represented in Figures 2.23 to

2.28.

Bivoltine breeds: The results on the susceptibility of bivoltine breeds to the isolated

microsporidia in respect of larval and pupal mortality, infection at moth stage, total

infection and effect on larval and cocoon characters are furnished in in Tables 2.20

and 2.21 respectively.

Larval mortality: The data as presented in Table 2.20 reveals that among the

bivoltine breeds, CSR2 has shown highest susceptibility to all the five microsporidia

tested showing a percent larval mortality of 49.3, 33.3, 57.3, 60.0 and 34.3 in NIK-

1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp inoculated batches respectively

followed by CSR4 with a larval mortality of 46.3, 30.3, 54.0, 56.3 and 31.0% in NIK-

1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp inoculated batches respectively

which in turn was followed by CSR19 with a larval mortality of 37.3, 23.0, 46.0, 48.7

and 24.3% in NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp inoculated

batches respectively. CSR18 was found least susceptible to all the microsporidia

tested showing a percent larval mortality of 35.3, 20.7 , 42.3 , 45.7 and 21.3%

respectively in NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp inoculated

batches. Results of the study also reveal that CSR2 breed was most susceptible to the

standard strain Nosema bombycis with a larval mortality of 95.3% followed by CSR4

(93.3%), CSR19 (87.7%) and CSR18 (85.3%).

Among the five microsporidia, the lowest larval mortality of 33.3, 30.3, 20.7

and 23.0% respectively was recorded in CSR2, CSR4, CSR18 and CSR19 batches

infected with the microsporidian NIK-1Cc which was closely followed by NIK-1Dp

infected batches with a larval mortality of 34.3, 31.0, 21.3 and 24.3%. Highest larval

mortality of 60.0, 56.3, 45.7 and 48.7% was recorded in CSR2, CSR4, CSR18 and

CSR19 batches respectively inoculated with NIK-1So. The other microsporidia viz.,

NIK-1Pr and NIK-1Cpy resulted in an intermediate level of larval mortality which

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was recorded as 49.3, 46.3, 35.3 and 37.3% (NIK-1Pr) and 57.3, 54.0, 42.3 and 46.0%

(NIK-1Cpy) in CSR2, CSR4, CSR18 and CSR19 breeds respectively. Compared to

the larval mortality caused due to infection with the isolated microsporidia, the

infection with the standard microsporidian strain, N. bombycis resulted in significantly

higher level of larval mortality in the bivoltine breeds tested (95.3, 93.3, 85.3 and

87.7% in CSR2, CSR4, CSR18 and CSR19 batches respectively).

Pupal mortality: The highest mortality at pupal stage was recorded in CSR2 (14.3,

9.7, 13.0, 10.3 and 12.3%) followed by CSR4 (11.7, 8.3, 10.7, 9.7 and 10.3%),

CSR19 with a pupal mortality of 7.7, 5.3, 8.3, 8.7 and 8.7% in NIK-1Pr, NIK-1Cc,

NIK-1Cpy, NIK-1So and NIK-1Dp inoculated batches respectively. The lowest

mortality at pupal stage was recorded in CSR18 batches (6.3, 4.3, 7.0, 8.0 and 7.3%)

due to the microsporidia NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp

respectively. In the batches inoculated with the standard strain, Nosema bombycis,

pupal mortality of 1.7, 2.0, 3.3 and 2.7% was recorded in CSR2, CSR4, CSR18 and

CSR19 breeds respectively (Table 2.20).

Percent infection at moth stage: The infection percent at moth stage due to the

isolated microsporidia ranged from 20.3 to 29.7% in CSR2, 20.7 to 35.3% in CSR4,

20.7 to 45.7% in CSR18 and 21.7 to 42.7% in CSR19.

Total percentage of infection: The total infection (from larval to moth stage) in the

batches inoculated with the microsporidia NIK-1Cpy and NIK-1So was 95.0% and

above in the four bivoltine breeds tested. The lowest total infection was recorded in

the batches infected with NIK-1Cc (63.3% in CSR2, 59.3% in CSR4, 45.7% in

CSR18 and 50.0% in CSR19). In the batches inoculated separately with NIK-1Pr and

NIK-1Dp, an intermediate level of total infection was recorded (84.7, 81.3, 69.0 and

72.0% due to NIK-1Pr and 67.7, 62.7, 51.0 and 55.0% due to NIK-1Dp in CSR2,

CSR4, CSR18 and CSR19 batches respectively). In the batches inoculated with N.

bombycis, a total infection of 100% was recorded in all the four bivoltine breeds

tested (Table 2.20 and Figures 2.29 to 2.34).

The data on the impact of infection caused by isolated microsporidia on larval

and cocoon characters of the four bivoltine breeds is furnished in Table 2.21.

Larval weight: Data indicates that there was a significant reduction in the V instar

6th day larval weight in the inoculated batches compared to the normal control batches

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and ranged from 30.75 to 37.85 g in CSR2, 28.32 to 36.37 g in CSR4, 27.21 to 34.07

g in CSR18 and 25.57 to 32.01 g in CSR19 batches. In normal control batches, the V

instar larval weight of 39.21g (CSR2), 38.02 g (CSR4), 36.01 g (CSR18) and 34.02 g

(CSR19) was recorded.

Larval duration: The larval duration was increased from 24 to 25 days in CSR2 and

23 to 24 days in CSR4, CSR18 and CSR19 batches inoculated with the isolated

microsporidia compared to the normal control. A similar trend was observed in

N.bombycis inoculated batches except in CSR4 wherein the larval duration was

increased from 23 to 25 days.

Single cocoon weight: In the inoculated batches, the single cocoon weight was

reduced significantly and ranged from 1.435 to 1.489 g in CSR2, 1.495 to 1.540 g in

CSR4, 1.490 to 1.533 g in CSR18 and 1.431 to 1.480 g in CSR19 compared to the

non inoculated control batches (1.552 g in CSR2, 1.600 g in CSR4, 1.570 g in CSR18

and 1.520 g in CSR19).

Single shell weight: The single shell weight in the batches inoculated separately with

the isolated microsporidia and Nosema bombycis was reduced due to infection and

ranged from 0.255 to 0.300 g in CSR2, 0.260 to 0.309 g in CSR4, 0.258 to 0.305 g in

CSR18 and 0.242 to 0.289 g in CSR19 compared to the normal control wherein the

same was recorded as 0.343 g in CSR2, 0.352 g in CSR4, 0.340 g in CSR18 and

0.320 g in CSR19.

Shell percentage (SR%): Infection with the isolated microsporidia significantly

reduced the shell percentage of the bivoltine breeds studied. The highest reduction in

the shell percentage was recorded in NIK-1So inoculated batches (19.63% in CSR2,

19.27% in CSR4, 19.18% in CSR18 and 18.47% in CSR19) followed by NIK-1Cpy

(19.70% in CSR2, 19.37% in CSR4, 19.25% in CSR18 and 18.62% in CSR19), NIK-

1Pr (19.86, 19.52, 19.46 and 18.76% in CSR2, CSR4, CSR18 and CSR19

respectively), NIK-1Dp inoculated batches (19.97, 19.76, 19.62 and 18.98% in CSR2,

CSR4, CSR18 and CSR19 respectively) which in turn was followed by NIK-1Cc

inoculated batches (20.14% in CSR2, 20.06% in CSR4, 19.89% in CSR18 and

19.52% in CSR19). Compared to the reduction in SR% by the isolated microsporidia,

the reduction in SR% was significantly higher in the batches inoculated with Nosema

bombycis and SR% was recorded as 17.77% in CSR2, 17.39% in CSR4, 17.31% in

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CSR18 and 16.91% in CSR19. The shell percentage recorded in the normal control

batches was 22.11, 22.00, 21.67 and 21.04% in CSR2, CSR4, CSR18 and CSR19

breeds respectively (Table 2.21).The percentage reduction in the SR% of the bivoltine

breeds inoculated separately with the isolated microsporidia viz., NIK-1Pr, NIK-1Cc,

NIK-1Cpy, NIK- 1So, NIK- 1Dp and the standard strain N.bombycis compared to that

of the non inoculated control batches is graphically represented in Figures 2.35 to

2.40.

Cross breeds: The results on the susceptibility of two popular cross breeds of

silkworm to the isolated microsporidia in terms of larval and pupal mortality, moths

infected, total infection and effect on larval and cocoon characters are furnished in

Tables 2.22 and 2.23 respectively.

Larval mortality: The data as presented in Table 2.22 shows that among the two

cross breeds screened, CSR2×CSR4 was found to be comparatively more susceptible

to infection by the isolated microsporidia with a larval mortality of 42.3, 26.3, 50.3,

52.7 and 27.3% in NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So and NIK-1Dp inoculated

batches respectively. Also, it was more susceptible to Nosema bombycis, which

caused a larval mortality of 90.3%. PM×CSR2 was recorded to be less susceptible to

the isolated microsporidia. A larval mortality of 11.0, 6.7, 14.7, 16.3 and 10.3% was

recorded in PM×CSR2 batches inoculated with NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-

1So and NIK-1Dp respectively. In Nosema bombycis inoculated batches, a larval

mortality of 38.7% was recorded. Among the five microsporidia, lowest larval

mortality of 6.7% in PM×CSR2 and 26.3% in CSR2 × CSR4 was recorded in batches

infected with NIK-1Cc closely followed by the batches infected with NIK- 1Dp with

a larval mortality of 10.3 and 27.3% in PM×CSR2 and CSR2×CSR4 batches

respectively. Highest larval mortality of 16.3 and 52.7% respectively in PM×CSR2

and CSR2×CSR4 was observed in the batches inoculated with the microsporidian

NIK-1So. The other microsporidia viz., NIK-1Pr and NIK-1Cpy resulted in

intermediate level of larval mortality (11.0 to 14.7% in PM×CSR2 and 42.3 to 50.3%

in CSR2×CSR4 batches respectively). When the mortality caused by the five

microsporidia is compared with that due to Nosema bombycis, which is a standard

microsporidian strain causing pebrine disease in silkworm, it is observed that

infection with Nosema bombycis caused significantly higher level of larval mortality

(38.7% in PM×CSR2 and 90.3% in CSR2×CSR4 batches).

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Pupal mortality: The data with regard to mortality at pupal stage in the two cross

breeds tested as presented in Table 2.22 shows that pupal mortality percent was more

in CSR2×CSR4 batches (10.0, 6.3, 9.7, 9.3 and 9.7% in NIK-1Pr, NIK-1Cc, NIK-

1Cpy, NIK-1So and NIK-1Dp inoculated batches respectively). In PM×CSR2, 7.3,

4.7, 6.0, 8.3 and 7.7% pupal mortality was recorded in NIK-1Pr, NIK-1Cc, NIK-

1Cpy, NIK-1So and NIK-1Dp inoculated batches respectively. In the batches

inoculated with N. bombycis, 9.7% pupal mortality in PM×CSR2 and 1.3% pupal

mortality in CSR2×CSR4 was observed.

Percent infection at moth stage: The infection percent at moth stage due to the

isolated microsporidia and Nosema bombycis ranged from 20.7 to 72.3% in

PM×CSR2 and 8.3 to 38.0% in CSR2×CSR4 batches.

Total percentage of infection: The total infection in the batches inoculated with

NIK-1Cpy and NIK-1So was 93.0% and above in both the cross breeds. The lowest

total infection was recorded in NIK-1Cc infected batches (32.3% in PM × CSR2 and

54.3% in CSR2 × CSR4). In NIK-1Pr and NIK-1Dp, total infection recorded was of

intermediate level (52.0 and 38.7% in PM×CSR2 and 77.7 and 58.7% in CSR2×CSR4

respectively). In the Nosema bombycis inoculated batches, which were kept as control

for comparison purpose, the total infection recorded was 99.0-100% (Table 2.22 and

Figures 2.41 to 2.46).

The effect of microsporidian infection on the larval and cocoon characters of the

two cross breeds of silkworm is presented in Table 2.23.

Larval weight: The V instar larval weight of the microsporidia inoculated batches

was lowered significantly and ranged from 37.01 to 37.20 g in PM×CSR2 batches and

43.42 to 44.01 g in CSR2×CSR4 batches compared to that of non-inoculated control

batches (38.21 g in PM×CSR2 and 45.02 g in CSR2×CSR4).

Larval duration: The larval duration was increased from 25 to 26 days in PM×CSR2

and from 23 to 24 days in CSR2×CSR4 batches infected separately with five

microsporidia when compared to normal control. Also, in the batches inoculated with

Nosema bombycis, the larval duration was increased from 25 to 26 days and 23 to 25

days in PM×CSR2 and CSR2×CSR4 batches respectively.

Single cocoon weight: Single cocoon weight of the microsporidia inoculated batches

was significantly reduced due to infection. In PM×CSR2, it ranged from 1.504 to

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1.534 g and in CSR2×CSR4, it ranged from 1.646 to 1.681 g compared to the non-

inoculated control batches (1.604 g in PM×CSR2 and 1.721g in CSR2×CSR4).

Similar trend was also noticed in terms of single shell weight and shell ratio in

microsporidia inoculated batches vis-à-vis normal control in both the hybrids.

Single shell weight: In the cross breeds inoculated separately with the isolated

microsporidia and Nosema bombycis, single shell weight was recorded to be

significantly reduced and ranged from 0.247 to 0.259 g in PM×CSR2 and 0.311 to

0.345 g in CSR2×CSR4 compared to the normal control batches where shell weight of

0.284 g in PM×CSR2 and 0.382 g in CSR2×CSR4 was recorded.

Shell percentage (SR%): The results in Table 2.23 show that the microsporidian

infection significantly reduced the shell percentage of the cross breeds studied. The

highest reduction in SR% was recorded in the batches inoculated with NIK-1So

(16.51 and 19.89% in PM×CSR2 and CSR2×CSR4 respectively) closely followed by

NIK-1Cpy (16.60 and 19.98% in PM×CSR2 and CSR2×CSR4 respectively), NIK-1Pr

(16.72 and 20.17% in PM×CSR2 and CSR2×CSR4 respectively), NIK-1Dp (16.77

and 20.31% in PM×CSR2 and CSR2×CSR4 respectively) which in turn was followed

by NIK-1Cc inoculated batches (16.86 and 20.50% in PM×CSR2 and CSR2×CSR4

respectively). In Nosema bombycis inoculated batches, the reduction in shell

percentage was comparatively more (16.39 and 18.89% in PM×CSR2 and

CSR2×CSR4 respectively) than that of the batches inoculated with the isolated

microsporidia. In the normal control batches, SR% of 17.72 and 22.21% was recorded

in PM×CSR2 and CSR2×CSR4 respectively. The percentage reduction in the SR% of

the cross breeds inoculated separately with the isolated microsporidia viz., NIK-1Pr,

NIK-1Cc, NIK-1Cpy, NIK-1So, NIK-1Dp and the standard strain N.bombycis

compared to that of the non inoculated control batches is graphically represented in

Figures 2.47 to 2.52.

DISCUSSION

Pebrine is deadliest disease of the silkworm, Bombyx mori L. The most

common microsporidian routinely encountered to cause this disease is Nosema

bombycis (Sprague, 1982). However, there are many other microsporidia which are

encountered from time to time in silkworm. These microsporidia differ from the

standard strain, Nosema bombycis in many ways including pathogenicity. Studies on

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the characterization of microsporidia isolated from insect pests of mulberry and some

other agricultural crops infecting silkworm and presented in the preceding Chapter-1

revealed certain specific characteristics that make them different from other

microsporidia isolated from silkworm. The isolation and purification of spores,

confirmation of their infectivity as per Koch’s postulates, spore morphology,

arrangement of polar filament, number of coils of polar filament, serological affinity,

artificial germination and rate of sporulation at different temperatures were discussed.

In the present chapter, results of the detailed studies on mode of infection, site of

infection, pathogenicity, rate of spread of infection in a fixed size population of

silkworm and susceptibility of different productive breeds of the silkworm, Bombyx

mori L. to these microsporidia have been reported with comparison to corresponding

characters in batches infected with Nosema bombycis, the standard strain causing

pebrine disease of silkworm.

Several microsporidia naturally occurring in other lepidopteran pests are also

found to infect silkworm (Kishore et al., 1994; Sharma et al., 1989, 2003). Kramer

(1976) reported that microsporidia invade insects through three natural portals of

entry viz., oral, cuticular and ovarial. Entrance by the oral and cuticular portals results

in horizontal transmission and by the ovarial portal in vertical transmission. The oral

portal is the most common route through which the microsporidian gains access to

host tissues. In case of honey bees, the microsporidian, Nosema apis has been

reported to infect only through the oral portal (Bailey, 1963). In the present study

also, the isolated microsporidia were capable of infecting the silkworm, B. mori L.

through the oral portal. Feeding of mulberry leaf smeared with the isolated

microsporidia resulted in varying degrees of larval and pupal mortality and also

infection at moth stage. Infection was also recorded in the treatment wherein the egg

surface was contaminated with the isolated microsporidia. The development of

infection by the said treatment also took place through the oral portal only as the

neonates eat a portion of egg shell while hatching. Canning et al. (1985) also have

reported that though the embryo within an egg is not infected but the larvae

subsequently become infected when the microsporidian spores are ingested along with

the egg shell as the larvae eat their way through the egg shell. Thus, the infection is

similar to that of larvae feeding on the mulberry leaf contaminated with the

microsporidian spores. Significantly, the isolated microsporidia as well as N.

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bombycis were not capable of infecting silkworm through the cuticular portal. Though

the infection was recorded in the treatments wherein the microsporidian spores were

smeared on the larval integument but not dusted with bed disinfectant (Vijetha), the

cause of infection appears to be secondary contamination. After 24 hours of treatment,

when the first feeding was provided, the treated larvae came in contact with the

mulberry leaf thereby contaminating the leaf with the microsporidian spores. As the

larvae fed on the contaminated leaf, the microsporidian infection developed. Thus, the

mode of infection in this treatment also was per oral only rather than cuticular. The

said results were confirmed by observing the treatments wherein the isolated

microsporidia were smeared on the larval integument followed by Vijetha dusting

after 24 hours of treatment. Microscopic examination of larvae, pupae, moths, dead

eggs and progeny larvae from the said treatment did not show the microsporidian

infection confirming that the isolated microsporidia are not capable of infecting

silkworm through the cuticular portal. As per Brooks (1973), the entry via the

cuticular portal involves the inoculation of the microsporidian by the ovipositor of a

parasitoid and only the lepidopteran hosts that have hymenopteran parasitoids are

invaded through cuticular portal. All the isolated microsporidia except NIK-1Cc were

capable of infecting silkworm through ovarial portal also. The progenies from NIK-

1Pr, NIK-1Cpy, NIK-1So and NIK-1Dp infected female moths were also infected.

However, the progenies from NIK-1Cc infected female moths were not infected

indicating that the said microsporidian does not infect silkworm through the ovarial

portal. Tanada and Kaya (1993) also reported that infection through ovarial portal is

commonly observed in lepidopteran and mosquito hosts and the microsporidian is

incorporated into the egg or embryo within the female’s reproductive tract and the

progeny from such infected females is also infected.

Lepidopteran pests of mulberry (sole food plant of silkworm, Bombyx mori L)

and other agricultural crops constitute a constant source of microsporidian infection in

silkworm. In light of the above, it is essential to understand the nature of infection,

target tissues and pathogenicity of microsporidia isolated from lepidopteran insect

pests in silkworm. Hence, in the present study, five different microsporidia were

studied for the intensity of infection in different target tissues of silkworm. Nosema

bombycis has been reported as occurring in nearly all tissues of all stages of the

silkworm. The fat tissue of the silkworm appears to be particularly involved.

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Furthermore, the cells of the silk glands and of the malpighian tubules are frequently

as much affected as are those of the fat body (Steinhaus, 1949). These three tissues

appear to be more susceptible to parasitization by N. bombycis than do the epithelial

cells of the gut, the blood and pericardial cells. The tissues comprising the muscular

and the nervous systems are also readily attacked. The muscular tissue of the infected

silkworm becomes liquefied and microscopic observation reveals to consist of a

homogenous pasty mass, in which are suspended the remaining muscle fibers and

nuclei (Stempell, 1909). Several authors have reported that the microsporidian spore

formation occurs in mid gut epithelium, malpighian tubules, silk gland, fat bodies,

adipose tissue, gonads and trachea (Fujiwara, 1980,1985; Kawarabata, 2003; Shabir

Ahmad Bhat and Nataraju, 2007a). Infection in all the tissues of the silkworm larvae

due to the microsporidia viz., NIK-1s and NIK-2r has been reported. The

microsporidian NIK-3h has been reported to infect all the tissues except the midgut

tissue whereas infection by yet another microsporidian strain NIK-4m has not been

observed in any of the tissues other than the cysts originated from the gut basement

membrane (Ananthalakshmi and Fujiwara, 1993). Observations as recorded in present

study also clearly indicate that spore formation takes place in the midgut, fat bodies,

malpighian tubules, trachea, silk gland, gonads and haemolymph. The highest spore

yield was found in midgut followed by fat bodies and comparatively low infection in

other tissues by all the microsporidia indicating clear difference in the preference of

tissues for multiplication and sporulation by the microsporidia. Sasidharan et al.

(1994) reported that the rate of sporulation of Nosema bombycis increased with age of

silkworm pupa and moth and the cephalothoracic region recorded highest spore

concentration especially around the wing and wing muscles among all the tissues

tested. Multiplication and spore yield of microsporidia in various host systems have

also been studied by Lai and Canning (1983). The microsporidia enter silkworm by

per os means and the process of infection initiates with the germination of spores in

the gut and later passes through the gut wall and invades various other tissues of the

host (Brooks, 1988). Pleistophora sp. form uninucleate environmental spores and

infect only mid gut epithelium (Fujiwara, 1984a) and Thelohania sp. form octospore

only in the larval muscle cells (Becnel and Andreadis, 1999). Based on the

observations recorded in the present study, it can also be said that time taken by

different microsporidia to infect different tissues in silkworm also varies greatly as the

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target tissues like midgut and fat bodies are infected first followed by other tissues

such as malpighian tubules, silk gland, gonads and trachea. In these late infected

tissues, the number of microsporidian spores is also comparatively low. Rate of

sporulation of five microsporidia in different tissues of silkworm was found to be low

when compared to that of the rate of sporulation of the standard strain, Nosema

bombycis.

Madana Mohanan et al. (2006) reported that difference in spore yield in

different tissues is a reflection of the host-parasite interaction and possibly difference

in the virulence of the microsporidia is an important factor which determines the

spore yield in different tissues. Results reported in the present study suggest that all

the tissues examined are the sites of multiplication of microsporidian spores but the

multiplication rate varies in different tissues. In an earlier study also, all the tissues of

the silkworm larvae were found to be infected with two microsporidia viz., NIK-1s

and NIK-2r (Ananthalakshmi and Fujiwara, 1993). Kellen and Lindegren (1974)

reported difference in virulence of Nosema heterosporum and Nosema plodia in the

larvae of Indian meal moth, Plodia interpunctella. Our results, therefore, are in

conformity with these studies and suggest that different microsporidia studied in

present investigation infect different tissues of silkworm such as gut, fat body,

malpighian tubules, tracheal tissues, silk gland, gonads and haemolymph with varying

degree of intensity. Gonadal infection with Nosema spp. has an important bearing on

the vertical transmission of the parasite. This aspect is important in sericulture

industry since silkworm eggs distributed to farmers are required to be microsporidian

free (Nageswara Rao et al., 2004). Interestingly, in the present study, one

microsporidian, NIK-1Cc did not show any infection in gonads even up to the end of

the observation period confirming our findings with regard to the mode of infection of

the said microsporidian through ovarial portal where this microsporidian has not

shown any transmission from infected moth to the eggs in the silkworm. This finding

can be considered significant as transovarial transmission poses a serious threat to

sericulture industry as a whole.

The observations on the mortality in silkworm due to the infection by different

microsporidia under inoculated condition reveals that all the five isolated

microsporidia exhibit low pathogenicity in silkworm compared to N.bombycis.

Kishore et al. (1994) reported that two microsporidia viz., MCcrB1 and MCpyB1

Chapter 2

93

isolated from the butterflies, Catopsilia crocale and Catopsilia pyranthe respectively

do not cause any mortality in silkworm but a significant level of infection has been

observed at moth stage. However, in our study, two microsporidia viz., NIK-1Cc and

NIK-1Cpy isolated from the same butterflies caused varying degree of mortality at

larval and pupal stages depicting that the isolated microsporidia are different from

those reported by Kishore et al. (1994). An increase in percent mortality at larval

stage and a decrease in percent adult eclosion was observed with an increase in spore

concentration of each microsporidian. The results were, therefore, found to be in

conformity with an earlier study reported by Choi et al. (2002) wherein a

microsporidian isolated from cabbage white butterfly inoculated to second instar

Pieris larvae at a dosage of 1×108 spores/ml resulted in death of all larvae prior to

adult eclosion, at a lower spore dosage of 1×107 spores/ml, a few adults successfully

emerged. At 1×104 spores/ml, many individuals survived to adulthood and only a few

of these adults were infected. The isolated microsporidian was virulent and caused

chronic disease at low concentration, whereas it was highly virulent and produced

acute disease at high concentration (Choi et al., 2002). As has been reported in the

result section of the chapter, the LC50 values of the isolated microsporidia for the

larvae of CSR2 breed of the silkworm, Bombyx mori L. are 1×108.1, 1×107.5 and

1×106.6 spores/ml (NIK-1Pr); 1×1014.6, 1×109.5 and 1×107.5 spores/ml (NIK-1Cc);

1×107.5, 1×106.7 and 1×105.9 spores/ml (NIK-1Cpy), 1×107.2, 1×106.1 and 1×105.4

spores/ml (NIK-1So) and 1×1011.6, 1×108.9 and 1×107.2 spores/ml (NIK-1Dp) for 12,

15 and 21 days PI respectively. On the other hand, the LC50 values of Nosema

bombycis are significantly low (1×106.9, 1×105.6 and 1×105.3 spores/ml for 12, 15 and

21 days PI respectively). The LC50 values, therefore, clearly suggest that all the five

isolated microsporidia are less pathogenic to silkworm compared to the standard

strain Nosema bombycis.

The spread of microsporidiosis within a healthy colony of silkworm after

introduction of specific number of carrier larvae takes place by way of horizontal

transmission i.e., the transmission of the pathogen from one individual to another of

the same generation in a population (Steinhaus and Martignoni, 1970). Microsporidia

are horizontally transmitted when a susceptible host ingests spores that are released

into the environment along with the faeces of infected individuals or via decomposed

cadavers (Becnel and Andreadis, 1999). Spores of enterogastric microsporidia are

Chapter 2

94

released in the faeces of the host insect throughout the larval stage (Weiser, 1961) and

are also found in the meconium of newly eclosed adults that survive larval infection

(Inglis et al., 2003). Vavra et al. (2006) reported that when microsporidian infection is

primarily restricted to the insect fat body, release of the spores via decomposing

cadavers may be the most important means of transmission. Several of the Nosema

isolates infect the silk glands of the host and have been shown to infect susceptible

larvae via spores isolated from faeces. Jeffords et al. (1987) suggested that spore-

containing silk is an important horizontal transmission route for the microsporidian

Nosema portugal infecting Lymantria dispar but not in other insects.

In an earlier study, the rate of spread of pebrine after introduction of

transovarially infected worms into healthy colony of silkworm has been investigated

(Baig et al., 1988) and the study reveals that after introducing 1, 3, 6, 9, 12 and 15

larvae transovarially infected with pebrine in colonies of 200 healthy larvae each,

there was no mortality at larval stage. However, at moth stage, pebrine infection was

recorded which increased proportionately with an increase in the number of infected

larvae introduced. In a recent study also, a microsporidian isolated from a north-

eastern silkworm breed (Lamerin) has been found to spread infection in a healthy

silkworm colony but at a low rate. After introducing 1, 3, 6 and 9 carrier larvae

infected with lamerin microsporidian into healthy colonies containing 99, 97, 94 and

91 larvae respectively, there was no mortality at larval and pupal stages in three

silkworm breeds tested viz., Lamerin, Pure Mysore and CSR2. However,

microsporidian infection was recorded at moth stage and the extent of infection was in

proportion to the number of infected carrier larvae introduced thus depicting the

spread of infection through secondary contamination (Shabir Ahmad Bhat, 2006).

The results of the present investigation also establish the spread of the disease

through secondary contamination. In the present study, the infected carriers after their

introduction into healthy colony of silkworm larvae resulted in lower level of larval

and pupal mortality, however, significant infection was recorded at moth stage,

thereby spreading the microsporidiosis among the healthy silkworm of the fixed size

colony. The extent of spread was in proportion to the number of infected larvae

introduced. The maximum spread of microsporidiosis of 35.0, 25.0, 50.3, 45.7, 27.3

and 74.0% was recorded when 7 carrier larvae infected separately with NIK-1Pr,

NIK-1Cc, NIK-1Cpy, NIK-1So, NIK-1Dp and Nosema bombycis respectively were

Chapter 2

95

introduced into healthy colonies of silkworm larvae. Ishihara and Fujiwara (1965)

and Baig et al. (1988) reported that the change of epizootic pattern corresponds to the

change in number of larvae excreting the spores. In the silkworm, Bombyx mori L.,

common sources of pathogens for infection and spread of diseases are the

contaminated rearing trays and seat papers (Ishikawa and Miyajima, 1964; Miyajima,

1979a; Baig et al., 1990) which are used for the rearing of domesticated silkworm.

The pathogens are extruded by infected silkworms through gut juice, faecal matter

and decomposed dead body contaminating rearing trays and fresh mulberry leaves

provided to silkworm as feed, leading to the spread of the disease in healthy larvae by

secondary contamination. The study also reveals that the five different microsporidia

isolated from insect pests of mulberry and other agricultural crops show different rate

of spread in a healthy colony of silkworm that depends on their pathogenicity and

virulence.

The studies on the susceptibility of different silkworm breeds to the isolated

microsporidia revealed that there was a distinct variation among the multivoltine,

bivoltine and cross breeds in their susceptibility to the isolated microsporidia and their

effect on larval and cocoon characters. In inoculated batches, both the larval weight

and larval duration were affected. The larval weight got reduced and the larval

duration got prolonged in inoculated batches compared to the control batches. This

variation due to microsporidian infection is attributed to the multiplication of

pathogen in the host tissues, deprivation of nutrients to host growth and mechanical

disruption of tissues (Jolly, 1986). Cocoon characters were also adversely affected

due to microsporidian infection in all the ten breeds screened. Similar results have

been reported in an earlier study by Baig (1994) where a significant alteration in

larval weight and larval duration of thirty silkworm races (pure/hybrids) was

recorded. The larval weight got reduced, larval duration got prolonged and the cocoon

characters viz., single cocoon weight, single shell weight and shell ratio were reported

to be significantly inferior to cocoons obtained from healthy silkworms. Kudo (1931)

reported that heavily infected larvae of Bombyx mori do not spin cocoons and die,

whereas mild infection allows the larvae to spin cocoons. As the larval weight

decreases due to progressive pebrine infection, the cocoon is rendered inferior

(Naomani et al., 1971, Patil and Geethabai, 1989). Due to increase in time duration

from the day of inoculation to spinning, the silk gland also gets infected (Naomani et

al., 1971, Patil and Geethabai, 1989) and due to its impaired function, the cocoon

characters viz., single cocoon weight, single shell weight and silk ratio are adversely

Chapter 2

96

affected when compared to the cocoons obtained from control batches. Jameson

(1922) and Ghosh (1944) have also reported that pebrine infected silkworms spin poor

quality and flimsy cocoons. The silk from the cocoons of pebrine-infected larvae is

inferior in strength and uniformity of thickness to that of healthy larvae (Steinhaus,

1949).

In the present study, the mortality percentage due to infection by the isolated

microsporidia during larval stages was comparatively higher than the pupal stage. In

the moth stage, the percent infection was significantly higher than that of larval and

pupal stages, which is probably due to longer time available to the pathogen for its

multiplication in the host. The results are in conformity with earlier studies (Baig,

1994; Shabir Ahmad Bhat, 2006) wherein similar observations have been recorded.

According to Tanada (1976), the susceptibility of insects to microsporidia may vary

with different larval instars and occasionally with the different stages viz., larval,

pupal and adult stage. Multivoltine races have been reported to be more tolerant to

pebrine than bivoltines (Devaiah, 1973; Patil and Geethabai, 1989). Resistance to

pebrine is comparatively greater in Chinese races, followed by Japanese and European

races (Govindan et al., 1998; Singh and Saratchandra, 2003; Nataraju et al., 2005).

Among them, polyvoltine breeds were relatively resistant followed by bivoltines,

while the univoltines show poor resistance (Lu Yup-Lian, 1991). In the present study

also, similar results were observed. Multivoltines exhibited lower levels of

microsporidian infection in comparison to bivoltine breeds. The percent larval

mortality in the treated multivoltine breeds viz., Pure Mysore, Nistari, ND7 and NP1

ranged from 12.3 to 21.3%, 11.3 to 18.3%, 17.7 to 27.0%, 19.7 to 28.7%, 12.0 to

20.7% and 40.7 to 54.3% compared to the percent larval mortality in Bivoltine breeds

(CSR2, CSR4, CSR18 and CSR19) where it ranged from 35.3 to 49.3%, 20.7 to

33.3%, 42.3 to 57.3%, 45.7 to 60.0%, 21.3 to 34.3% and 85.3 to 95.3% respectively in

NIK-1Pr, NIK-1Cc, NIK-1Cpy, NIK-1So, NIK-1Dp and Nosema bombycis inoculated

batches. The highest infection level in more susceptible races such as bivoltines may

be attributed to larger body size, higher consumption of mulberry leaf and slower

regeneration of tissues compared to the less susceptible races like multivoltines

(Weiser, 1963). Among the cross breeds, PM×CSR2 was found to be less susceptible

to microsporidian infection compared to CSR2×CSR4. Various reasons have been

attributed to this difference in susceptibility/resistance of insects to microsporidian

disease. Weiser (1969) attributed three factors for this difference in

susceptibility/resistance:

Chapter 2

97

1. Failure of spores to germinate in the digestive tract because of pH, enzymes and

inadequate digestion of the seal covering the polar filament.

2. Non-susceptibility of host tissues and

3. Host resistance to infection by actively destroying the microsporidian during its

migration to susceptible tissues or by elimination of infected cells by the host.

Resistance to microsporidiosis has been found to vary based on the portal of

entry of the pathogen also. In some cases, an insect species that is resistant through

oral inoculation may become susceptible when the microsporidian is inoculated into

the haemocoel (Fisher and Sanborn, 1962; Undeen and Maddox, 1973). In this case,

the midgut is an effective barrier to the microsporidian.

It is, therefore, concluded that the five microsporidia isolated from insect pests

of mulberry and other agricultural crops infect silkworm through oral and ovarial

portals (except the microsporidian NIK-1Cc which does not infect silkworm through

the ovarial portal) and not through the cuticular portal. However, the percentage of

infection in larvae, pupae, moths, dead eggs and progeny larvae due to the isolated

microsporidia is low compared to that observed due to the standard strain, N.

bombycis. The isolated microsporidia also infect different tissues of silkworm but the

intensity of infection in different tissues by the isolated microsporidia is low when

compared to the intensity of infection in those tissues due to N. bombycis. The

isolated microsporidia are low in pathogenicity and show a lower rate of spread

within a healthy colony of silkworm when compared with the standard strain Nosema

bombycis. Also, different productive breeds of silkworm show a less susceptibility to

infection by the isolated microsporidia when compared to their susceptibility to

infection by Nosema bombycis.

98

Table 2.1: Mode of infection of NIK-1Pr in silkworm

Mortality (%) Treatment

Larva Pupa Infected moths

(%) Fecundity

(No.) Hatching %

Dead eggs infected (%)

Progeny larvae infected (%)

Leaf contamination (T1) 42.0

(40.4)±2.0 9.7

(18.1)±1.2 44.7

(41.9)±1.5 384.0±2.1 80.3±1.0

86.9 (68.7)±1.6

97.4 (80.7)±0.9

Egg surface contamination (T2) 54.3

(47.4)±1.5 20.0

(26.5)±1.0 67.0

(54.9)±2.0 354.0±2.5 74.3±1.0

90.1 (71.6)±0.9

98.5 (82.9)±0.3

Body surface contamination followed by vijetha dusting (T3)

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

498.0 ± 4.0 95.5 ± 0.8 0.0

(0.0) ± 0.0 0.0(0.0) ± 0.0

Body surface contamination without vijetha dusting(T4)

30.7(33.6) ± 2.1

8.3(16.7) ± 0.6 40.0(39.2) ±

2.0 413.0 ± 5.7 84.5 ± 1.1

83.9 (66.3)±1.5

91.6 (73.1)±1.6

Control (T5) 0.0 (0.0)

± 0.0 0.0 (0.0)

± 0.0 0.0 (0.0)

± 0.0 505.0 2.0 97.0±0.4

0.0 (0.0)±0.0

0.0 (0.0)±0.0

CD at 5 % (1.6) (1.2) (1.6) 2.6 5.5 (1.6) (1.9)

Values are mean ± SD and the values in parenthesis are angular transformed.

99

Table 2.2: Mode of infection of NIK-1Cc in silkworm


Larva Pupa

Infected moths (%)

Fecundity (No.)

Hatching % Dead eggs

infected (%) Progeny larvae infected (%)


(34.4) ± 2.0 7.7

(16.1) ± 0.6 29.3

(32.7) ±1.2 417.0 ± 5.3 85.8 ± 0.6

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0


(39.0) ± 2.1 18.0

(25.1) ± 1.0 38.0

(38.0) ± 2.0 406.0 ± 3.0 81.4 ± 0.8

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0


0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

499.0 ± 1.5 96.4 ± 0.7 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0


21.3 (27.4) ± 1.5

7.0 (15.3) ± 1.0

23.7 (29.1) ± 1.5

429.0 ± 2.0 88.3 ± 0.4 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0

Control (T5) 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 509.0 ± 2.0 97.4 ± 0.5

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

CD at 5 % (1.6) (1.2) (1.4) 5.6 1.1 -- --


100

Table 2.3: Mode of infection of NIK-1Cpy in silkworm


Larva Pupa

Infected moths (%)

Fecundity (No.)




(56.7) ± 2.0 10.7

(19.0) ± 0.6 100.0

(90.0) ± 0.0 362.0 ± 2.0 77.5 ± 0.5

100.0 (90.0) ± 0.0

100.0 (90.0) ± 0.0


(60.6) ± 1.0 13.3

(21.3) ± 0.6 100.0

(90.0) ± 0.0 343.0 ± 2.0 72.7 ± 0.7

100.0 (90.0) ± 0.0

100.0 (90.0) ± 0.0


0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

498.0 ± 2.1 95.3 ± 1.0 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0


41.7 (40.2) ± 1.5

9.3 (17.7) ± 0.6

91.3 (72.8) ± 1.5

388.0 ± 3.1 80.9 ± 0.3 90.3

(71.8) ± 0.6 94.

(75.8) ± 1.0

Control (T5) 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 504.0 ± 1.5 97.0 ± 0.2

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

CD at 5 % (1.3) (0.8) (1.3) 4.0 1.1 (0.45) (0.9)


101

Table 2.4: Mode of infection of NIK-1So in silkworm


Larva Pupa

Infected moths (%)

Fecundity (No.)




(67.7) ± 0.6 11.3

(19.6) ± 0.6 100.0

(90.0) ± 0.0 359.0 ± 2.1 76.5 ± 0.5

100.0 (90.0) ± 0.0

100.0 (90.0) ± 0.0


(71.8) ± 0.6 3.7

(11.0) ± 0.6 100.0

(90.0) ± 0.0 340.0 ± 2.0 71.6 ± 0.5

100.0 (90.0) ± 0.0

100.0 (90.0) ± 0.0


0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

498.0 ± 2.0 95.7 ± 0.6 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0


45.7 (42.5) ± 1.2

10.7 (19.0) ± 0.6

95.0 (77.0) ± 1.0

385.0 ± 2.6 79.5 ± 0.6 94.

(75.8) ± 1.0 96.3

(78.9) ± 0.1

Control (T5) 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 503.0 ± 1.5 97.0 ± 0.2

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

CD at 5 % (0.8) (0.9) (1.1) 3.8 0.9 (0.9) (0.2)


102

Table 2.5: Mode of infection of NIK-1Dp in silkworm


Larva Pupa

Infected moths (%)

Fecundity (No.) Hatching % Dead eggs



(35.0) ± 1.0 8.3

(16.7) ± 0.6 32.7

(34.8) ± 0.6 407.0 ± 5.3 84.0 ± 0.4 85.3

(67.4) ± 1.0 84.1

(66.5) ± 0.7


(41.9) ± 1.5 18.7

(25.6) ± 0.6 40.7

(39.6) ± 3.2 395.0 ± 2.0 79.7 ± 0.4 88.0

(69.7) ± 0.6 86.5

(68.4) ± 0.2 Body surface contamination followed by vijetha dusting (T3)

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

499.0 ± 0.6 96.3 ± 0.9 0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0


24.0 (29.3) ± 2.0

8.0 (16.4) ± 1.0

28.0 (31.9) ± 1.0

420.0 ± 3.1 86.0 ± 0.6 81.8 (64.7) ± 1.5

78.5 (62.3) ± 1.4

Control (T5) 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 509.0 ± 1.0 97.4 ± 0.4 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0

CD at 5 % (1.4) (1.1) (1.5) 5.3 1.1 (1.2) (0.9)


103

Table 2.6: Mode of infection of N. bombycis in silkworm


Larva Pupa

Infected moths (%)

Fecundity (No.) Hatching % Dead eggs



(90.0) ± 0.0 -- -- -- -- -- --


(90.0) ± 0.0 -- -- -- -- -- --


0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0

499.0 ± 2.0 95.7 ± 0.4 0.0 (0.0) ± 0.0

0.0 (0.0) ± 0.0


52.0 (46.1) ± 2.0

17.0 (24.3) ± 1.0

100.0 (90.0) ± 0.0

360.0 ± 2.0 72.1 ± 0.4 100.0 (90.0) ± 0.0

100.0 (90.0) ± 0.0

Control (T5) 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0 501.0 ± 1.5 97.0 ± 0.4 0.0

(0.0) ± 0.0 0.0

(0.0) ± 0.0

CD at 5 % (0.93) (0.62) -- 2.6 0.6 -- --


104

Table 2.7: Site and intensity of infection in the host by the microsporidian NIK-1Pr

Site and intensity of infection (days post inoculation) Microsporidian isolate Host tissues

6 7 8 9 10 11 12 13

Midgut - - + ++ ++ ++ ++ ++

Fat bodies - - - + + ++ ++ ++

Malpighian tubules - - - - - + + ++

Trachea - - - - - + + +

Silk gland - - - - - - + +

Gonads - - - - - - + +

NIK-1Pr

Haemolymph - - - - - + + + - No infection; + Low (0.25 to 7.5×106spores/mg tissue); ++: High infection (7.75 to 15×106spores/mg tissue); for haemolymph the same is expressed as spores/ml.

Table 2.8: Site and intensity of infection in the host by the microsporidian NIK-1Cc

Site and intensity of infection (days post inoculation) Microsporidian isolate

Host tissues 6 7 8 9 10 11 12 13

Midgut - - - - + + ++ ++

Fat bodies - - - - - + + ++

Malpighian tubules - - - - - - + +

Trachea - - - - - - + +

Silk gland - - - - - - - +

Gonads - - - - - - - -

NIK-1Cc

Haemolymph - - - - - - + + - No infection; + Low (0.25 to 7.5×106spores/mg tissue); ++: High infection (7.75 to 15×106spores/mg tissue); for haemolymph the same is expressed as spores/ml.

105

Table 2.9: Site and intensity of infection in the host by the microsporidian NIK-1Cpy


Host tissues 6 7 8 9 10 11 12 13

Midgut - + ++ ++ ++ ++ ++ ++

Fat bodies - - + ++ ++ ++ ++ ++

Malpighian tubules - - - + + + ++ ++

Trachea - - - + + + + ++

Silk gland - - - + + + + +

Gonads - - - - + + + +

NIK-1Cpy

Haemolymph - - - - + + + +

- No infection; + Low (0.25 to 7.5×106spores/mg tissue); ++: High infection (7.75 to 15×106spores/mg tissue); for haemolymph the same is expressed as spores/ml.

Table 2.10: Site and intensity of infection in the host by the microsporidian NIK- 1So


Host tissues 6 7 8 9 10 11 12 13

Midgut - - + + ++ ++ ++ ++

Fat bodies - - - + + + ++ ++

Malpighian tubules - - - + + + + ++

Trachea - - - + + + + +

Silk gland - - - + + + + +

Gonads - - - + + + + +

NIK-1So

Haemolymph - - - - + + + +


106

Table 2.11: Site and intensity of infection in the host by the microsporidian NIK- 1Dp


Host tissues 6 7 8 9 10 11 12 13

Midgut - - - + + + ++ ++

Fat bodies - - - - + + ++ ++

Malpighian tubules - - - - - + + ++

Trachea - - - - + + + +

Silk gland - - - - - + + +

Gonads - - - - - - + +

NIK-1Dp

Haemolymph - - - - - - + +

- No infection; + Low (0.25 to 7.5×106spores/mg tissue); ++: High infection (7.75 to 15×106spores/mg tissue); for haemolymph the same is expressed as spores/ml. Table 2.12: Site and intensity of infection in the host by Nosema bombycis

Site and intensity of infection (days post inoculation) Microsporidian

isolate Host tissues

6 7 8 9 10 11 12 13 Midgut + ++ ++ +++ +++ +++ +++ +++

Fat bodies + + ++ ++ +++ +++ +++ +++

Malpighian tubules + + ++ ++ ++ +++ +++ +++

Trachea - + + ++ ++ +++ +++ +++

Silk gland - + + ++ ++ +++ +++ +++

Gonads - + + ++ ++ ++ +++ +++

Nosema bombycis

Haemolymph - + + + ++ ++ ++ +++


107

Table 2.13: Comparative pathogenicity of the isolated microsporidia and Nosema bombycis to the silkworm, Bombyx mori L.

Mortality due to

microsporidiosis (%) Microsporidian isolate

Spores/ml Larva Pupa

Moths emerged

(%)

Moths infected

(%)

Total infection %

1 × 103 0.0(0.0) 0.0(0.0) 100.0(90.0) 29.7(33.0) 29.7(33.0) 1 × 104 1.0(5.7) 1.7(7.5) 97.3(80.5) 33.3(35.2) 36.0(36.8) 1 × 105 7.3(15.6) 3.7(11.1) 89.0(70.6) 34.0(35.6) 45.0(42.1) 1 × 106 26.3(30.8) 9.0(17.4) 64.7(53.5) 28.3(32.1) 63.7(52.9) 1 × 107 43.0(40.9) 12.0(20.2) 45.0(42.1) 21.0(27.2) 76.0(60.6)

NIK-1Pr

1 × 108 66.0(54.3) 20.3(26.7) 13.7(21.7) 12.7(20.8) 99.0(84.2) 1 × 103 0.0(0.0) 0.0(0.0) 100.0(90.0) 14.3(22.2) 14.3(22.2) 1 × 104 0.0(0.0) 0.0(0.0) 100.0(90.0) 18.3(25.3) 18.3(25.3) 1 × 105 0.0(0.0) 0.0(0.0) 100.0(90.0) 21.3(27.5) 21.3(27.5) 1 × 106 19.7(26.3) 9.0(17.4) 71.3(57.6) 17.7(24.8) 46.3(42.8) 1 × 107 32.3(34.6) 11.0(19.3) 56.7(48.8) 17.0(24.3) 60.3(50.9)

NIK-1Cc

1 × 108 42.3(40.5) 14.0(21.9) 43.7(41.3) 15.3(23.0) 71.7(57.8) 1 × 103 0.0(0.0) 0.0(0.0) 100.0(90.0) 31.7(34.2) 31.7(34.2) 1 × 104 6.7(15.0) 3.7(11.1) 89.7(71.2) 36.0(36.8) 46.3(42.8) 1 × 105 16.3(23.8) 4.3(11.9) 79.3(62.9) 59.7(50.6) 80.3(63.6) 1 × 106 39.7(39.1) 7.3(15.6) 53.0(46.7) 49.3(44.6) 96.3(78.9) 1 × 107 70.0(56.8) 9.7(18.1) 20.3(26.7) 20.3(26.7) 100.0(90.0)

NIK-1Cpy

1 × 108 81.7(64.6) 12.0(20.2) 6.3(14.5) 6.3(14.5) 100.0(90.0) 1 × 103 0.0(0.0) 0.0(0.0) 100.0(90.0) 36.3(37.0) 36.3(37.0) 1 × 104 9.7(18.1) 4.7(12.5) 85.7(67.7) 32.0(34.4) 46.3(42.8) 1 × 105 21.0(27.2) 5.7(13.8) 73.3(58.8) 59.3(50.3) 86.0(68.0) 1 × 106 43.0(40.9) 9.7(18.1) 47.3(43.4) 46.3(42.8) 99.0(84.2) 1 × 107 85.7(67.7) 10.3(18.7) 4.0(11.5) 4.0(11.5) 100.0(90.0)

NIK-1So

1 × 108 97.0(80.0) 3.0(9.9) -- -- 100.0(90.0) 1 × 103 0.0(0.0) 0.0(0.0) 100.0(90.0) 17.7(24.8) 17.7(24.8) 1 × 104 0.0(0.0) 0.0(0.0) 100.0(90.0) 21.3(27.5) 21.3(27.5) 1 × 105 0.0(0.0) 1.7(7.5) 98.3(82.5) 25.0(30.0) 26.7(31.1) 1 × 106 22.7(28.4) 11.7(20.0) 65.7(54.1) 18.7(25.6) 53.0(46.7) 1 × 107 33.7(35.5) 14.0(21.9) 52.3(46.3) 17.3(24.5) 65.0(53.7)

NIK-1Dp

1 × 108 45.7(42.5) 17.3(24.5) 37.0(37.4) 14.3(22.2) 77.3(61.5) 1 × 103 3.7(11.1) 8.3(16.7) 88.0(69.7) 75.3(60.2) 87.3(69.1) 1 × 104 10.7(19.1) 10.3(18.7) 79.0(62.7) 68.7(55.9) 89.7(71.2) 1 × 105 21.7(27.7) 14.7(22.5) 63.7(52.9) 56.0(48.4) 92.3(73.9) 1 × 106 50.0(45.0) 18.0(25.1) 32.0(34.4) 32.0(34.4) 100.0(90.0) 1 × 107 100.0(90.0) -- -- -- 100.0(90.0)

Nosema bombycis

1 × 108 100.0(90.0) -- -- -- 100.0(90.0) A CD at 5% (0.6) (0.8) (0.7) (0.7) (0.5) B CD at 5% (0.6) (0.8) (0.7) (0.7) (0.5)

S.E. 0.51 0.70 0.63 0.60 0.44 Ax B

CD at 5% (1.4) (1.9) (1.8) (1.7) (1.2) Values in parenthesis are angular transformed

108

Table 2.14: LC50 and Fiducial limits of the isolated microsporidia and Nosema bombycis to the silkworm, Bombyx mori L. (12 days PI)

Treatment X mean Y mean Probit Equation Chi square S.E-b Fiducial limit LC50

NIK-1Pr 7.2297 4.2784 Y=-1.65+0.82X 3.369NS 0.1954 107.817-108.403 1×108.1

NIK-1Cc 6.2003 3.4014 Y= 2.22+0.19X 7.250NS 0.1108 109.621-1019.607 1×1014.6

NIK-1Cpy 6.6950 4.4338 Y= 0.22+0.63X 1.719NS 0.1166 107.317-107.870 1×107.5

NIK-1So 6.5788 4.5545 Y= 0.30+0.65X 4.308NS 0.1100 107.028-107.506 1×107.2

NIK-1Dp 6.3902 3.6017 Y= 1.91+0.27X 7.291NS 0.1081 109.430-1013.903 1×1011.6

N. bombycis 5.8336 4.2657 Y= 0.54+0.64X 1.722NS 0.0687 106.759-107.206 1×106.9 PI: Post inoculation; NS: Non-significant.



NIK-1Pr 6.7540 4.4824 Y=-0.04+0.67X 2.602NS 0.1227 107.275-107.781 1×107.5

NIK-1Cc 6.1868 4.0375 Y=2.24+0.29X 9.423NS 0.0834 108.459-1010.553 1×109.5

NIK-1Cpy 6.3482 4.7562 Y=0.71+0.63X 3.793NS 0.0911 106.529-106.932 1×106.7

NIK-1So 6.0098 4.8790 Y=0.58+0.72X 7.819NS 0.0976 105.998-106.359 1×106.1

NIK-1Dp 6.2184 4.1226 Y=2.10+0.33X 2.104NS 0.0830 108.125-109.711 1×108.9

N. bombycis 5.6593 4.9740 Y=0.36+0.82X 6.401NS 0.1068 105.526-105.856 1×105.6

PI: Post inoculation; NS: Non-significant.

109



NIK-1Pr 6.3301 4.7803 Y=-0.09+0.74X 2.600NS 0.1063 106.443-106.811 1×106.6

NIK-1Cc 6.0083 4.4530 Y=2.29+0.36X 8.728NS 0.0736 107.081-107.974 1×107.5

NIK-1Cpy 5.8194 4.9094 Y=0.70+0.72X 4.496NS 0.0953 105.767-106.122 1×105.9

NIK-1So 5.4504 4.9871 Y=2.80+0.92X 9.351NS 0.1178 105.312-105.616 1×105.4

NIK-1Dp 6.7380 4.6183 Y=-0.57+0.77X 8.716NS 0.1299 107.032-107.436 1×107.2

N. bombycis 5.4369 5.0390 Y=2.11+0.54X 4.452NS 0.0766 105.150-105.579 1×105.3

PI: Post inoculation; NS: Non-significant.

110

Table 2.17: Rate of spread of the microsporidian infection in a healthy colony of the silkworm, Bombyx mori L

Spread of infection Microsporidian

isolates No.of carrier larvae

introduced

Larval duration (D:H)

Larval weight (g) Dead Larvae (%)

Dead Pupae (%)

Moths infected (%)

Total spread (%)

1 24:00 33.4 1.0 (5.7) 1.0 (5.7) 19.7 (26.3) 21.7 (27.7) 3 24:00 34.1 3.0 (9.9) 1.0 (5.7) 21.7 (27.7) 25.7 (30.4) 5 24:00 33.9 5.7 (13.8) 2.3 (8.7) 22.3 (28.2) 30.3 (33.4)

NIK-1Pr

7 24:00 33.7 7.0 (15.3) 2.7 (9.4) 25.3 (30.2) 35.0 (36.2) 1 24:00 34.7 1.0 (5.7) 0.3 (3.1) 9.0 (17.4) 10.3 (18.7) 3 24:00 33.9 2.7 (9.4) 0.7 (4.8) 12.3 (20.5) 15.7 (23.3) 5 24:00 34.5 4.3 (11.9) 1.0 (5.7) 16.0 (23.6) 21.3 (27.5)

NIK-1Cc

7 24:00 34.5 5.7 (13.8) 1.7 (7.5) 17.7 (24.8) 25.0 (30.0) 1 24:00 33.4 3.7 (11.1) 0.7 (4.8) 30.7 (33.6) 35.0 (36.2) 3 24:00 34.6 5.7 (13.8) 1.7 (7.5) 33.0 (35.0) 40.3 (39.4) 5 24:00 34.8 7.0 (15.3) 3.0 (9.9) 35.7 (36.7) 45.7 (42.5)

NIK-1Cpy

7 24:00 33.8 9.0 (17.4) 3.7 (11.1) 37.7 (37.8) 50.3 (45.1) 1 24:00 34.3 3.3 (10.4) 0.7 (4.8) 23.0 (28.6) 27.0 (31.3) 3 24:00 34.5 4.7 (12.5) 1.3 (6.5) 25.7 (30.4) 31.7 (34.2) 5 24:00 34.4 6.3 (14.5) 2.7 (9.4) 29.7 (33.0) 38.7 (38.4)

NIK-1So

7 24:00 34.2 7.7 (16.1) 3.3 (10.4) 34.7 (36.1) 45.7 (42.5) 1 24:00 34.6 1.7 (7.5) 0.0 (0.0) 11.7 (20.0) 13.3 (21.4) 3 24:00 34.5 2.7 (9.4) 1.0 (5.7) 16.0 (23.6) 19.7 (26.3) 5 24:00 34.1 4.7 (12.5) 1.0 (5.7) 18.3 (25.3) 24.0 (29.3)

NIK-1Dp

7 24:00 34.2 7.0 (15.3) 1.0 (5.7) 19.3 (26.0) 27.3 (31.5) 1 24:12 33.8 4.3 (11.9) 2.7 (9.4) 40.0 (39.2) 47.0 (43.3) 3 24:12 34.3 6.7 (15.0) 3.7 (11.1) 43.0 (40.9) 53.3 (46.9) 5 24:12 34.1 7.7 (16.1) 4.7 (12.5) 45.0 (42.1) 57.3 (49.2)

Nosema bombycis

7 24:12 34.1 10.0(18.4) 5.0 (12.9) 59.0 (50.2) 74.0 (59.3) A CD at 5% -- -- (0.7) (0.8) (0.7) (0.8) B CD at 5% -- -- (0.6) (0.6) (0.5) (0.6)

S. E. ± -- 0.35 0.49 0.55 0.47 0.55 A × B

CD at 5% -- -- (1.4) (1.5) (1.3) (1.6) Values in parenthesis are angular transformed

111

Table 2.18: Mortality and infection level due to five different microsporidia in Multivoltine breeds of the silkworm, Bombyx mori L.

Mortality due to infection

(%) Microsporidian isolates

Silkworm breed

Larva Pupa

Moths infected (%)

Total Infection %

PM 12.3(20.5) 9.7(18.1) 34.7(36.1) 56.7(48.8) Nistari 15.3(23.0) 12.0(20.2) 33.3(35.2) 60.7(51.2) ND7 21.3(27.5) 18.0(25.1) 32.0(34.4) 71.3(57.6)

NIK-1Pr

NP1 18.3(25.3) 14.7(22.5) 32.3(34.6) 65.3(53.9) PM 11.3(19.6) 6.7(15.0) 22.7(28.4) 40.7(39.6)

Nistari 12.0(20.2) 8.7(17.1) 23.0(28.6) 43.7(41.3) ND7 18.3(25.3) 13.7(21.7) 24.7(29.8) 56.7(48.8)

NIK-1Cc

NP1 14.7(22.5) 10.3(18.7) 23.7(29.1) 48.7(44.2) PM 17.7(24.8) 7.7(16.1) 70.7(57.2) 96.0(78.4)

Nistari 20.3(26.7) 10.3(18.7) 67.3(55.1) 98.0(81.8) ND7 27.0(31.3) 17.0(24.3) 56.0(48.4) 100.0(90.0)

NIK-1Cpy

NP1 22.3(28.1) 13.0(21.1) 63.7(52.9) 99.0(84.2) PM 19.7(26.3) 8.7(17.1) 70.3(56.9) 98.7(83.4)

Nistari 22.3(28.1) 11.3(19.6) 66.3(54.5) 100.0(90.0) ND7 28.7(32.4) 18.7(25.6) 52.7(46.5) 100.0(90.0)

NIK-1So

NP1 25.3(30.2) 15.0(22.8) 59.7(50.6) 100.0(90.0) PM 12.0(20.2) 8.7(17.1) 22.3(28.1) 43.0(40.9)

Nistari 14.0(21.9) 9.7(18.1) 22.7(28.4) 46.4(42.9) ND7 20.7(27.0) 17.3(24.5) 22.3(28.1) 60.3(50.9)

NIK-1Dp

NP1 18.1(25.2) 13.3(21.4) 22.7(28.4) 54.2(47.4) PM 40.7(39.6) 11.3(19.6) 48.0(43.8) 100.0(90.0)

Nistari 48.0(43.8) 13.3(21.4) 38.7(38.4) 100.0(90.0) ND7 54.3(47.4) 21.7(27.7) 24.0(29.3) 100.0(90.0)

Nosema bombycis

NP1 50.3(45.1) 17.7(24.8) 32.0(34.4) 100.0(90.0) PM 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0)

Nistari 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) ND7 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0)

Normal control

NP1 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0)

A CD at 5% (0.9) (0.4) (0.4) (0.3)

B CD at 5% (0.6) (0.3) (0.3) (0.2)

S. E. ± 0.61 0.31 0.27 0.22 (A x B)

CD at 5% (1.7) (0.9) (0.7) (0.6) Values in parenthesis are angular transformed

112

Table 2.19: Effect of infection by the isolated microsporidia on larval and cocoon characters of Multivoltine breeds of the silkworm, Bombyx mori L.

Microsporidian isolates

Silkworm breed

Larval weight

(g)

Larval Duration (D: H)

Cocoon weight

(g)

Shell weight

(g) SR %

PM 19.30 28:00 0.760 0.103 13.54 Nistari 20.08 25:00 0.865 0.119 13.75 ND7 36.41 26:00 1.193 0.219 18.36

NIK-1Pr

NP1 33.83 25:00 1.194 0.210 17.55 PM 20.18 28:00 0.788 0.109 13.87

Nistari 21.31 25:00 0.875 0.122 13.97 ND7 37.48 26:00 1.197 0.222 18.57

NIK-1Cc

NP1 34.03 25:00 1.202 0.216 17.94 PM 18.96 28:00 0.752 0.101 13.38

Nistari 19.74 25:00 0.855 0.117 13.64 ND7 36.27 26:00 1.182 0.216 18.25

NIK-1Cpy

NP1 33.74 25:00 1.191 0.208 17.43 PM 18.72 28:00 0.750 0.100 13.32

Nistari 19.25 25:00 0.850 0.115 13.56 ND7 36.05 26:00 1.179 0.214 18.17

NIK-1So

NP1 33.71 25:00 1.184 0.206 17.39 PM 19.91 28:00 0.781 0.108 13.79

Nistari 20.58 25:00 0.870 0.121 13.91 ND7 36.95 26:00 1.184 0.219 18.46

NIK-1Dp

NP1 33.90 25:00 1.197 0.212 17.71 PM 16.18 29:00 0.730 0.085 11.64

Nistari 18.11 25:00 0.825 0.105 12.72 ND7 33.12 26:00 1.170 0.207 17.69

Nosema bombycis

NP1 31.49 25:00 1.173 0.193 16.45 PM 22.02 26:00 0.805 0.115 14.24

Nistari 24.41 23:00 0.911 0.133 14.64 ND7 38.92 25:00 1.220 0.246 20.13

Normal control

NP1 36.62 24:00 1.221 0.227 18.55

A CD at 5%

0.02 -- 0.002 0.001 0.04

B CD at 5%

0.02 -- 0.002 0.001 0.05

S. E. ± 0.02 -- 0.001 0.001 0.05 A×B CD at

5% 0.05 -- 0.004 0.002 0.13

113

Table 2.20: Mortality and infection level due to five different microsporidia in Bivoltine breeds of the silkworm, Bombyx mori L.

Mortality due to infection

(%) Microsporidian

isolates Silkworm

breed Larva Pupa

Moths infected (%)

Total infection %

CSR2 49.3(44.6) 14.3(22.2) 21.0(27.2) 84.7(66.9) CSR4 46.3(42.8) 11.7(20.0) 23.3(28.8) 81.3(64.3) CSR18 35.3(36.4) 6.3(14.5) 27.3(31.5) 69.0(56.1)

NIK- 1Pr

CSR19 37.3(37.6) 7.7(16.1) 27.3(31.5) 72.3(58.2) CSR2 33.3(35.2) 9.7(18.1) 20.3(26.7) 63.3(52.7) CSR4 30.3(33.4) 8.3(16.7) 20.7(27.0) 59.3(50.3) CSR18 20.7(27.0) 4.3(11.9) 20.7(27.0) 45.7(42.5)

NIK- 1Cc

CSR19 23.0(28.6) 5.3(13.3) 21.7(27.7) 50.0(45.0) CSR2 57.3(49.2) 13.0(21.1) 29.7(33.0) 100.0(90.0) CSR4 54.0(47.3) 10.7(19.1) 35.3(36.4) 100.0(90.0) CSR18 42.3(40.5) 7.0(15.3) 45.7(42.5) 95.0(77.0)

NIK- 1Cpy

CSR19 46.0(42.7) 8.3(16.7) 42.7(40.8) 97.0(80.0) CSR2 60.0(50.7) 10.3(18.7) 29.7(33.0) 100.0(90.0) CSR4 56.3(48.6) 9.7(18.1) 34.0(35.6) 100.0(90.0) CSR18 45.7(42.5) 8.0(16.4) 43.3(41.1) 97.0(80.0)

NIK- 1So

CSR19 48.7(44.2) 8.7(17.1) 40.0(39.2) 97.3(80.5) CSR2 34.3(35.8) 12.3(20.5) 21.0(27.2) 67.7(55.3) CSR4 31.0(33.8) 10.3(18.7) 21.3(27.5) 62.7(52.3) CSR18 21.3(27.5) 7.3(15.6) 22.3(28.1) 51.0(45.5)

NIK- 1Dp

CSR19 24.3(29.5) 8.7(17.1) 22.0(27.9) 55.0(47.8) CSR2 95.3(77.4) 1.7(7.5) 3.0(9.9) 100.0(90.0) CSR4 93.3(75.0) 2.0(8.1) 4.7(12.5) 100.0(90.0) CSR18 85.3(67.4) 3.3(10.4) 11.3(19.6) 100.0(90.0)

Nosema bombycis

CSR19 87.7(69.4) 2.7(9.4) 9.7(18.1) 100.0(90.0) CSR2 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) CSR4 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) CSR18 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0)

Normal control

CSR19 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) A CD at 5% (0.5) (0.5) (0.4) (0.3)

B CD at 5% (0.4) (0.4) (0.3) (0.3)

S. E. ± 0.33 0.34 0.30 0.25 (A x B)


114

Table 2.21: Effect of infection by the isolated microsporidia on larval and cocoon characters of Bivoltine breeds of the silkworm, Bombyx mori L.

Microsporidian isolates

Silkworm breed

Larval weight

(g)

Larval Duration (D: H)

Cocoon weight

(g)

Shell weight

(g) SR %

CSR2 36.19 25:00 1.483 0.295 19.86 CSR4 35.23 24:00 1.537 0.300 19.52 CSR18 33.00 24:00 1.529 0.298 19.46

NIK-1Pr

CSR19 31.35 24:00 1.473 0.276 18.76 CSR2 37.85 25:00 1.489 0.300 20.14 CSR4 36.37 24:00 1.540 0.309 20.06 CSR18 34.07 24:00 1.533 0.305 19.89

NIK-1Cc

CSR19 32.01 24:00 1.480 0.289 19.52 CSR2 35.47 25:00 1.480 0.292 19.70 CSR4 34.81 24:00 1.535 0.297 19.37 CSR18 32.50 24:00 1.527 0.294 19.25

NIK-1Cpy

CSR19 30.72 24:00 1.469 0.274 18.62 CSR2 35.18 25:00 1.478 0.290 19.63 CSR4 34.22 24:00 1.532 0.295 19.27 CSR18 32.14 24:00 1.524 0.292 19.18

NIK-1So

CSR19 30.21 24:00 1.465 0.271 18.47 CSR2 37.05 25:00 1.487 0.297 19.97 CSR4 35.87 24:00 1.539 0.304 19.76 CSR18 33.89 24:00 1.532 0.301 19.62

NIK-1Dp

CSR19 31.90 24:00 1.476 0.280 18.98 CSR2 30.75 25:00 1.435 0.255 17.77 CSR4 28.32 25:00 1.495 0.260 17.39 CSR18 27.21 24:00 1.490 0.258 17.31

Nosema bombycis

CSR19 25.57 24:00 1.431 0.242 16.91 CSR2 39.21 24:00 1.552 0.343 22.11 CSR4 38.02 23:00 1.600 0.352 22.00 CSR18 36.01 23:00 1.570 0.340 21.67

Normal control

CSR19 34.02 23:00 1.520 0.320 21.04 A CD at 5% 0.02 -- 0.001 0.001 0.05 B CD at 5% 0.03 -- 0.001 0.001 0.06

S. E. ± 0.02 -- 0.00 0.00 0.05 A×B

CD at 5% 0.06 -- 0.003 0.002 0.14

115

Table 2.22: Mortality and infection level due to five different microsporidia in Cross breeds of the silkworm, Bombyx mori L.

Mortality due to

infection (%) Microsporidian isolates

Silkworm breed

Larva Pupa

Moths infected

(%)

Total infection %

PM × CSR2 11.0(19.3) 7.3(15.7) 33.7(35.5) 52.0(46.1) NIK-1Pr

CSR2 × CSR4 42.3(40.5) 10.0(18.4) 25.3(30.2) 77.7(61.8)

PM × CSR2 6.7(15.0) 4.7(12.5) 21.0(27.2) 32.3(34.6) NIK-1Cc

CSR2 × CSR4 26.3(30.8) 6.3(14.5) 21.7(27.7) 54.3(47.4)

PM × CSR2 14.7(22.5) 6.0(14.2) 72.3(58.2) 93.0(74.6) NIK-1Cpy

CSR2 × CSR4 50.3(45.1) 9.7(18.1) 38.0(38.0) 98.0(81.8)

PM × CSR2 16.3(23.8) 8.3(16.7) 72.0(58.0) 96.7(79.5) NIK-1So

CSR2 × CSR4 52.7(46.5) 9.3(17.7) 37.0(37.4) 99.0(84.2)

PM × CSR2 10.3(18.7) 7.7(16.1) 20.7(27.0) 38.7(38.4) NIK-1Dp

CSR2 × CSR4 27.3(31.5) 9.7(18.1) 21.7(27.7) 58.7(50.0)

PM × CSR2 38.7(38.4) 9.7(18.1) 50.7(45.4) 99.0(84.2) Nosema bombycis CSR2 × CSR4 90.3(71.8) 1.3(6.5) 8.3(16.7) 100.0(90.0)

PM × CSR2 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0) Normal control

CSR2 × CSR4 0.0(0.0) 0.0(0.0) 0.0(0.0) 0.0(0.0)

A CD at 5% (0.6) (0.7) (0.5) (0.5)

B CD at 5% (0.3) -- (0.2) (0.3)

S. E. ± 0.29 0.36 0.22 0.24 (A x B)


116

Table 2.23: Effect of infection by the isolated microsporidia on larval and cocoon characters of Cross breeds of the silkworm, Bombyx mori L.

Microspo-ridian

isolates Silkworm hybrid

Larval weight

(g)

Larval duration (D: H)

Cocoon weight

(g)

Shell weight

(g) SR %

PM × CSR2 37.11 26:00 1.518 0.254 16.72 NIK-1Pr

CSR2 × CSR4 43.97 24:00 1.674 0.338 20.17

PM × CSR2 37.20 26:00 1.534 0.259 16.86 NIK-1Cc

CSR2 × CSR4 44.01 24:00 1.681 0.345 20.50

PM × CSR2 37.08 26:00 1.514 0.251 16.60 NIK-1Cpy

CSR2 × CSR4 43.94 24:00 1.671 0.334 19.98

PM × CSR2 37.04 26:00 1.510 0.249 16.51 NIK-1So

CSR2 × CSR4 43.89 24:00 1.667 0.332 19.89

PM × CSR2 37.16 26:00 1.530 0.257 16.77 NIK-1Dp

CSR2 × CSR4 43.99 24:00 1.677 0.341 20.31

PM × CSR2 37.01 26:00 1.504 0.247 16.39 Nosema bombycis CSR2 × CSR4 43.42 25:00 1.646 0.311 18.89

PM × CSR2 38.21 25:00 1.604 0.284 17.72 Normal control CSR2 × CSR4 45.02 23:00 1.721 0.382 22.21

A CD at 5% 0.02 -- 0.002 0.001 0.07

B CD at 5% 0.01 -- 0.002 0.001 0.06

SE ± 0.01 -- 0.001 0.001 0.05 A × B

CD at 5 % 0.04 -- 0.004 0.003 0.14

117

Midgut Fat bodies

Trachea Silk gland

Malpighian tubules Gonad

Fig. 2.1: Microphotographs of different tissues of silkworm infected with the

microsporidian, NIK-1Pr (600 X) [Arrows indicate the spores]

118

Midgut Fat bodies

Trachea Silk gland

Malpighian tubules Gonad (No infection)


microsporidian, NIK-1Cc (600 X)

119

Midgut Fat bodies

Trachea Silk gland


Fig. 2.3: Microphotographs of different tissues of silkworm infected with the microsporidian, NIK-1Cpy (600 X)

120

Midgut Fat bodies

Trachea Silk gland

Malpighian tubule Gonad


microsporidian, NIK-1So (600 X)

121

Midgut Fat bodies

Trachea Silk gland



microsporidian, NIK-1Dp (600 X)

122

Midgut Fat bodies

Trachea Silk gland


Fig. 2.6: Microphotographs of different tissues of silkworm infected with the standard

microsporidian strain, Nosema bombycis (600 X)

123

Midgut Fat bodies

Trachea Silk gland


Fig. 2.7: Microphotographs of different tissues of healthy silkworm larvae, without any

infection (600 X)

124

0

1

2

3

4

5

6

0 3 6 9Log concentration

Pro

bit o

f mor

talit

y

Y = -1.65+ 0.82XLC50 = 10 8.110

Microsporidian NIK-1Pr

0

1

2

3

4

5

6

0 3 6 9

Log concentration

Pro

bit o

f mor

talit

y

Microsporidian NIK-1Cc

Y = 2.22 + 0.19XLC50 = 1014.614

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y

Microsporidian NIK-1Cpy

Y = 0.22 + 0.63XLC50 = 107.594

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y

Microsporidian NIK-1So

Y = 0.30 + 0.65XLC50 = 107.267

0

1

2

3

4

5

6

0 3 6 9

Log concentration

Pro

bit o

f mor

talit

y

Microsporidian NIK- 1Dp

Y = 1.91 + 0.27XLC50 = 1011.667

0

1

2

3

4

5

6

0 3 6 9

Log concentration

Pro

bit o

f mor

talit

y

Microsporidian N. bombycis

Y = 0.54 + 0.64XLC50 = 106.983

Figure 2.8: Probit graphs for mortality due to isolated microsporidia and N.bombycis on

12th day PI

125

0

1

2

3

4

5

6

0 3 6 9

Log concentration

Pro

bit o

f mor

talit

y


Y = -0.04+ 0.67XLC50 = 107.528

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y

Microsporidian NIK-1 Cc

Y = 2.24 + 0.29XLC50 = 109.506

0

1

2

3

4

5

6

0 3 6 9

Log concentration

Pro

bit o

f mor

talit

y


Y = 0.71 + 0.64XLC50 = 106.730

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y


Y = 0.58 + 0.72XLC50 =106.179

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y

Microsporidian NIK-1Dp

Y = 2.10 + 0.33XLC50 = 108.918

0

1

2

3

4

5

6

0 3 6 9

Log concentration

Pro

bit o

f mor

talit

y


Y = 0.36 + 0.82XLC50 = 105.691

Figure 2.9: Probit graphs for mortality due to isolated microsporidia and N.bombycis on

15th day PI

126

0

1

2

3

4

5

6

7


Pro

bit o

f mor

talit

y


Y = 0.10+ 0.74XLC50 = 106.627

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y

Microsporidian NIK-1Cc

Y = 2.29 + 0.36XLC50 = 107.528

0

1

2

3

4

5

6

7


Pro

bit o

f mor

talit

y


Y = 0.70 + 0.72XLC50 = 105.945

0

1

2

3

4

5

6

7

8


Pro

bit o

f mor

talit

y


Y = 2.84 + 0.92XLC50 = 105.464

0

1

2

3

4

5

6


Pro

bit o

f mor

talit

y

Microsporidian NIK-1Dp

Y = -0.57 + 0.77XLC50 = 107.234

0

1

2

3

4

5

6

7


Pro

bit o

f mor

talit

y


Y = 2.11 + 0.54XLC50 = 105.364

Figure 2.10: Probit graphs for mortality due to isolated microsporidia and N.bombycis on 21st day PI

127

35.0

30.3

25.7

21.7

0

5

10

15

20

25

30

35

40

1 3 5 7

Number of carrier larvae introduced

To

tal s

pre

ad

%

Fig. 2.11: Total spread of microsporidiosis in healthy colonies of silkworm by the microsporidian, NIK-1Pr

25.0

21.3

15.7

10.3

0

5

10

15

20

25

30

1 3 5 7Number of carrier larvae introduced

To

tal s

pre

ad

%

Fig. 2.12: Total spread of microsporidiosis in healthy colonies of silkworm by the microsporidian, NIK-1Cc

128

35.0

40.345.7

50.3

0

10

20

30

40

50

60


Tot

al s

prea

d %

Fig. 2.13: Total spread of microsporidiosis in healthy colonies of silkworm by the microsporidian, NIK-1Cpy

45.7

38.7

31.7

27.0

0

10

20

30

40

50

1 3 5 7

Number of carrier larvae introduced

To

tal s

prea

d %

Fig. 2.14: Total spread of microsporidiosis in healthy colonies of silkworm by the microsporidian, NIK-1So

129

27.3

24.0

19.7

13.3

0

5

10

15

20

25

30


To

tal s

pre

ad

%

Fig. 2.15: Total spread of microsporidiosis in healthy colonies of silkworm by the microsporidian, NIK-1Dp

74.0

57.353.3

47.0

0

10

20

30

40

50

60

70

80


To

tal s

pre

ad

%

Fig. 2.16: Total spread of microsporidiosis in healthy colonies of silkworm by the standard microsporidian strain, Nosema bombycis

130

65.371.3

60.756.7

0

10

20

30

40

50

60

70

80

PM Nistari ND7 NP1Multivoltine breeds

To

tal i

nfe

ctio

n %

Fig. 2.17: Total infection % in Multivoltine breeds by the microsporidian NIK-1Pr

48.7

56.7

43.740.7

0

10

20

30

40

50

60


To

tal i

nfe

ctio

n %

Fig. 2.18: Total infection % in Multivoltine breeds by the microsporidian NIK-1Cc

131

96.0 98.0 100.0 99.0

0

15

30

45

60

75

90

105


Tot

al in

fect

ion

%

Fig. 2.19: Total infection % in Multivoltine breeds by the microsporidian NIK-1Cpy

100.0100.0100.098.7

0

15

30

45

60

75

90

105


To

tal i

nfe

ctio

n %

Fig. 2.20: Total infection % in Multivoltine breeds by the microsporidian NIK-1So

132

54.2

60.3

46.443.0

0

10

20

30

40

50

60

70


To

tal i

nfe

ctio

n %

Fig. 2.21: Total infection % in Multivoltine breeds by the microsporidian NIK-1Dp

100.0100.0100.0100.0

0

15

30

45

60

75

90

105

PM Nistari ND7 NP1Mult ivolt ine breeds

To

tal i

nfe

ctio

n %

Fig. 2.22: Total infection % in Multivoltine breeds by the standard microsporidian strain, Nosema bombycis

133

5.39

8.79

6.07

4.91

0

5

10

PM Nistari ND7 NP1

Multivoltine breeds

% R

ed

uct

ion

in S

R %

Fig. 2.23: Percentage reduction in SR% of Multivoltine breeds due to the microsporidian NIK-1Pr compared to healthy control

3.28

7.74

4.57

2.59

0

5

10


% R

ed

uct

ion

in S

R%

Fig. 2.24: Percentage reduction in SR% of Multivoltine breeds due to the microsporidian NIK-1Cc compared to healthy control

134

6.03

9.33

6.836.03

0

5

10


% R

educt

ion in

SR

%

Fig. 2.25: Percentage reduction in SR% of Multivoltine breeds due to the microsporidian NIK-1Cpy compared to healthy control

6.25

9.73

7.376.46

0

5

10

15

PM Nistari ND7 NP1

Multivoltine breeds

% R

educ

tion

in S

R %

Fig. 2.26: Percentage reduction in SR% of Multivoltine breeds due to the microsporidian NIK-1So compared to healthy control

135

4.52

8.29

4.98

3.16

0

5

10

PM Nistari ND7 NP1

Multivoltine breeds

% R

ed

uct

ion

in S

R %

Fig. 2.27: Percentage reduction in SR% of Multivoltine breeds due to the microsporidian NIK-1Dp compared to healthy control

11.3212.12

13.11

18.25

0

5

10

15

20


% R

ed

uct

ion

in S

R %

Fig. 2.28: Percentage reduction in SR% of Multivoltine breeds due to the standard microsporidian strain, N.bombycis compared to healthy control

136

72.369.0

81.384.7

0

10

20

30

40

50

60

70

80

90

CSR2 CSR4 CSR18 CSR19Bivoltine breeds

To

tal i

nfe

ctio

n %

Fig. 2.29: Total infection % in Bivoltine breeds by the microsporidian NIK-1Pr

50.045.7

59.363.3

0

10

20

30

40

50

60

70

CSR2 CSR4 CSR18 CSR19

Bivoltine breeds

Tot

al in

fect

ion

%

Fig. 2.30: Total infection % in Bivoltine breeds by the microsporidian NIK-1Cc

137

97.095.0100.0100.0

0

15

30

45

60

75

90

105


To

tal i

nfe

ctio

n %

Fig. 2.31: Total infection % in Bivoltine breeds by the microsporidian NIK-1Cpy

97.397.0100.0100.0

0

15

30

45

60

75

90

105


To

tal i

nfe

ctio

n %

Fig. 2.32: Total infection % in Bivoltine breeds by the microsporidian NIK-1So

138

55.051.0

62.767.7

0

10

20

30

40

50

60

70

80


To

tal i

nfe

ctio

n %

Fig. 2.33: Total infection % in Bivoltine breeds by the microsporidian NIK-1Dp

100.0100.0100.0100.0

0

15

30

45

60

75

90

105


To

tal i

nfe

ctio

n %

Fig. 2.34: Total infection % in Bivoltine breeds by the standard microsporidian strain, Nosema bombycis

139

10.8310.1911.27

10.17

0

5

10

15


% R

ed

uct

ion

in S

R %

Fig. 2.35: Percentage reduction in SR% of Bivoltine breeds due to the microsporidian NIK-1Pr compared to healthy control

7.22

8.218.818.91

0

5

10


% R

ed

uct

ion

in S

R %

Fig. 2.36: Percentage reduction in SR% of Bivoltine breeds due to the microsporidian NIK-1Cc compared to healthy control

140

11.5011.1611.95

10.90

0

5

10

15


% R

ed

uct

ion

in S

R %

Fig. 2.37: Percentage reduction in SR% of Bivoltine breeds due to the microsporidian NIK-1Cpy compared to healthy control

12.2111.49

12.4011.21

0

5

10

15


% R

ed

uct

ion

in S

R %

Fig. 2.38: Percentage reduction in SR% of Bivoltine breeds due to the microsporidian NIK-1So compared to healthy control

141

9.799.4610.18

9.67

0

5

10

15


Bivoltine breeds

% R

ed

uct

ion

in S

R %

Fig. 2.39: Percentage reduction in SR% of Bivoltine breeds due to the microsporidian NIK-1Dp compared to healthy control

19.6220.1120.9519.62

0

5

10

15

20

25


Bivoltine breeds

% R

ed

uct

ion

in S

R %

Fig. 2.40: Percentage reduction in SR% of Bivoltine breeds due to the standard microsporidian strain, Nosema bombycis compared to healthy control

142

77.7

52.0

0

10

20

30

40

50

60

70

80

90

PM×CSR2 CSR2×CSR4Cross breeds

Tot

al in

fect

ion

%

Fig. 2.41: Total infection % in Cross breeds by the microsporidian NIK-1Pr

54.3

32.3

0

10

20

30

40

50

60


To

tal i

nfe

ctio

n %

Fig. 2.42: Total infection % in Cross breeds by the microsporidian NIK-1Cc

143

93.098.0

0

15

30

45

60

75

90

105


Tot

al in

fect

ion

%

Fig. 2.43: Total infection % in Cross breeds by the microsporidian NIK-1Cpy

99.096.7

0

15

30

45

60

75

90

105

PM×CSR2 CSR2×CSR4

Cross breeds

To

tal i

nfe

ctio

n %

Fig. 2.44: Total infection % in Cross breeds by the microsporidian NIK-1So

144

38.7

58.7

0

10

20

30

40

50

60

70

PM×CSR2 CSR2×CSR4

Cross breeds

To

tal i

nfec

tion

%

Fig. 2.45: Total infection % in Cross breeds by the microsporidian NIK-1Dp

100.099.0

0

15

30

45

60

75

90

105


To

tal i

nfe

ctio

n %

Fig. 2.46: Total infection % in Cross breeds by the standard microsporidian strain, Nosema bombycis

145

9.18

5.64

0

5

10


% R

ed

uct

ion

in S

R %

Fig. 2.47: Percentage reduction in SR% of Cross breeds due to the microsporidian NIK-1Pr compared to healthy control

7.69

4.85

0

5

10

PM×CSR2 CSR2×CSR4

Cross breeds

% R

ed

uct

ion

in S

R %

Fig. 2.48: Percentage reduction in SR% of Cross breeds due to the microsporidian NIK-1Cc compared to healthy control

146

10.04

6.32

0

5

10

15


% R

ed

uct

ion

in S

R %

Fig. 2.49: Percentage reduction in SR% of Cross breeds due to the microsporidian NIK-1Cpy compared to healthy control

10.44

6.82

0

5

10

15

PM×CSR2 CSR2×CSR4

Cross breeds

% R

ed

uct

ion

in S

R %

Fig. 2.50: Percentage reduction in SR% of Cross breeds due to the microsporidian NIK-1So compared to healthy control

147

8.55

5.36

0

5

10


% R

ed

uct

ion

in S

R %

Fig. 2.51: Percentage reduction in SR% of Cross breeds due to the microsporidian NIK-1Dp compared to healthy control

14.94

7.50

0

5

10

15

20


% R

ed

uct

ion

in S

R %

Fig. 2.52: Percentage reduction in SR% of Cross breeds due to the standard microsporidian strain, Nosema bombycis compared to healthy control

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