Pebrine is a deadliest disease of mulberry silkworm, B. mori L. caused by the pathogen, N.bombycis Nageli, (Microsporidia: Nosematidae). It is transmitted primarily through eggs i.e., transovarian transmission and through the feeding of contaminated leaf, rearing tray, rearing bed, layings, as well as cross-transmission from alternate host secondarily i.e., transovum transmission (Chakrabarty et al., 2013a). Each oval spore measuring 3 - 4 µm × 1.5 - 2 µm includes a coiled polar filament that may be 100 µm in length. The disease is spread transovarially by environmental spore and secondarily by primary spores. Ultra-structures of the two type of spores are entirely different, primary spore contain short polar tube (ST) with thin wall (< 200 nm); whereas, the environmental spore contain long polar tube (LT) with thick wall (> 200 nm). Pasteur (1870) was the first to detect the transmission of pebrine through the eggs i.e., transovarian transmission and he advised for examination of mother moth. It is the standard method followed to control the disease throughout the country. Transovarian transmission has been reported to be sole mechanism by which transmission of the parasites occurs (Kellen et al., 1965, Chapman et al., 1966). It is reported that transmission of microsporidia horizontally by feeding spores produced in male larvae back to larval hosts have been unsuccessful (Kellen et al., 1965) as the size of the spore is larger than the sperm. However, transovum transmission of spore through external surface of genital organ of heavily infected male moths can’t be rule out. However, such type of secondary source of contamination of N.bombycis is lacking in the literature. Though, some preliminary studies on veneral transmission of spore have been done by some workers (Patil, 1993). However, we identified that N.bombycis is capable to specify gender of the Bombyx mori and multiplied in male very quickly to spread the infection through transovum transmission (Chakrabarty et al., 2013a).
Therefore, role of transovum transmission of pebrine spore through accessory sex organs / external body surface of male moths in successive generations for transmission of disease was undertaken to find out the sole mechanism of outbreak of disease.
One hundred live cocoons were collected from one farmer’s house at Barunighata village, Birbhum district, West Bengal, India. Fifty cocoons were subjected for isolation of N. bombycis, pebrine spore. Spores were isolated from live infected pupae and purified by centrifugation at 3000 rpm for 10 min following new method of pebrine isolation (Chakrabarty et.al, 2013b). After isolation, spores were suspended in 0.85% NaCl and stored at 4℃. Spores were counted using a Neubauer haemocytometer under light microscope (x 600) and determined the inoculum concentration following standard method (Undeen, 1997) and used as stock solution. Fresh spores with 3.0-4.0 × 106 spores. mL-1 concentration were inoculated (i.e., artificial infection) to 4th stage 1st d larvae (Race: M Con1). Other fifty cocoons were allowed for moth emergence in ambient condition (i.e., natural infection). Whole body tissues of both male and female moths were examined under light microscope for detection of pebrine spores after coupling for male moth and oviposition for female moth. Eggs were incubated properly maintaining temperature and humidity as per standard procedure and allowed for hatching in normal condition. Rearing was conducted as per standard procedure (1st generation). Again, eggs were prepared utilizing moths generated from 1st rearing and rearing of 2nd generation was conducted following previous procedure i.e., 1st generation.
Rearing of 2nd generation were conducted with fifty larvae (Race: Nistari) of 1st brushing (T1) and fifty larvae of 2nd brushing (T2) when eggs were prepared utilizing moths recovered from natural infection of 1st generation. Rearings of 2nd generation were also conducted with fifty larvae of 1st brushing (T3) and fifty larvae (Race: Nistari) of 2nd brushing (T4) when eggs were prepared utilizing moths recovered from artificial infection of 1st generation.
Rearing of 3rd generation was conducted with 500 larvae when eggs were prepared utilizing moths recovered from natural infection of 2nd generation (T2).
Rearing of 4th generation was conducted with 1000 larvae when eggs were prepared utilizing moths recovered from natural infection of 3rd generation (T2).
All the procedure was same as Rearing 2nd, 3rd and 4th generations. However, these experiments were conducted in two separate batches, one with protection using existing disease management systems, using ‘Labex’ as bed disinfectant and ‘5% Bleaching powder’ as room disinfectant and other with new disease management system using ‘Sericillin’ as bed disinfectant and ‘fumigant chemicals’ as room disinfectant.
Here, we have crossed the fully infected male moths (1.0-1.2 × 107 spores.mL-1) recovered from rearing 3rd generations with healthy female moths. We have continued the generations with recording survival percentage and intensity of infection.
Nosema could produce two spores from sporont (Ishiwara, 1969). Sporont of N.bombycis usually produced two sporoblast (Lai and Canning, 1983). The first populations of spores mainly cause the spread of the parasite in the epithelium. The second population of spore is formed later on for adapting to survive outside the host (Graaf et al., 1994). Early spores and environmental spores are immature and variants of the same spore type, normally occurs in different tissues in the host (Larson, 1999).
In rearing of 1st generation of infected silkworm (experiment-I) , all the larvae were died when the larvae were artificially infected with same inoculums concentration of pebrine spore harvested from the moths collected from the field (3.0-4.0 × 106 spores. mL-1). However, the larvae were survived harbouring the same concentration of pebrine spore harvested from the moths collected from the field (3.0-4.0 × 106 spores.mL-1) and larvae completed the life cycle though survival percentage were decreased to 62.3% where intensity of spore were remain almost same concentrations and constant (1.0 × 107 spores. mL-1) for all larval stages, pupal stage and moth (Table 1 and 2). Some of the new factor is responsible in the evolution of pathogens as well as variability in host specificity so that their multiplication rate and virulency are restricted in the same host and host of the same origin are only survived. Alternately, artificially infected larvae were died due to loosing the originality of host specificity.
In rearing of 2nd, 3rd and 4th generation of infected silkworm (experiment-II), all artificially infected larvae were died in 1st generation. But larvae were died in 4th generation when silkworm rearing was conducted in natural condition. In this case, intensity of infection (3.0-4.0 × 109 spores.mL-1) was increased and survival percentage was decreased till 3rd generation and at last all larvae were died in 4th generation. Two contrasting results were observed in this experiment. Intensity of infection was more in naturally infected 1st brushing larvae (3.0-4.0 × 109 spores.mL-1) whereas, it is more in 2nd brushing larvae for artificial infection (1.0 × 108 spores.mL-1) before their death. Alternately, survival percentage was more in 2nd brushing larvae (ERR~ 22.32 %) infected naturally whereas, it is more in 1st brushing larvae infected artificially (ERR ~ 17.34 %). It was very much interesting to observe that all the larvae were survived in 2nd generation when the larvae contain full of pebrine spore (3.0- 4.0 × 107 spores.mL-1) when larvae infected naturally and finally, all larvae were died in 3rd generation before resume to 3rd stage and contain full of pebrine spore (3.0-4.0 × 109 spores.mL-1) (Table 3).
In rearing of 2nd, 3rd and 4th generation of infected silkworm with protection (experiment-III), all the naturally infected larvae were died in 4th generation when rearing was conducted in natural condition and artificially infected larvae were died in 1st generation with existing disease management system. But the survival percentage were increased in all generations when rearing was conducted with new system of management. It was surprised that more than 98% larvae were survived during 4th generation when rearing was conducted with new system of management and no spore was observed in moth under microscope (Table 4).
In rearing of 3rd generation of infected male with healthy male (experiment-IV), when artificially infected male moth were mated with healthy female then all offspring were died resume from 1st moult at 6th generation and develop pebrine disease though very less spore harvest (3.0-10.0×105 spores.mL-1) was recorded. We have not recorded any spore till 5th generation. Besides, all the pre cocoon and post cocoon parameters at 5th generation revealed that the lot was healthy as well as robust considering Effective Rearing Rate (~75% ), single mature larval weight (~2.496 g), Shell % (~13.0), Filament Length (~271 m), Non-breakable Filament length (~261 m), Denier (1.91) Fitness Efficiency test i.e., Grainage performance (Table 5 and 6) . Besides, the lot performed better in all the favourable and nonfavourable seasons.
Time taken for establishment of the pathogen for completion of its life cycle and production of spores depends on inoculum load and other environmental factors (Steinhaus and Huges, 1949). We have observed in our previous study that spore harvest was more in male moth compared to that in female moth though the inoculum concentration, source of pathogen and the rearing were conducted in the same environment (Chakrabarty et al., 2013b). The physiological changes in the insect might have possibly influenced the developmental cycle of the parasite to switch on from a predominantly vegetative stage to sporogony, resulting in an increasing spore production. Spore production had reached a stationary phase and yielded similar amounts of spore of N. acridiophagus and N. cuneatum in larvae of Melanoplus asnguinipes after 20 d of inoculation with 104 and 106 spores compared to lower spore yields which were continued in spore multiplication stage when inoculated with 102 spores (Cali, 1970). If development is allowed to proceed to the stationary phase, multiplication of the parasite is greater with lower concentrations than with higher concentrations (Kawarabata and Ishihara, 1984). But there is an apparent increase in multiplication with increased concentrations during the exponential phase and the difference between the multiplications of spore with the high and low initial spore concentration is narrow. Multiplication of pathogens depends upon the age of silkworm, time and other indirect factors (Loubes, 1999). A particular concentration is effective for multiplication of spore and mortality (Graaf et al., 1994) and below that threshold level the concentrations do not cause any larval mortality (Larson, 1999). Therefore, the number of spores (intensity of infection) that a host can harbour and still function normally is important in determining the role of microsporidia as a parasite in nature (Graaf et al., 1994). We have also found in our previous study that spore production reached the stationary phase in female during pupal stage to moth stage where as it is continued in progress in male pupa to moth stage till the dead of the moth i.e., the time of harvest. It is an interesting phenomenon of N. bombycis which needs to instigate further for detailed understanding and the findings assume importance for taxonomic classification (Chakrabarty et al., 2013a).
From the study, it may be concluded that both male and female moths to be examined microscopically during hybrid laying preparation. We could observe only 4th generation from parental generation (P3) to commercial rearing in the field. As there is no chance for 6th generation study in the field, for that reason outbreak of pebrine disease from secondary transmission is not observed. However, we should take care where parental generation (P3, P2 and P1) is maintained to check the secondary transmission. Besides, new management system using ‘Sericillin’ as bed disinfectant with ‘fumigant chemicals’ as room disinfectant is required to be adopted, especially where parental generation is maintained i.e., seed production centre, to control the secondary transmission of pebrine disease.