Proc.Zool.Soc.India. 16 (1) : 85 - 94 : (2017)ISSN 0972 - 6683 : INDEXED AND ABSTRACTED 18

Received - 11.01.2017 Accepted - 10.04.2017

ABSTRACT

A microsporidum (M-Cfl) was isolated from Catopsilia florella caught from mulberry and vegetable fields in BBAU campus, Lucknow area. The spores were 4.09±0.03×2.19±0.02 μm in size and ovo-cylindrical in shape. In contrast, the spores of Nosema bombycis were oval in shape and were 3.32±0.02 μm in length, 1.97±0.01 in width. The bivoltine breed, CSR2×CSR4 of mulberry silkworm Bombyx mori L. was selected to the study the infectivity of M-Cfl microsporidian on silkworm larval and cocoon characters and it was found that M-Cfl spore is infective to silkworm but the intensity of infection was lower as compared to that of Nosema bombycis, the known pebrine causing agent of silkworm Bombyx mori L.

Key words : Cross infectivity, Microsporidia, Larva Cocoon, Silk worm.

INTRODUCTION

Pebrine is the most dreaded disease affecting the mulberry silkworm, Bombyx mori caused by the microsporidian Nosema bombycis Naegeli (Naegeli, 1857) which devastated the whole of silk industry in France and other countries during the 19th century. However, recent studies suggest that B. mori is not only infected by the Nosema sp. but also with microsporidians belonging to several other genera viz., Vairimorpha, Pleistophora, Endoreticulatus, Cystosporogenes, Orthosomella, Thelohania, Octosporea and Gurleya that have been reported from various insects (Abe and Fujiwara, 1979; Rao et al., 2004). Some of the lepidopteran insect species that are known to harbour microsporidia include Euproctis chrysorrhoea, Choristoneura fumiferana, Ostrinia nubilalis, Lymantria dispar, Diatraea saccharalis, Choristoneura pinus pinus, Operophtera brumata, Lacanobia oleracea, Plutella xylostella, Cnaphalocrocis medinalis, Pieris rapae, Pieris rapae, Crucivora and Pieris conidia sordid, Catopsilia crocale, Catopsilia pyranthe, Spilosoma oblique, Diaphania pulverulentalis, Spodoptera litura, Spodoptera exigua, Helicoverpa armigera etc. (Hylis et al., 2006; Sharma et al., 2003; Solter et al,. 2005; Solter et al., 2010; Simões et al., 2015; van Frankenhuyzen et al., 2011; Canning and Curry 2004; Tsai et al., 2003; Singh et al., 2008; Basir et al., 2011). Therefore, the perpetual incidence of microsporidian infection in silkworms other than N. bombycis may be due to various sources of secondary contamination including alternative hosts in and around mulberry elds (Kishore et al., 1994). Reports are also available on the cross infectivity of these microsporidia from insects to silkworm B. mori (Kishore et al., 1994 ; Sharma et al., 2003; Singh et al., 2008; Basir et al., 2011; Bhuvaneswari and Nath 2015a, 2015b). Presumably, insect feaces and scales containing microsporidian spores might stick to mulberry leaves and transmit the microsporidian infection to silkworm larvae (Kawarabata, 2003). In the present investigation, we have isolated microsporidian spores from African migrant buttery, Catopsilia florella Fabricius, 1775 and studied their morphology, infectivity and the impact of M-C microsporidian infection on the larval and cocoon parameters of silkworm B. mori L.

MATERIALS AND METHODS

The bivoltine breed, CSR2×CSR4 of mulberry silkworm B. mori L. was selected to the study the infectivity of M-C microsporidian on silkworm larval and cocoon characters. The third instar larvae of silkworm Bombyx mori were collected from the Rearing House of Babasaheb Bhimrao Ambedkar University, Lucknow.

Screening of Catopsilia orella for microsporidian infection

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MUKTA MAYEE KUMBHAR, KAMAL JAISWAL, SUMAN MISHRABabasaheb Bhimrao Ambedkar University, Lucknow-226025, U.P.

CROSS-INFECTIVITY OF MICROSPORIDIA (M-CFL) ISOLATED

FROM CATOPSILIA FLORELLA ON LARVAL AND COCOON

CHARACTERS OF THE SILKWORM, BOMBYX MORI L.

African migrant buttery, Fabricius, 1775 were collected from the in and around mulberry garden of Babasaheb Bhimrao Ambedkar University, Lucknow. The abdomen of each buttery was macerated individually in mortar pestle by adding sterilized distilled water. To check the presence of microsporidian infection a smear was prepared on the slide and viewed under a bright eld microscope. If the sample was found free of microsporidiian infection, the sample gets discarded whereas in case of positive infection, the crude homogenate ltered through triple layered muslin cloth followed by ltration through Whatmann lter paper. The suspensions were then centrifuged at 3000 rpm for 10 min. The spore pellet was percoll puried by gradient centrifugation as described by undeen and Alger (1971). The sediment obtained was suspended in minimal volume of sterilized distilled water and stored at -4º C.

The microsporidian isolates from buttery Catopsilia orella was designated as M-C based on its host name viz., M- Microsporidia and C- Catopsilia - orella. Similarly, the standard strain Nosema bombycis which is the pebrine causing agent of silkworm B.mori L. is designated as Nbo.

Preparation of different concentration M-C microsporidian inoculum: The spore concentration of the stock solution was estimated by following standard haemocytometer count method (Cantwell, 1970). Three different concentrations (1×105, 1×106, 1×107 Spores/ml) of M-C microsporidian spore and standard strain of Nosema bombycis spore suspension was obtained from the stock suspension of known spore concentration by serial dilution.

Infectivity of microsporidian infection on silkworm larva

Each inoculums dose i.e. 1 ×105, 1 ×106 and 1 ×107 spores/ml of both M-C and N. bombycis spore solution formed a treatment. The mulberry leaves were smeared with the respective spore suspension and fed to silkworms on day one of III instar (After II moult) larvae. For each treatment, two replications of 50 larvae each were maintained. A control batch of larvae fed on mulberry leaves smeared with distilled water was also maintained. Mortality rate was observed in all the doses after every 24 h up to the spinning and pebrine infection was also checked in the dead larvae under a phase contrast microscope. LC50 values were calculated by probit analysis (Finney, 1971).

The larval length, larval width and larval weight of the silkworm larva inoculated with M-C spores at a dose concentration of 1 ×107 spores/ml were recorded daily and compared one with batch inoculated with Nosema bombycis spores of the same concentration and another with control batch (reared without inoculation of any microsporidian pathogen). The data was statistically analyzed by one way ANOVA using IBM SPSS 20 Software.

Effect of microsporidian infection on cocoon characters

In the silkworm batches inoculated with C spores at a dose concentration of 1 ×107 spores/ml, the weight of the cocoon and that of the shell were recorded and the shell ratio % was calculated. The lament length of the cocoon was also recorded. The results were compared one with batch inoculated with Nosema bombycis of the same dose concentration (1 ×107 spores/ml) and another with control batch (reared without inoculation of any microsporidian pathogen). The data was statistically analyzed by one way ANOVA using IBM SPSS 20 Software.

RESULTS

External morphology of microsporidia

The fresh spores exihibit Brownian movement and were extremely refractile when viewed under phase contrast microscope. The morphological characteristics of the isolated M-C spores and Nosema bombycis spores are presented in Table 1 and Figure 1a and 1b. The microsporidian spore M-C, isolated from the Catopsilia florella buttery were ovo-cylindrical in shape and measured 4.09±0.03×2.19±0.02 μm in size. In contrast, the spores of N. bombycis were oval in shape and were 3.32±0.02 μm in length, 1.97±0.01 μm in width. Therefore, it is clear that M-C spore is different from that of N. bombycis strain in terms of both shape and size.

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Table 1: Morphological characterization of M-C microsporidian spores and Nosema bombycis spores

Figure 1 a): Light micrograph of M-C spores, b) Light micrograph of N. bombycis spores

Cross-infectivity of the microsporidia against B. mori

The infectivity of the microsporidian isolates M-C and Nbo were checked in B. mori and it was found that both the isolates were virulent to the lab host. At 15 days of post inoculation, the LC50 value of M-Cl against larval mortality of B. mori was calculated as 2.69×107 spores/ml whereas the LC50 value of Nosema bombycis (Nbo) against larval mortality of B. mori was calculated as 1.1×106 spores/ml (Table 2, Figure 2 and Figure 3). This indicates M-C spores has the ability to cause pebrine infection in B. mori but is less virulent than N bombycis.

Table 2: LC50 of M-C and Nosema bombycis to the silkworm, Bombyx mori L. (15 days PI)

Sl. No. Microsporidian isolates

Host Name Spore Shape Length (μm) Width (μm)

1 M-Cfl Catopsilia florella Ovo-cylindrical

4.09±0.03

2.19±0.02

2 Nosema bombycis (Nbo) Bombyx mori Oval 3.32±0.02 1.97±0.01

a b

ÇʼnśĂĊ▓ś■Ċ Batches

/ onc. ofMicrosporidia (Spores/ml)

[ ◘┼ Conc.

a śĂ■ [ ĂʼnōĂ▄ ▓◘ʼnĊĂ▄╜ĊŦ ŕĵ ś to microsporidiosis (%)

t ʼn◘Ľ╜Ċ ë Ă▄ĵ ś [ / 5 0Value (Spores/ml)

a -Cfl هو�و5يى وو ي 10×2.69 و

7

هو�و 6 ىىى يو ي

هو�و 7 ي هى يى ي

b ◘ℓś▓Ă bombycis

هو�و510×1.1 لآوى وو ي

6

هو�و 6 يى ىى ي و

هو�و7ي ي يي و ي

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Figure 2: Probit graphs for larval mortality due to M-C on Bombyx mori L. 15th day Post inoculation

Figure 3: Probit graphs for larval mortality due to N. bombycis on Bombyx mori L. 15th day Post inoculation

Effect of microsporidian infection larval growth of silkworm B. mori

The impact of infection of M-C and N. bombycis spores (dose of 1×107 spores/ml) on silkworm was determined by measuring the progressive increase in length, width and weight of the larva.

Larval length

The results with regard to the impact of infection caused by M-C and N. bombycis spores on the daily increase in larval length of silkworm (CSR2×CSR4 breed) is presented in Table 3 and Figure 4. The data revealed that there was no signicant reduction in larval length of silkworm was observed in the batches inoculated with M-c and Nosema bombycis upto 6th day of post inoculation., these microsporidia were not found pathogenic to the silkworm Bombyx mori L. However, from 7th day of PI onwards, the progressive increase in larval length was signicantly reduced in the batches inoculated with M-C (3.46 cm) and Nosema bombycis (33.32 cm) compared to the healthy control batches wherein the larval length was recorded as 3.62 cm on 7th day PI. The impact of pebrine infection on larval length was more pronounced with the progression of infection and on 15th day PI i.e. one day prior to the spinning, the larval length was recorded as 6.02 cm and 5.54 cm in the batches inoculated with M-C and N. bombycis respectively compared to healthy control batches wherein a larval length of 6.60 cm was recorded.

Table 3: Effect of M-C and N. bombycis (Nbo) infection on larval length of the silkworm, Bombyx mori L. (CSR2×CSR4 breed )

Microsporidian Isolates

Larval length (cm) Days post Inoculation (1× 107

Spores/ml inoculated batches)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

M-Cfl 1.80 ± 0.07

2.12 ± 0.05

2.24 ± 0.05

2.28 ± 0.11

2.36 ± 0.05

3.14 ± 0.05

3.46 ± 0.05*

3.52 ± 0.04*

3.64 ± 0.05*

3.66 ± 0.08*

3.70 ± 0.07*

4.66 ± 0.05*

5.10 ± 0.07*

5.70 ± 0.07*

6.02 ± 0.04*

Nosema bombycis

1.80 ± 0.07

2.14 ± 0.05

2.18 ± 0.04

2.22 ± 0.04

2.30 ± 0.07

3.14 ± 0.05

3.32 ± 0.08 *

3.44 ± 0.05*

3.62 ± 0.04*

3.62 ± 0.04*

3.64 ± 0.05*

4.42 ± 0.08*

4.86 ± 0.05*

5.48 ± 0.04*

5.54 ± 0.05*

Healthy Control

1.80 ± 0.04

2.16 ± 0.05

2.24 ± 0.05

2.30 ± 0.07

2.40 ± 0.07

3.18 ± 0.04

3.62 ± 0.04

3.7 ± 0.07

3.78 ± 0.04

3.86 ± 0.05

4.00 ± 0.07

5.28 ± 0.04

5.64 ± 0.05

6.14 ± 0.11

6.60 ± 0.07

Data are expressed as Mean ±SD (n=5), *statistically signicant (P<0.05) compared to healthy control larva

by One way ANOVA using IBM SPSS 20 Software

Figure 4: Graph showing the effect of M-C and N. bombycis (Nbo) on larval length of the silkworm,

Bombyx mori L.

Larval width

The impact of infection caused by M-C and N. bombycis spores on the daily increase in larval width of silkworm (CSR2×CSR4 breed) is presented in Table 4 and Figure 5. From 7th day PI onwards there was signicant reduction in larval width of silkworm in batches inoculated with Nosema bombycis where as in the silkworm batches inoculated with M-C spore, the signicant reduction in larval width was observed from 9th day PI onwards as compared to healthy control batches. The impact of infection on larval width was more pronounced with the progression of infection and on 15th day PI, the larval width was recorded as 0.96 cm and 0.84 cm in the batches inoculated with M-C and N. bombycis respectively compared to healthy control batches where larval width was recorded as 1.20 cm.

Table 4: Effect of M-C and N. bombycis (Nbo) on larval width of the silkworm, Bombyx mori L. (CSR2× CSR4 breed)

Data are expressed as Mean ±SD (n=5), *statistically signicant (P<0.05) compared to healthy control larva by One way ANOVA using IBM SPSS 20 Software

Figure 5: Graph showing the effect of M-C and N. bombycis (Nbo) on larval width of the silkworm, Bombyx mori L.

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Larval weight

The impact of infection on larval weight of silkworm caused by M-C and N. bombycis spores on the daily increase in larval width of silkworm (CSR2×CSR4 breed) is presented in Table 5 and Figure 6. From 7th day PI onwards there was signicant reduction in larval weight was observed in the batches inoculated with Nosema bombycis where as in the silkworm batches inoculated with M-C spore, the signicant reduction in larval weight was observed from 10th day PI onwards as compared to healthy control batches. The effect of infection on larval weight was more pronounced with the progression of infection and one day prior to spinning i.e. on 15th day PI, the larval weight was recorded as 17.73 cm and 13.97 cm in the batches inoculated with M-C and N. bombycis respectively compared to healthy control batches where larval weight was recorded as 19.25 cm.

Table 5: Effect of M-C and N. bombycis (Nbo) on larval weight of the silkworm, Bombyx mori L. (CSR2× CSR4 breed)

Data are expressed as Mean ±SD (n=5), *statistically signicant (P<0.05) compared to healthy control larva by One way ANOVA using IBM SPSS 20 Software

Figure 6: Graph showing the effect of M-C and N. bombycis (Nbo) on larval weight of the silkworm, Bombyx mori L.

Effect of microsporidian infection on larva and cocoon parameters of silkworm B. mori

The impact of infection of M-Cfl and N. bombycis spores on larval and cocoon parameters at different inoculation doses is furnished in Table 6 and Figure 7. In the M-C inoculated batches the average weight of 5 mature larvae ranged between 17.73 g to 18.07 g where as in N. bombycis inoculated batches the value ranged from 13.97 to 15.02 g and it was found that in both the batches the larval weight get signicantly reduced as compare to the weight of healthy larva where the value was recorded as 19.25 g. This suggested that the larval weight was affected by the inoculums of all the three (1× 105, 1× 106 and 1× 107 spore/ml) concentrations.

Table 6: Effect of M-C spores on larva and cocoon characters of the silkworm, B. mori L.

Data are expressed as Mean ±SD (n=5), *statistically signicant (P<0.05) compared to healthy control larva by One way ANOVA using IBM SPSS 20 Software.

Table 7: Effect of N. bombycis on larva and cocoon characters of the silkworm, B. mori L.

Figure 7: Graph showing the lament length of cocoon from respective batches

Cocoon weight, shell weight and Shell ratio %, Filament length

The weight of the cocoon was not affected in the silkworm batches inoculated with M-C spores of doses concentration 1× 105 spores/ml and 1× 106 spores/ml where as the weight of cocoon was signicantly affected in the batches inoculated with M-C spore of dose concentration 1× 107 spores/ml when compared to the weight of healthy cocoon from control batch. Again, the shell weight was affected in the M-C batches inoculated with doses of 1× 106 spores/ml and 1× 107 spores/ml. Furthermore, it was observed that, the shell ratio % (SR %) and lament length was greatly affected in all the batches inoculated with different dose concentration of M-C spores. However, the cocoon weight, shell weight, shell ratio % and lament length gets affected signicantly in all the batches of silkworm inoculated with three different doses of Nosema bombycis as compared to healthy control batches.

DISCUSSION

Microsporidia are ubiquitous and diversed parasite having a broad host range. Microsporidiosis is the deadliest disease of the silkworm, Bombyx mori L. The microsporidia commonly known to cause this disease in B. mori L. is Nosema bombycis (Sprague, 1982). However, the microsporidia naturally occurring in other lepidopteran pests are also found to cross infective to silkworm causing considerable problem in

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sericulture industries (Kishore et al., 1994; Sharma et al., 2014; Bhuvaneswari and Nath 2015a, 2015b). Therefore, in the present investigation, a detailed study was carried out on the M-C spore morphology, their infectivity to silkworm B. mori L., their effect of larval development and cocoon parameters and the results compared to the corresponding characters in the batches of silkworm infected with Nosema bombycis, the standard strain causing pebrine disease of silkworm. The isolated M-C microsporidian spore is clearly different from that of N. bombycis strain in terms of both shape and size. M-C spore is ovo-cylindrical in shape whereas N. bombycis spore is oval in shape with a smaller size than that of M-C spores.

It was found that, the isolated microsporidia M-C from buttery Catopsilia florella was capable of infecting the silkworm, B. mori L. through the oral route by feeding contaminated mulberry leaf smeared with the isolated microsporidia. Again, with increase in dose concentration of M-C and N. bombycis spores, increase larval mortality was observed which suggest that mortality rate was dose depended. In M-C inoculated silkworm batches, the larval mortality increased from 12% to 40% as the concentration of dose increased from 1×105 spores/ml to 1×107 spores/ml where as in N. bombycis inoculated batches, the larval mortality increased from 21 % to 81 % as the dose of N. bombycis increased from 1×105 spores/ml to 1×107 spores/ml. The result was supported by a number of studies conducted by various researches who suggested that larval mortality are always dose depended (Choi et al., 2002; Sharma et al., 2014; Bhuvaneswari and Nath 2015a, 2015b). The silkworm larval mortality due to N. bombycis infection increased from 11.33 % to 50.67 % as the inoculums dose increased from 1×103 spores/ml to 1×106 spores/ml (Sharma et al., 2014). In 2002, Choi et al. isolated microsporidia from cabbage white buttery and 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 median lethal concentration (LC50) of the M-C and N. bombycis for larval mortality was calculated as 2.69×107 spores/ml and 1.1×106 spores/ml at 15 days of post inoculation in the respective batches. However, Xing et al., (2014) and Bashir (2008) have reported the LC50 value of N. bombycis for silkworm larval mortality respectively as 0.85×105 spores/ml for 12 days PI and 1×105.6 spores/ml for 15 days PI. The LC value of microsporidia isolated from buttery Catopsilia crocale and Catopsilia pyranthe against 50

larval mortality of B. mori at 15 days PI was recorded as 1×109.5 and 1×106.7 spores/ml respectively (Bashir 2008). The LC50 values, therefore, clearly suggest that isolated M-C microsporidia are less pathogenic to silkworm compared to the standard strain Nosema bombycis. In the present study, it was observed that the M-C microsporidia signicantly affect the normal growth (length, width, weight ) of larva and also the cocoon parameters viz. cocoon weight, shell weight, shell ratio % and lament length but to a lesser extent as compared to that of N. bombycis inoculated batches of silkworm. However, Sharma et al. (2014) reported that, no signicant differences in larval weight was observed in the batches of silkworm larva inoculated with a new microsporidium and the batches inoculated with N. bombycis as compared to larval weight of healthy control batches. Further, they have reported that a signicant reduction was observed in the cocoon weight, shell weight and lament length of both the infected batches compared to healthy batches of silkworm.

CONCLUSION

The present study revealed that under normal sericultural practices, the microsporidiosis of silkworm might not be exclusively caused by N. bombycis, it is also possible that the pathogens may transmitted by wild insects, such as C. orella, through horizontal cross-infection to silkworm B. mori. It is concluded that the isolated M-C microsporidia from buttery Catopsilia florella infect silkworm through oral portals. However, the intencity of infection caused by M-C spores in larvae is low as compared to that observed due to the standard strain, N. bombycis, the pebrine causing microsporidia of silkworm B. mori L.

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