K.P. Kiran Kumar and S. Sankar Naik ISSN

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1 International Journal of Integrative sciences, Innovation and Technology (A Peer Review E-3 Journal of Science Innovation Technology) Section A Basic Sciences; Section B Applied and Technological Sciences; Section C Allied Sciences Available online at Research Article IDENTIFICATION OF SUITABLE BMNPV (BOMBYX MORI) NUCLEAR POLYHEDROSIS VIRUS) TOLERANT SILKWORM BREEDS FOR DISEASE RESISTANCE BREEDING K.P. KIRAN KUMAR* AND S. SANKAR NAIK 1 * Silkworm Pathology Laboratory, Central Tasar Research & Training Institute, Central Silk Board, Piska nagari, Ranchi, Jharkhand, India. 1 Department of Sericulture, S. K. University, Anantapur, Andhra Pradesh, India. kiran_ctrti@rediffmail.com RUNNING TITLE: Identification of Suitable BmNPV Tolerant Silkworm Breeds ABSTRACT Present investigation deals with the screening and identification of suitable BmNPV tolerant silkworm breeds for the utilization of disease resistance breeding. A total of 100 breeds which included 50 polyvoltine and 50 bivoltine breeds, whose tolerance level was unknown among the stocks available at Andhra Pradesh State Sericulture Research and Development Institute (APSSRDI), Hindupur, India were selected for study and their screening was conducted through virus exposer by per oral inoculation. Out of selected breeds, 55 breeds were completely susceptible to the BmNPV, 11 breeds showed apparent tolerance and 34 breeds were real tolerant, which included 18 bivoltines and 16 polyvoltine breeds. Real tolerant populations were multiplied for two generations and evaluated for the major economic traits such as survival, productivity and disease resistance by employing the evaluation index method. The suitable silkworm breeds identified for BmNPV tolerance included 03, 04, 16, 06 and 09 among bivoltines breeds and NPV9, NPV3, NPV8, NPV7 and 4 among polyvoltine breeds based on overall performance. These suitable breeds are being utilized in conventional breeding for the development of BmNPV tolerant silkworm breeds/hybrids. KEY WORDS: Bombyx mori, Identification, Germplasm, Nuclear polyhedrosis, Tolerance INTRODUCTION The domestication of mulberry silkworms for the past several thousands of years has render them most susceptible to diseases, which is main factors that seriously affect the cocoon production 1. The disease in silkworms is primarily due to the pathogens and certain stress factors, which influence the disease development during silkworm rearing. The environmental factors such as temperature and humidity largely determine the growth and success of the silkworm rearing 2,3. Silkworms adapted to a temperature of 25 o C, any increase or decrease in the temperature acts as a stress and influence the susceptibility of silkworm to various diseases 4,5. Survey reports from different sericultural areas in India have revealed that the crop loss is often due to the diseases 6,7,8. Hence, prevention of silkworm diseases is the most important aspect in the success of commercial sericulture. In order to obtain high and stable cocoon yield, it is necessary to make efforts firstly to decrease the pathogen load, pathogenicity and secondly to strengthen the larval constitution by increasing its disease resistant ability. It is generally recommended to disinfect the rearing house and the appliances before the commencement of rearing, but it is not necessarily adequate to prevent the occurrence of diseases. Moreover, among many measures of silkworm disease control and prevention, the utilization of disease resistant / tolerant silkworm variety along with the disinfection would be the most effective step in the direction of the disease prevention. Among all the silkworm diseases that cause damage, viral diseases are the most serious 6,8,9. According to the survey conducted in sericulture areas of Karnataka, grasserie disease caused by BmNPV accounts for about 50 of total crop loss caused due to viral diseases 10. BmNPV, which belongs to Baculoviridae, causes nuclear polyhedrosis in silkworms. Nuclear polyhedrosis is the most common viral disease and 6

2 is prevalent in almost all the sericulture areas in India. Inheritance to BmNPV tolerance is controlled by polygenes. Screening of the breeds for their relative tolerance / susceptibility for BmNPV would be helpful in identifying the silkworm breeds that are less susceptible and they could be exploited commercially to evolve tolerant breeds or hybrids. So, the present study has been carried out to screen the silkworm breeds for BmNPV tolerance and to identify the suitable breeds among them for the utilization of disease resistant breeding programme. MATERIALS AND METHODS Propagation of BmNPV: The BmNPV was propagated in a susceptible breed (NB18) of silkworm by per oral inoculation of BmNPV polyhedra during 4 th instar. The haemolymph of the infected larvae was collected and BmNPV polyhedra were purified as described by Sugimori et al. (1990) 11. Purification: The haemolymph was collected on ice from the BmNPV infected larvae, allowed for bacterial degradation for 24 hrs and centrifuged at 5000 rpm for 10 minutes for pellating the BmNPV polyhedra. The polyhedra were washed thrice in 1M NaCl followed by distilled water and quantified by haemocytometry. The purified polyhedra were stored at 4 o C for further use. Screening of the Germplasm: Before taking up disease resistant breeding programme in this direction, it would be essential to access the tolerant level / susceptibility status of the available breeding resource materials for the utilization. In this contest, 50 Bivoltine and 50 Polyvoltine silkworm breeds that are unknown for their tolerance were collected as source materials from the germplasm bank of APSSRDI, Hindupur, India. Screening was conducted against BmNPV and evaluation of suitable breeds was done for further research programme. The silkworm larvae out of second moult (3 rd instar) were per orally inoculated with BmNPV polyhedra (1 x 10 7 /ml) by smearing onto the mulberry leaf (0.25 ml / 25 cm 2 leaf / 25 larvae). The larvae were allowed to feed on to the inoculum-smeared leaf for six hours. The larvae were reared under standard rearing conditions in three replications of 100 larvae each. The mortality due to the nuclear polyhedrosis was recorded during larval and pupal stages. A mock-inoculated batch of each breed was also maintained as control and a known susceptible breed NB18 also maintained as a check variety. The BmNPV infection was confirmed by light microscopy. Accordingly the tolerant population in each batch was collected. The moths emerged from the survivors were processed for egg laying and the eggs were utilized for further evaluation. The degree of tolerance was estimated from the mortality recorded during larval and pupal stage. The relative tolerance was categorized in to (1) Apparent tolerance, the inoculated larvae complete the larval period and forms cocoons but do not metamorphose into pupae (2) Real tolerance, the inoculated larvae complete larval period, forms cocoons, metamorphoses into pupae and moths emerge from the cocoons. (3) Susceptibility, the inoculated larvae succumb to death due to nuclear polyhedrosis during larval period itself. The morphological characters like larval markings, cocoon type and colour, survival (effective rate of rearing and pupation rate) and reproductive (moth emergence and reproductive fitness index) characters of tolerant populations were recorded. The survival and reproductive characters were estimated on the basic larval number tested. The reproductive fitness index was calculated based on the number of layings produced to the basic larval number. The data was subjected to statistical analysis for calculation of mean, standard deviation and co-efficient of variation (C.V) of bivoltine and polyvoltine breeds. Evaluation of BmNPV tolerant breeds: The silkworm stocks showing tolerance to BmNPV were multiplied for two generations for increasing the base populations. These batches were reared under standard conditions with out exposer to BmNPV. Composite layings of these stocks were prepared and subjected to virus exposer following the procedure as described earlier. Silkworm larvae out of II molt were inoculated with BmNPV at the concentration of 1X10 7 /ml and the mortality was recorded up to egg laying. The breeds were evaluated for their overall performance for various characteristics such as cocoon yield by number, cocoon yield by weight, pupation rate, cocoon weight, shell weight and shell ratio, moth emergence and reproductive fitness index by employing evaluation index method 12 and the evaluation indices values are calculated. RESULTS The results indicate wide range of variability among different silkworm breeds screened and evaluated for their tolerance / susceptibility to BmNPV. I. Screening of the Germplasm stock A. Bivoltine silkworm breeds a) Susceptibility to BmNPV: Twenty-seven bivoltine silkworm breeds out of 50 breeds 7

3 were found to be completely susceptible to BmNPV and died during larval period. They included 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 breeds. The known susceptible breed, NB18 also was recorded 100 mortality by the end of IV instar. b) Apparent tolerance to BmNPV: Five silkworm breeds showed apparent tolerance to BmNPV among the 50 breeds screened (Table 1). The apparent tolerance exhibited in 19, 20, 21, 22 and 23 was 14.33, 13.66, 19.00, and respectively. Table 1: Characteristics and degree of tolerance of bivoltine silkworm breeds, which have shown apparent tolerance when screened for BmNPV S. N o Breed L.M Coc. col or Coc.Ty pe Plain W J Plain W J Mark ed F C Plain W C Plain W C D.T by larval stage 14.33± ± ± ± ± 2.00 by pupal stage c) Real tolerance to BmNPV: Among the 50 breeds screened for their tolerance / susceptibility, 18 showed real tolerance (Table 2). They include 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, and 18. The degree of tolerance ranged from ± 4.73 (07) to ± 3.51 (03) and it revealed > 40 in 04 (48.33 ± 4.04), 09 (47.66 ± 4.51), 16 (43.00 ± 3.61), 12 (42.33 ± 4.51) and lowest was observed in the 07 (19.66 ± 4.73). Highest SR was observed in 09 with and followed by 06 (19.84), 16 (19.78), 17 (19.63) and lowest observed in 11 (16.64). Highest moth emergence was observed in case of 04, 06 with 21, remaining breeds were ranged from 3 to 20 and least is in the case of 18. The reproductive fitness Index () recorded was highest observed in 04, 06, 12, and 16 with 4. The performance of remaining breeds did not exceed 3 and least was observed in 10, 13, 15, 17 and 18 with 1. B. Polyvoltine silkworm breeds a) Susceptibility to BmNPV: Twenty-eight bivoltine silkworm breeds were found to be completely susceptible to BmNPV, during larval period itself died among the 50 breeds screened. They included NPV23, NPV24, NPV25, NPV26, NPV27, NPV28, NPV29, NPV30, NPV31, NPV32, NPV33, NPV34, NPV35, NPV36, NPV37, NPV38, NPV39, NPV40, NPV41, NPV42, NPV43, NPV44, NPV45, NPV46, NPV47, NPV48, NPV49 and NPV50. S. No Table 2: Characteristics and performance of bivoltine silkworm breeds, which have shown real tolerance when screened for BmNPV Breed L.M Coc. color Coc. Type D.T M.NP V S.C wt.(g) S.S wt. (g) S.R M.E 1 01 Plain F J 29.66± Plain F J 34.66± Plain W C 50.66± Plain W C 48.33± Plain W J 27.33± Plain W C 39.00± Plain W J 19.66± R.F.I 8

4 8 08 Plain W J 31.00± Plain W C 47.66± Plain W J 37.00± Plain W J 33.33± Plain Y J 42.33± Plain W J 23.33± Plain W J 36.33± Plain W J 23.33± Plain W C 43.00± Marke d Y J 26.33± Marke d Y J 24.33± Mean S.D C.V L.M: Larval markings D.T: Degree of tolerance R.F.I: M.E: Moth emergence Reproductive fitness index J: Japanese; C: Chinese W: White; F: Flesh; Y: Yellow Values represent average of 3 replications ± S.D M.NPV: Mortality due to BmNPV b) Apparent tolerance to BmNPV: Six silkworm breeds showed apparent tolerance to BmNPV among 50 breeds screened (Table 3). The apparent tolerance exhibited by the 7, 8, 9, NPV20, NPV21 and NPV22 was 22.00, 15.00, 23.00, 23.00, and respectively. c) Real tolerance to BmNPV: Among the 50 breeds screened for their tolerance / susceptibility, 16 breeds showed real tolerance (Table 4). They included, NPV2, NPV3, NPV4, NPV5, NPV6, NPV7, NPV8, NPV9, 0, 1, 2, 3, 4, 5 and 16. The degree of tolerance ranged from ± 2.08 (NPV4) to ± 5.03 (NPV7) and it was more than 30 in NPV3 (36.33 ± 4.51), 4 (35.00 ± 3.46), NPV5 (30.33 ± 3.79) and least observed in the case of NPV4 (15.66 ± 2.08). (Table 4) Highest S.R is observed in NPV3 with followed by NPV6 (17.07), NPV2 (16.78), NPV7 (16.77) and least observed in 0 (14.37). Highest moth emergence was observed in case of NPV5 with 21 followed by NPV7 and 4 (17), NPV3 (16), (15) and lowest observed in the case of NPV4 and 0 with 5. The reproductive fitness Index () ranged from 1.33 (NPV4, NPV9, 0, 3) to 5.67 (NPV5). Table 3: Characteristics and degree of tolerance of polyvoltine silkworm breeds, which have shown apparent tolerance when screened for BmNPV S. Breed L.M Coc. color Coc. Type D.T No By larval stage By pupal stage 1 7 Plain White C 22.00± Plain White C 15.00± Marked White C 23.00± NPV20 Plain White C 23.00± NPV21 Plain White C 15.33± NPV22 Plain White J 21.66±5.51 9

5 Table 4: Characteristics and performance of polyvoltine silkworm breeds, which have shown real tolerance when screened for BmNPV S. No Breed L.M Coc. color Coc. Typ D.T M.NPV S.C wt.(g) S.S wt. (g) S.R M.E R.F.I e 1 Plain W C 26.33± NPV2 Plain W C 19.33± NPV3 Plain GY C 36.33± NPV4 Plain W C 15.66± NPV5 Plain GY C 30.33± NPV6 Plain GY C 28.00± NPV7 Plain GY C 37.66± NPV8 Plain GY C 28.33± NPV9 Plain GY C 21.33± Plain W C 18.66± Plain Y C 23.67± Plain Y C 28.00± Plain Y C 21.00± Plain GY C 35.00± Marked W J 18.00± Marked Y J 23.33± Larval markings (L.M) M.E: Moth emergence Mean S.D C.V D.T: Degree of tolerance R.F.I: Reproductive fitness index J: Japanese; C: Chinese W: White; GY: Greenish yellow; Y: Yellow Values represent average of 3 replications ± S.D M.NPV: Mortality due to BmNPV II. Evaluation of BmNPV Tolerance The BmNPV tolerant stocks isolated during screening were multiplied for two generations under normal conditions. These breeds were further evaluated by per oral inoculation with BmNPV by employing an evaluations index method based on nine quantitative traits to identify the most promising BmNPV tolerant breeds. The overall performance of breeds was expressed in the terms of total and mean evaluation indices (E.I). A. Bivoltine silkworm breeds Among the bivoltines highest mean E.I was recorded in 03 and followed by NPV4, 16 with 62.13, respectively (Fig. 1). > 50 is observed in 06, 09, 12, 14 in other breeds E.I was < 50; the promising bivoltine breeds whose E.I is > 55 are 03, NPV 104, NPV 116, NPV 106 and NPV 109 they recorded cocoon weight of 1.624g, 1.417g, 1.621g, 1.419g and 1.538g and shell weight of 0.307g, 0.271g, 0.329g, 0.285g and 0.313g and shell ratio of 18.93, 19.15, 20.28, and respectively (Table 5). Among the other breeds, 18, 17, 14, 15 excelled for cocoon weight; 18, 17, 05, 14 for shell weight; 17, 05, 18, 14 for shell ratio. 10

6 S. No Breed Table 5: Evaluation of bivoltine silkworm breeds for tolerant to BmNPV Pupation ERR Yield/10,000 larvae By By Wt. No. (Kg) S.C Wt.(g) S.S Wt (g) S.R M.E R.F.I Avg E.I Average S.D C.V Values represent average of 3 replications B. Polyvoltine silkworm breeds Among the Polyvoltine, (Table 6) highest mean E.I (62.83) was recorded in NPV9 and followed by NPV3 with (Fig. 2). > 50 is observed in NPV8, NPV7, 4, NPV5, 1, 5, NPV6 other breeds E.I was < 50; The promising polyvoltine breeds whose E.I is above 55 are NPV9, NPV3, NPV8, NPV7 and 4 they recorded cocoon weight of 1.625g, 1.472g, 1.665g, 1.518g and 1.542g and shell weight of 0.304g, 0.286g, 0.276g, 0.244g and 0.217g and shell ratio of 18.73, 19.42, 16.56, and respectively (Table 6). Among the other breeds, 1, 5, NPV4, 2 excelled for cocoon weight; 5, NPV4, 1, NPV2 for shell weight;, NPV2, 5, NPV6 for shell ratio. S.N o Breed Table 6. Evaluation of polyvoltine silkworm breeds for tolerant to BmNPV Pupation ERR Yield/10,000 larvae By No. By Wt.(Kg) S.C Wt.(g) S.S Wt (g) S.R M.E R.F.I Avg E.I NPV NPV NPV NPV NPV

7 7 NPV NPV NPV Average S.D C.V Values represent average of 3 replications DISCUSSION As silkworms are susceptible to various diseases caused by pathogenic microorganisms, silkworm breed having many qualitative and quantitative characters, it is weak against diseases resulting in poor cocoon yield cannot be considered as a good breed. In this situation, breeding for disease resistance / tolerance stock has become very vital for the sericulture industry. Disease resistance is an important complementary objective of silkworm breeding because high degrees of susceptibility will generally result in decreased yield and quality. The mechanism involved in disease resistance largely center on the immune responses exhibited by an organism. Knowing the importance of disease resistant breeding, in the present study an attempt is made to screen for their relative susceptibility by artificially disease induced method in order to get desired breed / line which could be utilized in the evolution of disease resistant breeds/ hybrids. The crop loss due to the incidence of nuclear polyhedrosis caused by BmNPV is quite frequent under tropical conditions 13. The status of germplasm stocks to specific pathogens for their relative resistance / tolerance / susceptibility provides an insight into the performance of the parental stocks as well as their hybrids. The possibility of the epizootic level of the infection becomes limited with the use of the non-susceptible hosts. The polygenic inheritance of tolerance to silkworm viruses poses several questions as how stable is the tolerance isolated and how long the tolerance lost as environmental and nutritional factors and their interaction influences the disease tolerance / susceptibility in silkworms. As inheritance to BmNPV tolerance is controlled by polygenes, identification of silkworm breeds by direct selection increases chances of retaining tolerance for BmNPV. The identification of silkworm breeds tolerant to the BmNPV by per oral inoculation tests were conducted for the silkworm germplasm stocks at NISES (Japan) by Furuta, (1995) 14, Liu (1984) 15 in China, Nataraju (1995) 16, Sen et al., (1997) 17 and Sivaprasad and Chandrasekharaiah (2003) 18 in India. Among the 50 bivoltine and 50 polyvoltine silkworm breeds screened for their relative tolerance / susceptibility, 18 bivoltine and 16 polyvoltine breeds showed real tolerance to BmNPV. The breeds showing apparent tolerance to BmNPV included 5 bivoltines and 6 polyvoltines. A total of 55 silkworm breeds included 27 bivoltines and 28 polyvoltines were completely susceptible to BmNPV. The degree of tolerance in real tolerance bivoltine breeds ranged from ± 4.73 (07) to ± 3.51 (03). Where as in real tolerance polyvoltine breeds, it was ± 2.08 (NPV4) to ± 5.03 (NPV7). The expression of tolerance to BmNPV infection differed among the batches. The expression of tolerance after two normal multiplicative generations all the bivoltine and polyvoltine breeds taken for the study could be retained. In the above contest, the identification of BmNPV tolerant silkworm breeds by the present study offers an exciting prospect for the management of BmNPV. Among the bivoltines five breeds, 03 (62.87), 04 (62.13), 16 (60.84), 06 (59.42) and 09 (58.68) and among polyvoltine five breeds NPV9 (62.83), NPV3 (60.14), NPV8 (57.38), NPV7 (56.78) and 4 (56.16), whose mean evaluation index value is more than 55 indicates their superiority in economic traits in multiple trait evaluation and they are most superior stocks in BmNPV tolerance. Hence, it is concluded that the potential BmNPV tolerance breeds 03, 04, 16, 06 and 09 in 12

8 bivoltines, NPV9, NPV3, NPV8, NPV7 and 4 in polyvoltines, are identified as most suitable breeds and could be utilized for disease resistance breeding programme. ACKNOWLEDGMENTS The authors express their sincere thanks to Dr. Chandrasekharaiah, founder & former Director of APSSRDI for providing facilities and encouragement to carry out research programme. REFERENCES 1. Watanabe, H., (1986) Resistance of the silkworm, Bombyx mori to viral infection. Agric. Ecosyst. Environ 15, Kenten, J., (1955) The effect of photoperiod and temperature on reproductive in Acyrthosiphonpism (Harris) and forms produced. Bull. Entomol. Res 49, Tazima, Y., (1978). Spermatogenesis: The Silkworm an Important Laboratory Tool. Kodensha Ltd., Tokyo, Japan, pp: Steinhaus, E.A., (1958) Stress as a factor in insect disease. Proc. 10 th Int. Comp. Entomol., 4: Watanabe, H., 1964) Temperature effects on the manifestation of susceptibility to per oral infection with Cytoplasmic polyhedrosis in silkworm, Bombyx mori L. J. Seric. Sci. Jpn., 33, Samson, M.V., M. Baig, S.D. Sharma, M. Balavenkatasubbaiah, T.O. Shashidharan and M.S. Jolly, 1990) Survey on the relative incidence of silkworm diseases in Karnataka, India. India. J. Seric 29, Savanurmath, C.J., S.S. Ingalgalli, K.K. Sing and R.D. Sanakal, (1995) Predisposing factor for viral diseases of the silkworm, <I>Bombyx mori</i> under agro climatic conditions of north Karnataka. Ind. J. Seric., 34, Sivaprakasham, N. and R.J. Rabindra, 1995) Incidence of grasserie in silkworm Bombyx mori L. in selected districts of Tamilnadu. India. J. Seric 34, Subba Rao, G., A.K. Chandraand and J. Battacharya (1991) Incidence of crop loss from adapted rearers level in west Bengal due to silkworm diseases. India. J. Seric 30, 167S. 10. Samson, M.V., 1992) Silkworm crop protection. Base paper presented in National conference on mulberry sericulture research, December 10-11, Central Sericulture Research and Training Institute, Mysore, India. 11. Sugimori, H., T. Nagamine and M. Kobayashi, (1990) Analysis of structural polypeptides of Bombyx mori (Lepidoptera: Bombycidae) nuclear polyhedrosis virus. Appl. Entomol. Zool., 25, Mano, Y., S. Nirmal Kumar, H.K. Basavaraja, N. Malreddy and R.K Datta, (1993). A new method to select promising silkworm hybrid combination. Indian Silk Sivaprasad, V., Chandrasekharaiah, C. Ramesh, S. Misra, K. P. K. Kumar and Y. U. M. Rao (2003) Screening of silkworm breeds for tolerance to Bombyx mori nuclear polyhedrosis virus (BmNPV). Int. J. Indust. Entomol 7, Furuta, Y., 1995) Susceptibility of the races of the silkworm, Bombyx mori, preserved in NISES to the nuclear polyhedrosis virus and densonucleosis viruses. Bull. Natl. Inst. Seric. Entomol. Sci., 15, Liu, S.X., 1984) Identification on the resistant of the silkworm, Bombyx mori races to six types of diseases. Sericologia, 24: Nataraju, B., 1995) Studies on nuclear polyhedrosis in silkworm, Bombyx mori. Ph.D. Thesis, Mysore University, Mysore, India. 17. Sen, R., A. K. Patnaik, M. Maheswari and R. K. Datta (1997) Susceptibility status of the silkworm (Bombyx mori) germplasm stocks in India to Bombyx mori nuclear polyhedrosis virus. Ind. J. Seric 36, Sivaprasad, V. and Chandrashekharaiah (2003) Strategies for breeding disease resistance silkworms. Mulberry Silkworm Breeders Summit, APSSRDI, Hindupur, India. 13