Bivoltine Silkworm Research Articles

Response of Silkworm (Bombyx mori L.) Breeds to Temperature and BmNPV Stress

Silkworm Bombyx mori L. is one of the most important domesticated insects, which produces luxuriant silk thread in the form of cocoon by consuming mulberry leaves during larval period. The growth and development of silkworm is greatly influenced by environmental conditions. The seasonal differences in the environmental components considerably affect the genotypic expression in the form of phenotypic output of silkworm crop. Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes great economic losses in sericulture. In the present investigation, twelve bivoltine silkworm breeds (WM, ND5, NB4D2, U-4, PO1, ND3, U-6, CSR2, SH6, SPO, U-3 and NSP) were studied for temperature and BmNPV stress. After third and fourth moult silkworm larvae were treated with BmNPV inoculum at 25±1⁰C and 30±1⁰C temperature respectively. Increased mortality was observed at 30±1⁰C when compared to 25±1⁰C. However, with respect to breeds the highest total mortality was recorded in breed PO1 and least in breed U-3. Silkworm breed U-3 may have the ability to tolerate BmNPV incidence and can be exploit for evolving disease-tolerant silkworm hybrids.

Evaluation of elite bivoltine mulberry silkworm (Bombyx mori L.) double hybrids suitable for North West India

Nine bivoltine silkworm double hybrids along with control were developed and evaluated at Central Sericultural Research and Training Institute, Central Silk Board, Pampore, Jammu and Kashmir (J&K) during summer (July-August) and autumn (August-September) 2020. The main aim is to develop silkworm double hybrids locally by utilising the silkworm breeds developed by different research Institutes. The bivoltine silkworm breeds developed by CSB research institutes were procured initially for preparation and evaluation of foundation crosses. Based on the performance, out of twenty four foundation crosses three oval and three constricted foundation crosses were shortlisted for further development of 09 double hybrids. On the basis of the evaluation index (E.I) values 05 double hybrids during summer and 05 double hybrids during autumn recorded E.I value above 50. During summer 05 silkworm double hybrids viz., (CSR50×PAM114) X (PAM117×APS4), (CSR50×PAM114) X (PAM117× SK7), (PAM114× CSR27) X (PAM117×APS4), (PAM114×CSR50) X (PAM117 ×APS4) and (PAM114×CSR50) X (SK6×SK7) recorded E.I values 50.09, 55.86, 57.95, 61.35 and 52.88, respectively over control FC2× FC1 (60.67) whereas in autumn 05 silkworm double hybrids viz., (CSR50×PAM114) X (SK6×SK7), (CSR50×PAM114) X (PAM117× SK7), (PAM114× CSR27) X (PAM117×APS4), (PAM114×CSR50) X (PAM117 ×APS4) and (PAM114×CSR50) X (SK6×SK7) recorded E.I values 56.20, 53.13, 54.62, 55.52 and 59.80, respectively over control FC2× FC1 (57.65). Based on results one double hybrid (PAM114×CSR50) X (PAM117 ×APS4) for summer and one double hybrid (PAM114× CSR50) X (SK6×SK7) for autumn identified for North West India.

Evaluation of elite bivoltine mulberry silkworm (Bombyx mori L.) double hybrids suitable for North West India

Nine bivoltine silkworm double hybrids along with control were developed and evaluated at Central Sericultural Research and Training Institute, Central Silk Board, Pampore, Jammu and Kashmir (J&K) during summer (July-August) and autumn (August-September) 2020. The main aim is to develop silkworm double hybrids locally by utilising the silkworm breeds developed by different research Institutes. The bivoltine silkworm breeds developed by CSB research institutes were procured initially for preparation and evaluation of foundation crosses. Based on the performance, out of twenty four foundation crosses three oval and three constricted foundation crosses were shortlisted for further development of 09 double hybrids. On the basis of the evaluation index (E.I) values 05 double hybrids during summer and 05 double hybrids during autumn recorded E.I value above 50. During summer 05 silkworm double hybrids viz., (CSR50×PAM114) X (PAM117×APS4), (CSR50×PAM114) X (PAM117× SK7), (PAM114× CSR27) X (PAM117×APS4), (PAM114×CSR50) X (PAM117 ×APS4) and (PAM114×CSR50) X (SK6×SK7) recorded E.I values 50.09, 55.86, 57.95, 61.35 and 52.88, respectively over control FC2× FC1 (60.67) whereas in autumn 05 silkworm double hybrids viz., (CSR50×PAM114) X (SK6×SK7), (CSR50×PAM114) X (PAM117× SK7), (PAM114× CSR27) X (PAM117×APS4), (PAM114×CSR50) X (PAM117 ×APS4) and (PAM114×CSR50) X (SK6×SK7) recorded E.I values 56.20, 53.13, 54.62, 55.52 and 59.80, respectively over control FC2× FC1 (57.65). Based on results one double hybrid (PAM114×CSR50) X (PAM117 ×APS4) for summer and one double hybrid (PAM114× CSR50) X (SK6×SK7) for autumn identified for North West India.

Identification of superior bivoltine mulberry silkworm (Bombyx mori L.) double hybrids suitable for North West India

The present research investigation was carried out at Central Sericultural Research and Training Institute, Central Silk Board, Pampore, Jammu & Kashmir (J&K) during summer (July-August) and autumn (August-September) 2021. The main objective is to develop silkworm double hybrids by utilising the silkworm breeds developed by different research Institutes. The silkworm breeds procured from different research institutes were utilised for preparation and evaluation of foundation crosses. Based on the performance, out of twenty four foundation crosses three oval and three constricted foundation crosses were shortlisted for further development of double hybrids. A total of nine bivoltine silkworm double hybrids were developed and evaluated along with control (CSR2×CSR27) X (CSR6×CSR26) for identification of superior bivoltine silkworm double hybrid suitable for North West India. On the basis of the evaluation index (E.I) values 04 double hybrids during summer and 04 double hybrids during autumn recorded E.I value above 50. During summer 04 silkworm double hybrids viz., (CSR50×PAM114) X (PAM117× SK7), (PAM114× CSR27) X (PAM117×APS4), (PAM114×CSR50) X (PAM117 ×APS4) and (PAM114×CSR50) X (SK6×SK7) recorded E.I values 57.88, 53.46, 60.37 and 54.38 respectively over control FC2× FC1 (59.59) whereas in autumn 04 silkworm double hybrids viz., (CSR50×PAM114) X (PAM117× SK7), (PAM114× CSR27) X (PAM117×APS4), (PAM114×CSR50) X (PAM117 ×APS4) and (PAM114×CSR50) X (SK6×SK7) recorded E.I values 56.56, 54.88, 58.02 and 61.93 respectively over control FC2× FC1 (59.50). Based on results one double hybrid (PAM114×CSR50) X (PAM117 ×APS4) for summer and one double hybrid (PAM114× CSR50) X (SK6×SK7) for autumn identified for North West India.

M6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori).

Research has shown that epigenetic modification are involved the regulation of diapause in bivoltine silkworms (Bombyx mori), but it remains unclear how epigenetic modification in response to environmental signals precisely to regulate the diapause processing of bivoltine B. mori. In this study, the diapause terminated eggs of bivoltine B. mori, Qiufeng (QF) were divided into two groups: a QFHT group incubated at 25°C with a natural day/night cycle to produce diapause eggs, and a QFLT group incubated at 16.5°C in darkness to produce non-diapause eggs. On the 3rd day of the pupal stage, the total RNAs of the eggs were extracted and their N6-adenosine methylation (m6A) abundances were analyzed to explore the effects of m6A methylation on diapause in the silkworm. The results showed that 1984 m6A peaks are shared, 1563 in QFLT and 659 in QFHT. The m6A methylation level of the QFLT group was higher than that of the QFHT one in various signaling pathways. The m6A methylation rate of mevalonate kinase (MK) in the insect hormone synthesis pathway was significantly different between the two groups. The knockdown of MK by RNA interference in the pupae of QFLT resulted in females laying diapause eggs rather than non-diapause eggs after mating. m6A methylation involves in the diapause regulation of bivoltine B. mori by changing the expression levels of MK. This result provides a clearer image of the environmental signals on the regulation of diapause in bivoltine silkworms.

Study of Uzi Fly, <i>Exorista bombycis</i> (Louis) Infestation during Rearing of Mulberry Silkworm in Different Seasons in Karnataka

Present investigation was carried out during summer and rainy seasons under laboratory conditions at the Department of Sericulture, Chintamani UAS (B) during 2012-13. Rearing of bivoltine silkworm hybrid CSR2 X CSR4 in different seasons reveals that rainy season open rearing is found to be susceptible for Uzi infestation as compared to summer season under protected and open rearing. Uzi fly incidence on top position tray was observed significantly maximum in rainy season open rearing as compared to summer season open batch and least observed in summer season protected rearing. Longevity of the uzi fly is reported to be negatively correlated with weather parameters such as temperature; higher the temperature lower the adult longevity. Experimental observation revealed that rearing of silkworm in summer with all the precautionary measures under protection is suitable for more yield of cocoon with least uzi fly infestation in Karnataka.

Studies of the silkworm enzyme activity and their correlations with economic variables

The experiment was conducted to analyse the larval performance and economic traits of bivoltine silkworm breeds of silkworm (SK-1, SK-6, SK-22, SK-28, SK-33, CSR4, CSR2, NB4D2, DUN6 and APS4) during spring season. The haemolymph total protein, succinate and gultamate dehydrogenase activities were estimated and their correlation with economic traits were also worked out. The results of the study confirmed that among ten bivoltine silkworm breeds, highest haemolymph SDH activity of 3.47 µmoles/ml/mgprotein/min was recorded in the silkworm breed SK1 and least SDH activity of 1.58 µmoles/ml/mg protein/min was recorded in the breed APS4. The highest peak of succinate dehydrogenase activity of 2.65 µmoles/ml/mg protein/min was observed on 7th day of the 5th instar and lowest peak of succinate dehydrogenase activity of 2.39 µmoles/ml/mg protein/min was observed on 4th day of the 5th instar. GDH activity of 0.46 µmoles/ml/mg protein/min was recorded highest in the silkworm breed SK1 and lowest of 0.15 µmoles/ml/mg protein/min was recorded in silkworm breed APS4. The highest peak of haemolymph GDH of 0.36 µmoles/ml/mg protein/min was recorded on 7th day of 5th instar and lowest peak of 0.26 µmoles/ml/mg protein/min was recorded on 4th day of 5th instar. The correlational studies revealed that haemolymph total protein, SDH and GDH were found to be positively corelated with yield by weight and number (cocoon), weight of mature larvae, shell weight ,cocoon weight, shell ratio percent, silk productivity, rate of pupation, fecundity, raw silk percentage and length of filament. Thus, the study revealed that silkworm breeds like SK1, SK6, SK22 and SK28 as productive breeds and hence may be used for future breeding programmes for evolution of new robust silkworm breeds

Evaluation of Thermotolerant Bivoltine Silkworm Breeds through Cocoon Yield and Filament Characteristics against Beauveria bassiana (Bals-Criv.) Vuill. Inoculation

Silkworm, Bombyx mori L. being a poikilothermic insect is susceptible to both biotic and abiotic factors. The present study was carried to assess the performance of few thermotolerant bivoltine silkworm breeds under high temperature treatment (36 ± 1⁰ C for 6 hours) and Beauveria bassiana (Bals-Criv.) Vuill. inoculation (6.86 ̕X 104 spores / ml @ 0.5 ml / silkworm). Seven thermotolerant bivoltine silkworm breeds viz., B1, B2, B4, B6, B8, APS12, and APS45 and two popular bivoltine breeds, CSR2 and CSR4 breeds and one popular multivoltine breed Pure Mysore were utilized for the study. Among the thermotolerant breeds studied, the breed B4 was found to perform better with respect to cocoon yield by number, cocoon yield by weight, average filament length, filament weight under both high temperature treatment and B. bassiana inoculation. CSR4 breed was found to be least performer for all the parameters. Therefore, the breed B4 can be a better parent for hybridization and improvement under both the stress conditions.

Studies on the digestive juice protein in various bivoltine silkworm breeds and correlations with economic variables

Studies on the digestive juice protein in various bivoltine silkworm breeds and correlations with economic variables

Genome-wide screening of genes involved in programming diapause in the next generation in silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Maternal silkworms (Bombyx mori) of bivoltine strains are destined to produce either diapause or non-diapause eggs depending on environmental factors, such as, temperature and photoperiod experienced during the egg and larval stages. However, the molecular mechanisms that program diapause, which depend on information about the environment, remain unclear. We aimed to identify genes that are involved in programming diapause in the next generation in bivoltine silkworms. We therefore screened differentially expressed genes (DEGs) in the larval brains of diapause- and non-diapause-egg producers kept under three different diapause-inducing conditions using cap analysis of gene expression. Under each condition, only temperature, illumination or photoperiod was changed during the egg or larval stage as a diapause-controlling stimulus to induce the production of diapause or non-diapause eggs. We then verified the expression of DEGs that were common to all the three conditions using real-time quantitative PCR. We investigated the functional involvement of candidate genes in programming diapause using double-stranded RNA interference (RNAi) for gene knockdown. The results showed more abundant juvenile hormone acid methyltransferase (Jhamt) and proton-coupled folate transporter (Pcft) gene expression in the brains of fifth instar larvae of producers of diapause eggs than those of non-diapause eggs under the three conditions. Furthermore, RNAi against either of these genes significantly decreased the incidence of diapause in the next generation. These findings indicate that both Jhamt and Pcft are involved in the programming of diapause in the silkworm brain. These genes could function in retaining information that leads to diapause in the next generation.

YTHDF3 Is Involved in the Diapause Process of Bivoltine Bombyx mori Strains by Regulating the Expression of Cyp307a1 and Cyp18a1 Genes in the Ecdysone Synthesis Pathway.

The variable diapause features of bivoltine silkworm (Bombyx mori) strains regulated by environmental signals in the embryonic stage are closely related to epigenetics. Previously, we showed that the expression of YTHDF3 is significantly different in the pupae of the bivoltine silkworm Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, nondiapause egg producer), indicating that the expression of diapause-associated genes is regulated by the m6A modification level. However, how YTHDF3 regulates the expression of diapause-related genes remains unclear. In this study, we observed that the knockdown of B. mori YTHDF3 resulted in delayed embryo development, while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs. Further studies showed that YTHDF3, as a reading protein, can recognize the m6A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway (ESP), and the overexpression of YTHDF3 affects the diapause traits of the silkworm by decreasing the stabilities of mRNAs of Cyp307a1 and Cyp18a1 and inhibiting their translation. The above results demonstrate that m6A modification mediates YTHDF3 to affect the expression levels of its target genes, Cyp307a1 and Cyp18a1, in the ESP to regulate diapause in bivoltine B. mori. This is the first report of the m6A methylation regulation mechanism in diapause in B. mori and provides new experimental data for clarifying the diapause regulation network.

ESTIMATION OF GENETIC PARAMETERS AND VARIABILITY IN THE BIVOLTINE SILKWORM BOMBYX MORI L GERMPLASM

Genetic variability is a fundamental and an essential requirement of crop species for the development of superior breeds of high commercial value with strong genetic base. Evaluation and estimation of genetic potentials of silkworm breeds are of prime importance in order to identify promising silkworm genotypes for its commercial exploitation through breeding to enhance the silk productivity for sustainable use. The objective of this study was to evaluate the genetic variability of vast silkworm germplasm and to estimate the genetic parameters of important productive traits. 368 bivoltine silkworm accessions from the germplasm are considered for assessment of genetic variability with five year data on important productive traits. Wide range of variability was observed based on Coefficient of Variation (%) values in the productive parameters viz., fecundity (11.316), weight of the larva (9.285), cocoon yield (7.892), single cocoon weight (8.272 ), shell weight and (12.289) shell ratio (7.082). Based on the multiple trait analysis by Mano index values, 20 top performing accessions were selected and genetic parameters such as heritability, genetic advance and phenotypic & genotypic coefficient of variances were estimated. Pooled ANOVA of 20 selected silkworm accessions indicates significant variations @1% level over the five generations in all the parameters. Higher heritability associated genetic advance was recorded in cocoon weight, shell ratio, shell weight and larval weight, besides, higher genotypic and phenotypic variances in cocoon weight. The results enumerate the importance of evaluation of silkworm germplasm and estimation of genetic parameters for selection of parental stocks for subsequent breeding programme in development of desired breeds.

Comparative Transcriptome Analysis Reveals bmo-miR-6497-3p Regulate Circadian Clock Genes during the Embryonic Diapause Induction Process in Bivoltine Silkworm.

Simple SummaryDiapause is a type of seasonal polyphenism and is induced by multiple cues, such as light and temperature. However, the molecular mechanisms of how the environment induces diapause remains unclear. We are interested in the epigenetic regulation, especially the non-coding RNA, involved in environmentally induced diapause. The progeny eggs of bivoltine silkworm strains undergoing diapause or not depends on the incubation temperature during the maternal embryonic stage. In this research, we compared the differentially expressed microRNAs in embryos incubated under diapause-inducing temperature (25 °C) and non-diapause-inducing temperature (15 °C) in silkworm. Our results indicate that miRNAs respond to diapause-inducing temperature and regulate the expression of circadian clock genes. Our research not only provides reference for the study of the diapause induction process, but also gives insights into the role of epigenetic modification in diapause.Diapause is one of the survival strategies of insects for confronting adverse environmental conditions. Bombyx mori displays typical embryonic diapause, and offspring diapause depends on the incubation environment of the maternal embryo in the bivoltine strains of the silkworm. However, the molecular mechanisms of the diapause induction process are still poorly understood. In this study, we compared the differentially expressed miRNAs (DEmiRs) in bivoltine silkworm embryos incubated at diapause- (25 °C) and non-diapause (15 °C)-inducing temperatures during the blastokinesis (BK) and head pigmentation (HP) phases using transcriptome sequencing. There were 411 known miRNAs and 71 novel miRNAs identified during the two phases. Among those miRNAs, there were 108 and 74 DEmiRs in the BK and HP groups, respectively. By the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the predicted target genes of the DEmiRs, we found that aside from metabolism, the targets were also enriched in phototransduction-fly and insect hormone biosynthesis in the BK group and the HP group, respectively. Dual luciferase reporter assay illustrated that bmo-miR-6497-3p directly regulated Bmcycle and subsequently regulated the expression of circadian genes. These results imply that microRNAs, as vitally important regulators, respond to different temperatures and participate in the diapause induction process across species.

Design and evaluation of eco-friendly mountages for the silkworm, Bombyx mori L.

The silkworm Bombyx mori L. ends its larval stage with construction of silken armour called ‘cocoon’. The management of spinning larvae demands much care to maximize profit to the farmer, which necessitates manual picking of the ripened worms and provide proper anchorage to construct cocoons and the process is called as mounting. More than 40 % of the labour force is dedicated towards mounting (15 out of total 35 mandays/100 DFLs) that draws attention of the researchers striving hard to double farmer’s income by designing self mounting cocooning structures. Though corrugated plastic mountages are available, they are suitable only for Bivoltine (BV) silkworms that have a notable crawling habit. Ourefforts to design and develop new ecofriendly self mounting structures for mounting both crossbreeds (CB) and BV silkwormsevolved four new type of mountages viz., Spiral, Zig-zag, Square mountages made of bamboo and the Ribbon mountage. The lab tests of these mountages in comparison with the regular bamboo mountage and the plastic self mounting structures clearly indicated that the spiral mountages are comparable with the regular bamboo mountage with respect to various cocoon and reeling parameters among both CB (PM X CSR2) and BV double Hybrid (FC1 X FC2). Also these are self mounting cocooning frames that reduce the cost on labour during ripe worm management.

Studies on Nutrigenetic Traits of Silkworm, Bombyxmori L. for Determining Growth and Development for Identifying Parental Breeds for Breeding under Subtropical Region of North West India

Study was conducted during spring and autumn season (2018 & 2019) to screen and identify rich nutrigenetic breeds from the selected breed’s for their nutrigenetic traits in silkworm, BombyxmoriL.(Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds under Subtropical condition of Jammu based on the breeds which shows less food consumption with higher efficiency conversion based on leaf to cocoon and leaf to shell ratio. Highly significant variations were found among all nutrigenetic traits of bivoltine silkworm breeds in the study. The nutritionally efficient silkworm breeds were shortlisted by utilizing nutrition consumption index and efficiency for conversion of ingesta/cocoon traits as the index for selection of highly promising breeds. Furthermore, based on the average of data from both the seasons, the overall rearing nutrigenetic traits utilized as index, eight bivoltine silkworm breeds (B.con 1, B.con 4, BHR 2, ATR 16, BHR 3, CSR 50, RSJ 14 and NB4D2) were identified as havingthe potential for nutrition efficiency conversion and can be utilized for further breeding programme. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds/hybrids and their effective commercial utilization in the sericulture industry.

Production Activities and Value Chain Analysis of Sericulture in Western Inner Terai Region of Nepal

The study was conducted in Nov 2019 – Feb 2020 with the objective of assessing the whole value chain of sericulture products in the Western inner terai region of Nepal. The beginning of sericulture in the region was due to some personal interest but gained popularity and huge return and turned out to achieve the objectives of food security and source of employment. As per the finding and research, Bi-voltine Silkworm (Bombyx mori) was mainly reared which feeds upon the leaves of the mulberry plant. The interview result indicates that producers produce cocoon at an average of 30.05 kg per ropani in one production round and production was made twice a year. In this region, three marketing channels were identified (producer to the primary collector/small hand processor) and (producer to primary processor or reelers) and the producer himself as the processor and distributor. Producers sold the cocoon to primary collectors at NRs 500per kg cocoon which was 15% more than percent sales while selling to the government, fetching an average BC ratio of 1.3. Problems recorded as per farmers were lack of appropriate technology to widen the scope of sericulture and modern silkworm rearing techniques, lack of proper irrigation facilities, and governmental aids and support. This research will address various problems and make an emphasis to make regional sericulture production mature and profitable.

Prospectus for commercialization and sustenance of sericulture at high altitude Kargil-Ladakh

Sericulture has great potential to alleviate poverty and unemployment in the areas where sericulture has not been introduced. High altitude belts of the J & K, like Kargil where scope of other agricultural crops is limited due to short growing seasons, sericulture has the scope to be introduced for long term basis to ensure the livelihood security and better socio-economic status of the people from this tribal region. To see how economic activities in Kargil district can be given right focus, a study on silkworm rearing cum demonstration program was launched by CoTS, SKUAST-K, Mirgund at high altitude Kargil-Ladakh during July-August 2018 and 19. In order to assess the feasibility and sustainability of sericulture in the region, a popular bivoltine silkworm double hybrid (FC2 x FC1) was procured from Nodal Silkworm Seed Agency, NSSO, CSB, GoI, Bangalore which was reared for two consecutive years in three different border villages of Kargil viz, Akchamal, Mangbour and Shilikchey. Although prior to this, small scale silkworm rearing trails were conducted at Kargil during 2012-13, 2015-17 and 2018-19 by CoTS, SKUAST-K, Mirgund and CSRTI, CSB, Pampore respectively and have reported encouraging results which laid the basis of this study for further expansion and strengthening of sericulture activities in new far flung border areas of Kargil.. As such, the present investigation was aimed to test the validity of the results obtained in previous study trails for commercialization and popularization of sericulture in the region. The results obtained in the current investigation were encouraging and revealed highest hatching percentage of 98.70 and 97.80 during the year 2018 and 2019 respectively. Average Cocoon yield of 48.20 and 47.40 Kg/100 DFLs’ was obtained during 2018 and 19 respectively. Average Single Cocoon Weight (SCW) and average Single Shell Weight (SSW) of 1.78g and 0.38g was obtained during 2018. whereas in 2019 it was 1.76g and 0.39g respectively. Average Shell Ratio (SR%) of 21.34% and 22.15% was also obtained during the year 2018 and 19 respectively. Hence, results of the present investigation attest the validity of the previous study trails conducted in the region from time to time and established that sericulture can be promoted at high altitude Kargil region of Ladakh on sustainable basis, the prospectus of which are underlined in the study.

A 14-amino acids deletion in BmShadow results to non-moult on the 2nd instar in the bivoltine silkworm, Bombyx mori.

The Bombyx mori Shadow gene (BmShadow) belongs to the superfamily of cytochrome P450 genes. To elucidate the function of the BmShadow gene and its association with diapause, we employed the CRISPR/Cas9 system to knock out the BmShadow gene in the bivoltine strain Qiufeng. The mutant (BmShadow-/-) was obtained in G2, exhibiting a 42-base deletion corresponded exactly to the amino acids regions from positions 155 to 168. The larvae of BmShadow-/- cannot moult at the pre-moulting stage of the 2nd instar. When the BmShadow-/- larvae were fed with 20E analogue at the late stage of the 2nd instar, they were rescued and developed into the 3rd instar. Rescue experiments indicated that the 20E concentration of BmShadow-/- larvae was significantly lower than that in WT larvae, and the 20E concentration of BmShadow-/- larvae which fed 20E analogue was restored to normal levels. Interestingly, the BmShadow-/- larvae could not moult on the 1st instar when they hatched from eggs after being stored at 5°C for 40days or after hibernation, suggesting that the 20E transported from the mother was partially consumed in the diapause maintenance phase. Our study confirmed that BmShadow is involved in 20E synthesis and a 14-amino acids region from position 155 to 168 was essential for its function, also there appears to be no other compensation pathway in vivo, which offered an important potential target locus for the control of silkworm development and the biological control of agricultural and forestry pests.

Atypical voltinism in mulberry silkworm Bombyx mori L., 1758 (Lepidoptera: Bombycidae) races Barapat and Barapolu

The mulberry silkworm, Bombyx mori L., 1758 (Lepidoptera: Bombycidae) is classified as univoltine, bivoltine, and polyvoltine based on the annual brood frequency. Voltinism in B. mori is characterized by the occurrence of embryonic diapause (facultative and obligatory diapause). Univoltine silkworms suited to cold and temperate regions lay diapausing eggs and polyvoltine silkworms suited to tropical regions lay non-diapausing eggs. In contrast, the occurrence of diapause in bivoltine silkworms is influenced by the environment. In addition to the difference in the occurrence of embryonic diapause, larval duration, and cocoon characters show distinct variations. In the present correspondence, we are presenting an interesting observation from two silkworm races Barapath and Barapolu originating from the North-East region of India that display divergence in voltinism. The unique characteristic features of univoltine, bivoltine, and polyvoltine observed in the races are discussed explaining the occurrence.

Elucidating genetic divergence of silkworm (Bombyx mori) using internal transcribed Spacer1 (ITS1)

The silkworm Bombyx mori is one of the prominent molecular model system, being continuously evaluated through genetic approaches, since decades to better the best in silk production. DNA profiles provide reliable estimates of genetic diversity within and between genotypes. This is important both for the maintenance of genotypes as well as selection of parents for the development of elite hybrids. To ascertain genetic diversity and phylogenetic relationship among 23 bivoltine silkworm strains, DNA barcoding of ITS1 region and Mahalanobis D2 analysis was carried out. Neighbour-joining analysis grouped 23 strains into five clusters based on sequence variation of ITS1 region. Based on seven quantitative traits, the D2 values were obtained and the selected strains were grouped into three clusters using Tocher’s method. Cocoon yield by number (44.27%) and shell ratio (41.5%) recorded maximum contribution towards divergence. The clustering pattern revealed agreement for divergence as SKUAST-1 and SKUAST-22 formed an outgroup cluster at both molecular as well as genetic level. The current study clearly demonstrated that ITS1 sequences of B. mori were useful in studying intra specific nucleotide variations and can be used as DNA barcode for future B. mori studies. The divergent strains identified in this study could be used in future breeding programmes for quality silk production.