Pebrine disease, caused by Nosema bombycis (Nb) infection in silkworms, is a severe and long-standing disease that threatens sericulture. As parasitic pathogens, a complex relationship exists between microsporidia and their hosts at the mitochondrial level. Previous studies have found that the translocator protein (TSPO) is involved in various biological functions, such as membrane potential regulation, mitochondrial autophagy, immune responses, calcium ion channel regulation, and cell apoptosis. In the present study, we found that TSPO expression in silkworms (BmTSPO) was upregulated following Nb infection, leading to an increase in cytoplasmic calcium, adenosine triphosphate, and reactive oxygen species levels. Knockdown and overexpression of BmTSPO resulted in the promotion and inhibition of Nb proliferation, respectively. We also demonstrated that the overexpression of BmTSPO promotes host cell apoptosis and significantly increases the expression of genes involved in the immune deficiency and Janus kinase-signal transducer and the activator of the transcription pathways. These findings suggest that BmTSPO activates the innate immune signalling pathway in silkworms to regulate Nb proliferation. Targeting TSPO represents a promising approach for the development of new treatments for microsporidian infections.

The silkworm Bombyx mori (Lepidoptera: Bombycidae) is a lepidopteran model insect of great economic importance. The parasitoid Exorista sorbillans (Diptera, Tachinidae) is the major pest of B. mori and also a promising candidate for biological control. However, the molecular interactions between hosts and dipteran parasitoids have only partially been studied. Gene expression analysis by reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is indispensable to characterise their interactions. Accurate normalisation of RT-qPCR-based gene expression requires the use of reference genes that are constantly expressed irrespective of experimental conditions. In this study, the expression stability of 13 traditionally used reference genes was estimated by five statistical algorithms (ΔCt, geNorm, Normfinder, BestKeeper, and RefFinder) to determine the best reference genes for gene expression studies in different tissues of B. mori under E. sorbillans parasitism. Specifically, TATA-box-binding protein was the best reference gene in epidermis and testis, while elongation factor 1α was the most stable gene in prothoracic gland and midgut. Elongation factor 1γ, ribosomal protein L3, actin A1, ribosomal protein L40, glyceraldehyde-3-phosphate dehydrogenase and eukaryotic translation initiation factor 4A were the most suitable genes in head, silk gland, fat body, haemolymph, Malpighian tubule and ovary, respectively. Our study offers a set of suitable reference genes for gene expression normalisation in B. mori under the parasitic stress of E. sorbillans, which will benefit the in-depth exploration of host-dipteran parasitoid interactions, and also provide insights for further improvements of B. mori resistance against parasitoids and biocontrol efficacy of dipteran parasitoids.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

Many parasites manipulate host behaviour to enhance their transmission. Baculoviruses induce enhanced locomotory activity (ELA) combined with subsequent climbing behaviour in lepidopteran larvae, which facilitates viral dispersal. However, the mechanisms underlying host manipulation system are largely unknown. Previously, larval locomotion during ELA was summarized as the distance travelled for a few minutes at several time points, which are unlikely to characterize ELA precisely, as ELA typically persists for several hours. In this study, we modified a recently developed method using time-lapse recording to characterize locomotion of Bombyx mori larvae infected with B. mori nucleopolyhedrovirus (BmNPV) for 24 h at 3 s resolution. Our data showed that the locomotion of the mock-infected larvae was restricted to a small area, whereas the BmNPV-infected larvae exhibited a large locomotory area. These results indicate that BmNPV dysregulates the locomotory pattern of host larvae. Furthermore, both the mock- and BmNPV-infected larvae showed periodic cycles of movement and stationary behaviour with a similar frequency, suggesting the physiological mechanisms that induce locomotion are unaffected by BmNPV infection. In contrast, the BmNPV-infected larvae exhibited fast and long-lasting locomotion compared with mock-infected larvae, which indicates that locomotory speed and duration are manipulated by BmNPV.

The Murshidabad District of West Bengal, India has practised traditional mulberry sericulture since time immemorial. One of the most important aspects for sustainable development of the sericulture industry is the control and prevention of silkworm diseases. The main objective of this study was to determine silkworm disease incidence over the period 1992–2011 in Murshidabad District and how environmental factors have contributed towards their occurrence. Data were collected from a crop-wise survey of silkworm diseases from 25 farmers (five farmers from five villages), who each had a separate rearing house, were progressive and had a capacity of rearing at least 100 disease free layings (dfls) in each crop. Disease incidence was expressed in percentage and calculated taking into consideration 40,000 larvae per 100 dfls. Crop-wise silkworm disease mortality data were correlated with meteorological data. Data collected from the continuous survey conducted in the district during 1992–2011 revealed that there was no set pattern of occurrence of silkworm diseases. However, maximum mortality (up to 30%) of silkworm due to diseases in all the seasons occurred mainly due to grasserie, as relative humidity remains high for most part of the year in this area. However, other than grasserie, for most of the period, disease incidence was below 10%. This observation of the dominance of grasserie over other diseases in causing silkworm mortality calls for renewed emphasis on the preventive measures and development of disease forecasting models, for disease control. Another observation was that since 1993, pebrine, the deadliest disease of the silkworm, has not been reported from the farmers’ fields.

The present study was undertaken to determine the nutritional value of the fifth instar larva of Bombyx mori, locally referred to as ‘Niang ryndai’, which is a popular edible silkworm among the local inhabitants of East and West Khasi Hills and Ri-Bhoi district. Larvae were collected from the Iewduh market of Shillong in the months of October and November as these larvae are available during these months. The composition of lipids, amino acids and the fat-soluble vitamins A and E in the frozen and oven-dried samples of B. mori was estimated and compared with that of conventional food items. The detection of the fatty acids eicosapentaenoic acid and docosahexaenoic acid in the larva indicates its potential to support a healthy cardiovascular function. Because of their use for the production of silk yarn, silkworms have economic potential for the people of the State and north-eastern region of India. Increased appreciation of this insect as an alternative and cheap source of nutrition would contribute to the socio-economic development of the indigenous people.

Infection of the commercially important silkworm, Bombyx mori by a tachnid parasitoid, Exorista bombycis induced activation of genes and cellular responses associated with apoptosis in integumental epithelial cells. Composite cellular profile showed initial autophagy, intermediate endoplasmic reticulum degranulation and deformed nucleus as well as later DNA fragmentation indicating apoptosis. Two cell death-associated proteins, autophagy 5-like (Atg5L) and apoptosis-inducing factor (AIF), in addition to caspase, are identified from the infected integumental epithelium through mass spectrometric analysis. Genes encoding these proteins showed age-dependent activation after the infection as revealed by quantitative expression analysis. Atg5 showed early upregulation in association with signs of autophagy whereas AIF showed late upregulation in association with DNA condensation and fragmentation. Expression of AIF showed negative correlation with that of Atg5 after the infection. On the other hand, in control, caspase expression showed positive correlation with AIF expression indicative of regulated expression in normal larval epithelium, which was absent after infection. Activation of Atg5, AIF and caspase genes in close association with different cell death events revealed the synchronized differential expression of apoptosis-associated genes in response to the macroparasitism. Enhanced expression of Atg5, AIF and caspase genes coupled with the appearance of cell death symptoms indicate parasitism-induced activation of genetic machinery to modulate cell death events in the epithelium, which was hither to unknown in invertebrate systems.

Pebrine caused by Nosema bombycis in the silkworm Bombyx mori L. causes severe economic loss to the sericulture industry. Several species of microsporidia and strains of N. bombycis have been isolated from infected silkworms. The study of the developmental stages of any parasite is important, as it leads to the identification of stages that may be susceptible to control measures. For this study, five microsporidian isolates from B. mori were collected from five different locations in West Bengal, India and a study of the developmental stages from mid-gut smears and histological techniques was undertaken. The observations of mid-gut smears and histological specimens revealed differences in the morphometry and duration of different developmental stages of the parasites.

Inter simple sequence repeats (ISSR) and random amplified DNA polymorphism (RAPD) markers were used to estimate the genetic diversity among 14 tropical silkworm races (Bombyx mori L.) for identifying potential parents to be used for hybrid preparation for commercial exploitation. High polymorphism (70.91 and 74.70%) was revealed by ISSR and RAPD markers. The dendrograms generated, using unweighted pair group method using arithmetic average from these markers, grouped the 14 silkworm races into two major groups, which corroborates the differences in cocoon characteristics. Discriminant function analysis of ISSR and RAPD markers identified three functions for cocoon weight and two functions for shell weights, respectively. Step-wise multiple regression analysis identified six ISSR markers (834500, 8841700, 8841850, 8271500, 8401500 and 7891250) and seven RAPD markers (834500, 885900, 8101400, 884900, 8361500, 7891250 and 7621700) significantly associated with cocoon and shell weights. The genetically divergent parents, identified through this study, can be used for the preparation of hybrids for commercial utilization. Similarly, the DNA markers identified can be utilized for marker-assisted selection along with those identified through quantitative trait locus mapping by others. Thus the information generated in this study is useful in silkworm breeding programmes in the tropical sericulture industry.

In this study, silkworm moth (Bombyx mori L.) larvae were regarded as an animal protein source for astronauts in the bioregenerative life support system during long-term deep space exploration in the future. They were fed with mulberry and stem lettuce leaves during the first three instars and the last two instars, respectively. In addition, this kind of environmental approach, which utilised inedible biomass of plants to produce animal protein of high quality, can likewise be applied terrestrially to provide food for people living in extreme environments and/or impoverished agro-ecosystems, such as in polar regions, isolated military bases, ships, submarines, etc.

Respiration characteristics of the larvae during development under two main physiological conditions, namely eating and not-eating of leaves, were studied. Nutrient compositions of silkworm powder (SP), ground and freeze-dried silkworms on the 3rd day of the 5th instar larvae, including protein, fat, vitamins, minerals and fatty acids, were measured using international standard methods.

Silkworms’ respiration rates, measured when larvae were eating mulberry leaves, were higher than those of similar larvae that hadn't eaten such leaves. There was a significant difference between silkworms fed on mulberry leaves and those fed on stem lettuce in the 4th and 5th instars (P<0.01). Amounts of CO2 exhaled by the silkworms under the two physiological regimes differed from each other (P<0.01). There was also a significant difference between the amount of O2 inhaled when the insects were under the two physiological statuses (P<0.01). Moreover, silkworms’ respiration quotient under the eating regime was larger than when under the not-eating regime. The SP was found to be rich in protein and amino acids in total; 12 essential vitamins, nine minerals and twelve fatty acids were detected. Moreover, 359 kcal could be generated per 100 gram of SP (dry weight).

Diapause hormone (DH) and pheromone biosynthesis-activating neuropeptide (PBAN) code for the diapause gene (DH-PBAN) of Bombyx mori Linnaeus. The DH-PBAN gene sequence from the BLAST database was searched against B. mori genomic gene sequences for similarity. Results showed maximum homology with a genomic contig (accession no. D16230). Primers were designed for the intron, exon and promoter regions of the DH-PBAN gene sequence. The corresponding regions were amplified using genomic DNA as template and products of diapause and non-diapause silkworm races were compared. There was no variation in the 425 bp intron and 254 bp exon regions. However, 1.3% nucleotide sequence variation was observed in selected silkworm races. The promoter region showed 4.2% single nucleotide variation in the diapause and non-diapause silkworm races. The promoter region of the diapause gene showed variations in the sequence of the POU (Pit 1, Oct 1 and Unc-86)-binding site of non-diapause and diapause races of B. mori. In phylogenetic analysis, B. mori formed a cluster with other silkworms of the Bombycidae family, whereas the Noctuidae family formed a separate cluster, indicating that the diapause gene is conserved across the insect taxa.

The performance of the Bombyx mori Linnaeus bivoltine hybrid, Shaanshi BV-333, was evaluated on six mulberry cultivars of Morus spp. based on economic characters in rearing and mulberry leaf quality. The growth rate and morphological characteristics for all the cultivars were studied using several parameters. Specific characters such as disease resistance, survival percentage, cocoon weight, cocoon shell weight and single cocoon filament length were recorded during the rearing of silkworm larvae. Tested cultivars significantly differed in all parameters. Cultivars Kanva-2/M5, Thailand, Thika and S-36 exhibited superiority in rearing performance over other tested cultivars. Silkworms fed with cv. Thailand had the highest survival percentage of larvae in the short (S1) and long (S2) rainy seasons as compared with other cultivars. However, cv. Embu produced the lowest mortality during the dry season (S3), while S-41 produced the lowest survival rate in all seasons. Embu had significantly higher filament length and cocoon yield compared with the other cultivars during S3. However, S-41 performed poorly in survival percentage, cocoon yield, silk reeling and cocoon shell weight.

An amplification fragment length polymorphism (AFLP) molecular linkage map with a relatively high density for location of quantitative trait loci (QTLs) controlling the quantitative traits of silkworm (Bombyx mori) cocoons, was constructed using 91 individuals of the F2 generation. Among the 692 effective loci, 550 were allocated to subgroups a and b, of which 21 linkage groups in subgroup a had 233 molecular markers and 28 linkage groups in subgroup b had 317 markers. The number of markers on each linkage group in subgroups a and b ranged from 4 to 43 and 3 to 35, respectively. The total length of linkage groups for subgroup a was 1868.10 cM, and 2677.50 cM for subgroup b. The length of linkage group varied from 22.3 to 424.3 cM in subgroup a and from 2.4 to 366.5 cM in subgroup b. The average variation in the distance between markers was 3.39–17.43 cM in subgroup a and 0.8–26.96 cM in subgroup b. The average distance between the markers was 8.81 cM in subgroup a and 9.26 cM in subgroup b. There were 14 linkage groups, with an average distance below 10 cM, in subgroup a and 18 linkage groups in subgroup b. There were seven linkage groups with an average distance between 10 and 20 cM in subgroup a and ten linkage groups in subgroup b. Each linkage group in subgroup a had 11.1 loci on average, while there were 11.31 loci on each linkage group on average in subgroup b. The mean length for linkage groups in subgroups a and b was 89 and 95.6 cM, respectively. The total average length for both a and b subgroups was 2272.8, and 9.06 cM for the average marker distance, fulfilling the basic known requirements for locating QTLs.

Rearing temperature has an important influence on the efficiency of food utilization by larvae of the silkworm Bombyx mori L. To assess food intake and utilization efficiency in B. mori reared at different temperature combinations, larvae of Pure Mysore race were fed ad libitum on M5 mulberry leaves and reared at four different constant temperatures, viz. 26, 28, 30 and 32°C during instars I to III and combinations of these temperatures during later instars (IV and V). The larvae reared at 28°C (T2) when young and combinations of temperatures 26–26, 28–26, 30–26 and 32–26°C later were taken as controls. The amount of dry food consumed was significantly higher, but a significant decrease in conversion efficiencies (K1 and K2) was noticed in silkworms reared young at 28°C than at the other temperatures. However, the dry food consumed, food converted, assimilation rate and assimilation efficiency increased with the increasing temperature, and larval duration and conversion efficiency decreased significantly with the rise in temperature during both young- and late-instar rearing. Comparisons between these two ages with regard to food utilization parameters were carried out and the factors responsible for these changes are discussed.

Fluoride accumulation and the relative fluoride loading of different body parts in the wild mulberry silkworm (Bombyx mandarina M.), mulberry geometrid (Pathonandria atrineata B.) and mulberry silkworm (Bombyx mori L.) near fluoride pollution sources in the main sericultural region of China were studied. The results showed that the insects had an increased fluoride accumulation if compared to their feed, and that there were wide differences between the different insect species with the same feed and within the same polluted site. The digestive tract of the insect retained much higher fluoride content than other body parts. Moreover, a highly significant correlation was found between fluoride content in the studied insects and that of the mulberry leaves on which they were collected. Of the three insects studied, the mulberry geometrid appears to be most adapted to environmental fluoride, while the domestic silkworm is the least able to tolerate high fluoride levels, experiencing high mortalities at 94.3 mg/kg.

Mulberry (Morus spp.) was found to be the most sensitive plant to air-borne fluoride pollution among six plants studied in polluted areas, and could be a useful bioindicator to monitor environmental pollution. The highest fluoride content was found in the oldest leaves, and in plants growing closest to and downwind from the pollution source.

The hormetic influence on silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) of the juvenile hormone mimic ω-formyl longifolene oxime propargyl ether (NL13) was assayed. Emulsions containing 2.5, 5.0 and 10.0 ppm of the compound were administered topically as a single dose, to two popular commercial silkworm hybrids, viz. KA × NB4D2 and PM × NB4D2, at 24,48, 72 and 96 h into the 5th instar, and economic characters of the larvae and resultant cocoons measured. A medium and absolute control were maintained in parallel. It was found that administration of 5 ppm of NL13 to 48-h-old 5th instars resulted in the maximum improvement in commercial traits. The possible role of exogenous JH-like compounds in eliciting this response in silkworm is discussed.

The effect of fluoride on the food and dietary water intake and utilisation were studied in final instars of silkworm, Bombyx mori L. race NB4D2 fed ad libitum with mulberry leaves treated with sodium fluoride at concentrations of 25 ppm, 50 ppm and 75 ppm. The larval duration increased by two days while food consumption, assimilation rate, assimilation efficiency, feeding and metabolic rates increased significantly in all the treated larvae. On the other hand, the amount of faeces produced, food conversion, conversion rate and conversion efficiencies decreased significantly in all the treated batches when compared to controls. The water intake and utilisation parameters increased significantly while rate of water loss through faeces, water retained in the body and retention efficiency decreased in all the fluoride-treated silkworms. The factors responsible for these changes in treated batches are discussed in relation to the effect of fluoride on cocoon and shell weights.

Experiments on the effects of KK-42, an anti-juvenile hormone imidazole derivative, on the rearing and reproduction of Bombyx mori L. were carried out in a hot, wet summer in West Bengal, India. A single dose of 3 μg KK-42/larva induced precocious metamorphosis in a bivoltine hybrid (KPGB × P5) race of B. mori when applied topically to freshly moulted (less than 1-h old) fourth instars, and increased their rearing performance over control tetramoulters. However, cocoon and shell weights, shell ratio and filament length were reduced by the anti-JH treatment. Final body weight and head capsule width of the treated fourth instars were significantly higher than those of the controls. Bivoltine trimoulter males also showed good capacity for mating with multivoltine Nistari females. The possibility of obtaining three-way multi-bi hybrid eggs during the wet part of summer in a tropical climate is discussed.