Mechanism of programmed cell death in the posterior silk gland of the silkworm, Bombyx mori, during pupation based on Ca2+ homeostasis.

The silkworm, Bombyx mori, is a complete metamorphosed economic insect, and the silk gland is a significant organ for silk protein synthesis and secretion. The silk gland completely degenerates during pupation, but the regulatory mechanism of programmed cell death (PCD) has not yet been understood. In the present study, we investigated the non-genetic pathway of 20E-induced PCD in the posterior silk gland (PSG) based on intracellular Ca2+ levels. Silk gland morphology and silk gland index indicated rapid degeneration of silk gland during metamorphosis from mature silkworm (MS) to pupal day 1 (P1), and Ca2+ levels within the PSG were found to peak during the pre-pupal day 1 (PP1) stage. Moreover, the results of autophagy and apoptosis levels within the PSG showed that autophagy was significantly increased in MS-PP1 periods, and significantly decreased in PP2 and P1 periods. Apoptosis was almost absent in MS-PP1 periods and significantly increased in PP2 and P1 periods. Additionally, western blotting results showed that autophagy preceded apoptosis, and the autophagy-promoting ATG5 was cleaved by calpain to the autophagy-inhibiting and apoptosis-promoting NtATG5 since PP1 period, while decreased autophagy was accompanied by increased apoptosis. Collectively, these findings suggest that Ca2+ is a key factor in the shift from autophagy to apoptosis.

Effects of artificial diet breeding on intestinal microbial populations at the young stage of silkworm (Bombyx mori).

The silkworm (Bombyx mori) is an economically important insect and serves as a model organism for Lepidoptera. To investigate the effects of the intestinal microbial population on the growth and development of larvae fed an artificial diet (AD) during the young stages, we analyzed the characteristics of the intestinal microbial population using 16S rRNA gene sequencing technology. Our results revealed that the intestinal flora of the AD group tended to be simple by the 3rd-instar, which Lactobacillus accounting for 14.85% and leading to a decreased pH in the intestinal fluid. In contrast, the intestinal flora of silkworms in the mulberry leaf (ML) group showed continuous growth of diversity, with Proteobacteria accounting for 37.10%, Firmicutes accounting for 21.44%, and Actinobacteria accounting for 17.36%. Additionally, we detected the activity of intestinal digestive enzymes at different instars and found that the activity of digestive enzymes in the AD group increased by larval instar. Protease activity in the AD group was lower during the 1st- to 3rd-instars compared to the ML group, while α-amylase and lipase activities were significantly higher in the AD group during the 2nd- and 3rd-instar compared to the ML group. Furthermore, our experimental results indicated that changes in the intestinal population decreased the pH and affected the activity of proteases, which might contribute to the slower growth and development of larvae in the AD group. In summary, this study provides a reference for investigating the relationship between artificial diet and intestinal flora balance.

Determination of suitable reference genes for RT-qPCR normalisation in Bombyx mori (Lepidoptera: Bombycidae) infected by the parasitoid Exorista sorbillans (Diptera, Tachinidae).

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.

Low concentration chlorantraniliprole-promoted Ca2+ release drives a shift from autophagy to apoptosis in the silk gland of Bombyx mori.

The novel pesticide chlorantraniliprole (CAP) is widely used for pest control in agriculture, and the safety for non-target organisms of trace residues in the environment has received widespread attention. In the present study, exposure to low concentrations of CAP resulted in abnormal silk gland development in the B. mori, and induced the release of intracellular Ca2+ in addition to the triggering of Ca2+-dependent gene transcription. Moreover, the CAP treatment group exhibited down-regulation of oxidative phosphorylation and antioxidant enzyme-related genes in the silk gland, resulting in peroxide accumulation. Furthermore, transcript levels of autophagy-related genes were significantly up-regulated and protein levels of LC3-I and LC3-II were up-regulated, indicating an increase in autophagy. The protein levels of ATG5 and NtATG5 were also significantly up-regulated. While the protein levels of caspase3 and active caspase3 were significantly up-regulated consistent with the transcript levels of key genes in the apoptotic signaling pathway, ultimately affecting silk protein synthesis. Overall, these findings indicate that low concentration CAP induced abnormal development in the silk gland of B. mori by causing intracellular Ca2+ overload, which inhibits oxidative phosphorylation pathway and the removal of reactive oxygen species, leading to a driving a shift from autophagy to apoptosis. The findings herein provided a basis for evaluating the safety of CAP environmental residues on non-target organisms.

JH degradation pathway participates in hormonal regulation of larval development of Bombyx mori following λ-cyhalothrin exposure.

λ-Cyhalothrin (λ-cyh), a widely utilized pyrethroid insecticide, poses serious threats to non-target organisms due to its persistence nature in the environment. Exposure to low concentrations of λ-cyh has been observed to result in prolonged larval development in Bombyx mori, leading to substantial financial losses in sericulture. The present study was undertaken to elucidate the underlying mechanisms for prolonged development caused by λ-cyh (LC10) exposure. The results showed that the JH Ⅲ titer was significantly increased at 24 h of λ-cyh exposure, and the JH interacting genes Methoprene-tolerant 2, Steroid Receptor Co-activator, Krüppel-homolog 1, and JH binding proteins were also up-regulated. Although the target of rapamycin (Tor) genes were induced by λ-cyh, the biosynthesis of JH in the corpora allata was not promoted. Notably, 13 JH degradation genes were found to be significantly down-regulated in the midgut of B. mori. The mRNA levels and enzyme activity assays indicated that λ-cyh had inhibitory effects on JH esterase, JH epoxide hydrolase, and JH diol kinase (JHDK). Furthermore, the suppression of JHDK (KWMTBOMO01580) was further confirmed by both western blot and immunohistochemistry. This study has offered a comprehensive perspective on the mechanisms underlying the prolonged development caused by insecticides, and our results also hold significant implications for the safe production of sericulture.

Parasitism by the Tachinid Parasitoid Exorista japonica Leads to Suppression of Basal Metabolism and Activation of Immune Response in the Host Bombyx mori.

The dipteran tachinid parasitoids are important biocontrol agents, and they must survive the harsh environment and rely on the resources of the host insect to complete their larval stage. We have previously demonstrated that the parasitism by the tachinid parasitoid Exoristajaponica, a pest of the silkworm, causes pupation defects in Bombyx mori. However, the underlying mechanism is not fully understood. Here, we performed transcriptome analysis of the fat body of B. mori parasitized by E. japonica. We identified 1361 differentially expressed genes, with 394 genes up-regulated and 967 genes down-regulated. The up-regulated genes were mainly associated with immune response, endocrine system and signal transduction, whereas the genes related to basal metabolism, including energy metabolism, transport and catabolism, lipid metabolism, amino acid metabolism and carbohydrate metabolism were down-regulated, indicating that the host appeared to be in poor nutritional status but active in immune response. Moreover, by time-course gene expression analysis we found that genes related to amino acid synthesis, protein degradation and lipid metabolism in B. mori at later parasitization stages were inhibited. Antimicrobial peptides including Cecropin A, Gloverin and Moricin, and an immulectin, CTL11, were induced. These results indicate that the tachinid parasitoid perturbs the basal metabolism and induces the energetically costly immunity of the host, and thus leading to incomplete larval-pupal ecdysis of the host. This study provided insights into how tachinid parasitoids modify host basal metabolism and immune response for the benefit of developing parasitoid larvae.

Comparison of a novel MPN method against the yeast extract agar (YEA) pour plate method for the enumeration of heterotrophic bacteria from drinking water.

This study compared the Quanti-Disc most probable number (MPN) test for heterotrophic bacteria from drinking water with the widely used yeast extract agar (YEA) pour plate method. The Quanti-Disc test module contains 50 reaction wells in which a medium has been pre-deposited. The medium contains a suite of three fluorogenic enzyme substrates selected for the detection of enzymes expressed widely by heterotrophic bacteria. The MPN of heterotrophic bacteria is calculated from the number of fluorescing reaction wells after incubation of a sample. Quanti-Disc and the YEA pour plate method were compared according to guidance on comparing methods given in United Kingdom national guidance and ISO 17994:2004. The two methods were also challenged with reference strains and isolates of heterotrophic bacteria from drinking water. This indicated that heterotrophic bacteria commonly encountered in drinking water are detected by both the YEA pour plate method and Quanti-Disc. Analysis of data from split water samples (723 for 37 degrees C tests and 872 for 22 degrees C tests) from nine geographically diverse laboratories in England and Wales demonstrated that the Quanti-Disc method is equivalent to the YEA pour plate method for the analysis of heterotrophic bacteria from drinking and similar waters at 37 degrees C, and superior to YEA for the analysis at 22 degrees C. The Quanti-Disc method is a simple and efficient alternative method for the enumeration of heterotrophic bacteria from drinking water.