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Diapause is an important characteristic of insects used to adapt to extreme changes in environmental conditions. Embryonic diapause of the bivoltine silkworm (Bombyx mori) is determined by environmental conditions experienced by the mother while in the embryo stage. If they are incubated at 25°C with natural light, their progenies will be diapause‐destined. If they are incubated at 17°C in darkness, their progenies will be non‐diapause‐destined. The molecular mechanism of diapause remains unknown. In the present study, we analyzed two downregulated genes (BGIBMGA003835, BGIBMGA012335) and two upregulated genes (BGIBMG012996, BGIBMG002426) related to carbohydrate metabolism, verified differentially expressed in ovaries and heads of 1‐day‐old fifth‐instar larvae to 6‐day pupae by quantitative real‐time reverse transcription polymerase chain reaction (qRT‐PCR). In line with published data, the expression level of these genes in larvae were generally lower than in pupae. We further analyzed the expression levels of the four genes in BmN cells that had been treated with various concentrations of diapause hormone (DH). It demonstrated that the expression of these genes was affected by DH. Knockdown of the selected genes in non‐diapause‐destined female pupae changed the fate of the progeny from non‐diapause‐ to daipause‐destined, as seen by the appearance of diapause eggs. Our study provides insight into the molecular mechanism of diapause in B. mori. The expression levels of BGIBMGA003835, BGIBMGA012335, BGIBMG012996 and BGIBMG002426 were different at diverse developmental stages. They can also be influenced by Bmo‐DH. RNAi if several genes can turn over the fate of non‐diapause eggs. [ABSTRACT FROM AUTHOR]

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant. [ABSTRACT FROM AUTHOR]

In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene’s structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7—dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition protein (PGRP) binds specifically to peptidoglycan and plays an important role as a pattern recognition receptor in the innate immunity of insects. The cDNA of a short-type PGRP, an open reading frame of 588 bp encoding a polypeptide of 196 amino acids, was cloned from Bombyx mori. A phylogenetic tree was constructed, and the results showed that BmPGRP-S2 was most similar to Drosophila melanogaster PGRP ( DmPGRP-SA). The induced expression profile of BmPGRP-S2 in healthy Escherichia coli- and Bacillus subtilis-challenged B. mori was measured using semiquantitative reverse transcriptase polymerase chain reaction analysis. The expression of BmPGRP-S2 was upregulated at 24 h by E. coli and Ba. subtilis challenge. In addition, in the integument of B. mori, RNAi knockdown of BmPGRP-S2 caused an obvious reduction in the transcription expression of the transcription factor Relish and in antibacterial effector genes Attacin, Gloverin, and Moricin. The results indicated that BmPGRP-S2 participates in the signal transduction pathway of B. mori. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are an abundant family of endogenous noncoding small RNA molecules. They play crucial roles on regulation of life processes both in plants and animals. Fibroin modulator binding protein-1 (FMBP-1) is a silk gland transcription factor of Bombyx mori, which is considered as a trans-activator of fibroin genes. And bioinformatics prediction showed that at the 3' untranslated region (3' UTR) of BmFMBP-1 there were binding sites for three bmo-miRNAs, bmo-miR-2b*, bmo-miR-305, and bmo-miR-2758, separately. In order to validate whether these bmo-miRNAs involved in the regulation of BmFMBP-1 expression, the expression levels of three bmo-miRNAs and BmFMBP-1 in the middle silk gland (MSG) and posterior silk gland (PSG) during the fourth- and fifth-larval stages of B. mori were measured by semi-quantitative reverse transcription polymerase chain reaction. The results revealed that the expression level of bmo-miR-2758 was the highest in the three, and it expressed higher in the PSG than in the MSG with a similar expression pattern as BmFMBP-1, implying that bmo-miR-2758 may involved in regulation of BmFMBP-1. To validate the regulation function of bmo-miR-2758 on BmFMBP-1, recombinant plasmids pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] and pGL3 [A3-luc-FMBP-1 3' UTR-SV40] were constructed and co-transfected in BmN cells. The dual-luciferase reporter assay system was used for assay of transient expression. The results showed that the expression of the luciferase reporter was significantly decreased when pGL3 [A3-luc-FMBP-1 3' UTR-SV40] co-transfected with pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] (P <  .01). Furthermore, when the artificial antisense RNA of bmo-miR-2758 (inhibitor) was added to the above co-transfection, the expression of the luciferase reporter was recovered significantly (P < 0.01). These results suggest that bmo-miR-2758 represses the expression of BmFMBP-1 in vitro., (© The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.)

The heat shock cognate 70-4 protein gene promoter (HSC70-4p) from Bombyx mori (BmHSC70-4p) is an ideal candidate for the transgenic silkworm due to its high transcriptional activity, and the homologous region 3 from Bombyx mori nucleopolyhedrovirus (BmNPVhr3) functions as an enhancer for several promoters. Using luciferase as a reporter gene and transient expression system in vivo and in vitro, we found that BmNPVhr3 can significantly increase the transcriptional activity of BmHSC70-4p and HSC70-4p from Bombyx mandarina (BmandHSC70-4p). Moreover, the transcriptional activity of the combination of BmHSC70-4p and BmNPVhr3 changed with developmental stages and hormone titers. These results suggest that the combination of BmHSC70-4 promoter and BmNPVhr3 enhancer is more effective candidate for the transgene or stable cell expression system in Bombyx mori.