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 2^nd 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 5^th 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 4^th 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 2^nd instar.

Molting is an important physiological process in the larval stage of Bombyx mori and is controlled by various hormones and peptides. The silkworm mutant that exhibits the phenotype of non-molting in the 2^nd instar (nm2) is incapable of molting in the 2^nd instar and dies after seven or more days. The ecdysone titer in the nm2 mutant is lower than that in the wildtype, and the mutant can be rescued by feeding with 20E and cholesterol. The results of positional cloning indicated that structural alteration of BmCPG10 is responsible for the phenotype of the nm2 mutant. To explore the possible relationship between BmCPG10 and the ecdysone titer as well as the genes affected by BmCPG10, digital gene expression (DGE) profile analysis was conducted in the nm2 mutant, with the wildtype strain C603 serving as the control. The results revealed 1727 differentially expressed genes, among which 651 genes were upregulated and 1076 were downregulated in nm2. BLASTGO analysis showed that these differentially expressed genes were involved in various biological processes, cellular components and molecular functions. KEGG analysis indicated an enrichment of these differentially expressed genes in 240 pathways, including metabolic pathways, pancreatic secretion, protein digestion and absorption, fat digestion and absorption and glycerolipid metabolism. To verify the accuracy of the DGE results, quantitative reverse transcription PCR (qRT-PCR) was performed, focusing on key genes in several related pathways, and the results were highly consistent with the DGE results. Our findings indicated significant differences in cuticular protein genes, ecdysone biosynthesis genes and ecdysone-related nuclear receptors genes, but no significant difference in juvenile hormone and chitin biosynthesis genes was detected. Our research findings lay the foundation for further research on the formation mechanism of the nm2 mutant.

A new purple quail-like (q-l^p) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-l^p 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-l^p mutant in the 4^th 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-l^p. 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-l^p mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-l^p mutant; 2) more cuticle proteins are expressed in q-l^p 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-l^p mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-l^p mutant.

Neuropeptides are endogenous active substances that play important roles in a number of physiological processes and are ubiquitous in the nervous tissue in vivo. The gene encoding pedal peptide/orcokinin-type (PP/OK-type) neuropeptide is an important member of the neuropeptide gene family and is ubiquitous in invertebrates of Bilateria; orcokinin (OK) is mainly found in Arthropoda, while pedal peptide (PP) is mainly found in Mollusca. OK and PP are also present in other animals. PP/OK-type neuropeptides are a kind of multifunctional neuropeptides predominantly expressed in the nervous tissue and play important roles in the nerve regulation of movement. Moreover, OK has a number of other physiological functions. This review describes the distribution, expression, function and maturation of PP/OK-type neuropeptides to facilitate investigations of new functions and receptors of PP/OK-type neuropeptides, providing the theoretical foundation for the potential use of PP/OK-type neuropeptides in the prevention and control of agricultural and forestry pests, as an additive for skin care products and in the screening of drugs for the treatment of diabetes.