Overexpression of Ultrabithorax Changes the Development of Silk Gland and the Expression of Fibroin Genes in Bombyx mori.

Ultrabithorax (Ubx) is a member of the Hox gene group involved in cell fate decisions, cell proliferation and organ identity. Its function has been extensively researched in Drosophila melanogaster but little is known about it in Lepidoptera. To uncover the function of Ubx in the development of lepidopterans, we constructed the Ubx overexpression (UbxOE) strain based on the Nistari strain of Bombyx mori. The UbxOE strain showed a small body size, transparent intersegmental membrane and abnormal posterior silk gland (PSG). In the current study, we focused on the effect of Ubx overexpression on the posterior silk gland. As the major protein product of PSG, the mRNA expression of fibroin heavy chain (Fib-H) and fibroin light chain (Fib-L) was upregulated three times in UbxOE, but the protein expression of Fib-H and Fib-L was not significantly different. We speculated that the overexpression of Ubx downregulated the expression of Myc and further caused abnormal synthesis of the spliceosome and ribosome. Abnormalities of the spliceosome and ribosome affected the synthesis of protein in the PSG and changed its morphology.

Transcriptome reveal the response to Cry1Ac toxin in susceptible Bombyx mori.

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain.

Transcriptomic dissection of termite gut microbiota following entomopathogenic fungal infection.

Termites are social insects that live in the soil or in decaying wood, where exposure to pathogens should be common. However, these pathogens rarely cause mortality in established colonies. In addition to social immunity, the gut symbionts of termites are expected to assist in protecting their hosts, though the specific contributions are unclear. In this study, we examined this hypothesis in Odontotermes formosanus, a fungus-growing termite in the family Termitidae, by 1) disrupting its gut microbiota with the antibiotic kanamycin, 2) challenging O. formosanus with the entomopathogenic fungus Metarhizium robertsii, and finally 3) sequencing the resultant gut transcriptomes. As a result, 142531 transcripts and 73608 unigenes were obtained, and unigenes were annotated following NR, NT, KO, Swiss-Prot, PFAM, GO, and KOG databases. Among them, a total of 3,814 differentially expressed genes (DEGs) were identified between M. robertsii infected termites with or without antibiotics treatment. Given the lack of annotated genes in O. formosanus transcriptomes, we examined the expression profiles of the top 20 most significantly differentially expressed genes using qRT-PCR. Several of these genes, including APOA2, Calpain-5, and Hsp70, were downregulated in termites exposed to both antibiotics and pathogen but upregulated in those exposed only to the pathogen, suggesting that gut microbiota might buffer/facilitate their hosts against infection by finetuning physiological and biochemical processes, including innate immunity, protein folding, and ATP synthesis. Overall, our combined results imply that stabilization of gut microbiota can assist termites in maintaining physiological and biochemical homeostasis when foreign pathogenic fungi invade.

Identification and Characterization of Genes Related to Resistance of Autographa californica Nucleopolyhedrovirus Infection in Bombyx mori.

In Bombyx mori, as an important economic insect, it was first found that some strains were completely refractory to infection with Autographa californica nucleopolyhedrovirus (AcMNPV) through intrahemocelical injection; whereas almost all natural strains had difficulty resisting Bombyx mori nucleopolyhedrovirus (BmNPV), which is also a member of the family Baculoviridae. Previous genetics analysis research found that this trait was controlled by a potentially corresponding locus on chromosome 3, but the specific gene and mechanism was still unknown. With the help of the massive silkworm strain re-sequencing dataset, we performed the Genome-Wide Association Studies (GWAS) to identify the gene related to the resistance of AcMNPV in this study. The GWAS results showed that the Niemann-Pick type C1 (NPC-1) gene was the most associated with the trait. The knockdown experiments in BmN cells showed that BmNPC1 has a successful virus suppression infection ability. We found a small number of amino acid mutations among different resistant silkworms, which indicates that these mutations contributed to the resistance of AcMNPV. Furthermore, inhibition of the BmNPC1 gene also changed the viral gene expression of the AcMNPV, which is similar to the expression profile in the transcriptome data of p50 and C108 strains.

Bombyx mori Vps13d is a key gene affecting silk yield.

Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.