The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

The insect midgut secretes a semi-permeable, acellular peritrophic membrane (PM) that maintains intestinal structure, promotes digestion, and protects the midgut from food particles and pathogenic microorganisms. Peritrophin is an important PM protein (PMP) in the PM. Here, we identified 11 peritrophins with 1–16 chitin binding domains (CBDs) comprising 50–56 amino acid residues. Multiple CBDs in the same peritrophin clustered together, rather than by species. The CBD contained six highly conserved cysteine residues, with the key feature of amino acids between them being CX11-15CX5CX9-14CX11-12CX6-7C. Peritrophins with 2 and 4 CBDs (Bm09641 and Bm01504, respectively), and with 1, 8, and 16 CBDs (Bm11851, Bm00185, and Bm01491, respectively) were mainly expressed in the anterior midgut, and throughout the midgut, respectively. Survival rates of transgenic silkworms with Bm01504 overexpression (Bm01504-OE) and knockout (Bm01504-KO) infected with B. morinucleopolyhedrovirus (BmNPV) were significantly higher and lower, whereas expression of the key viral gene, p10, were lower and higher, respectively, compared with wild type (WT). Therefore, Bm01504-OE and Bm01504-KO transgenic silkworms were more and less resistant, respectively, to BmNPV. Bm01504 plays important roles in resisting BmNPV invasion. We provide a new perspective for studying PM function, and reveal how the silkworm midgut resists invasive exogenous pathogenic microorganisms.

Bombyx mori, an insect with significant economic importance in China contains a large amount of lipids among different developmental stages, especially the pupal stage. Silkworms are fed with a base diet and fish oil supplemented diet, and the fatty acid (FA) compositions of different strains, life stages, and sexes are determined by GC‐MS. No C20 or C22 polyunsaturated fatty acids (PUFAs) are detected in silkworms fed with the base diet. Females accumulates more unsaturated fatty acids (USFAs) than males in the pupal stage, and the relative amount of USFAs decreases from the wandering stage to the moth stage, especially in females, suggesting that silkworms prefer the utilization of USFAs. In addition, when silkworms are fed with a diet supplemented with fish oil, which contained C20 and C22 PUFAs, they accumulates C20 and C22 PUFAs in proportion to the concentration of fish oil added to the diet. Practical Applications: Currently, the main dietary source of C20 and C22 PUFAs is marine fish, however, of which the stocks are declining because of environmental pollution and overfishing. Because of its excellent capability to accumulate C20 and C22 PUFAs, silkworm could be developed as a potential new source of these long‐chain FAs by transferring ?5 and ?6 desaturases to silkworm or its primary food, mulberry. The silkworm (Bombyx mori) can accumulate C20 and C22 polyunsaturated fatty acids (PUFAs) from diet supplemented with these PUFAs (B). The accumulation efficiency of eicosapentaenoic acid (EPA) in silkworm is particularly outstanding. This capacity may make silkworm a potential new source of C20 and C22 PUFAs.