Disruption of egg-specific protein causes female sterility in Bombyx mori.

Yolk proteins are the main source of nutrients during embryonic and early larval development in oviparous animals. Therefore, vitellogenesis is crucial for reproduction. The silkworm, Bombyx mori, is a model lepidopteran insect in which there are three yolk proteins: vitellin, 30-kDa protein, and egg-specific protein (Esp). In this study, we explored the gene function of Esp through transgenic clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 technology-mediated mutations in the silkworm. We found that Esp mutation resulted in female sterility but had no effect on male fertility. Female mutants could lay eggs after mating, but the eggs were smaller and lighter colored than those laid by wild-type females. The most important finding is that the eggs laid by female mutants did not hatch. Furthermore, we observed stable inheritance of female sterility caused by Esp mutation through successive generations. Thus, Esp encodes a yolk protein that is crucial for female reproductive success and is a potential target for pest control.

Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication.

The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

MicroRNA-2738 regulates gene expression in the sex determination pathway in Bombyx mori.

MicroRNAs (miRNAs) are a class of short, non-coding transcripts that bind to 3'-untranslated regions to trigger messenger RNA degradation or translational inhibition. Here we explored how miRNAs regulate sex determination in Bombyx mori, a lepidopteran model insect. Genes known to be involved in sex determination, BmPSI, Bmdsx, and BmMasc, are predicted targets of the species-specific miR-2738. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-2738 suppressed transcription of BmPSI, Bmdsx, and BmMasc. The levels of BmPSI and BmMasc were significantly down-regulated in B. mori miR-2738 overexpression. In contrast, the genetic disruption of miR-2738 using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 transgenic system increased the levels of BmPSI and BmMasc transcripts, whereas splicing of Bmdsx was unaltered by miR-2738 depletion or overexpression. Taken together, this study implicates miR-2738 as a minor regulator of sex determination genes in the silkworm.

CRISPR disruption of TCTP gene impaired normal development in the silkworm Bombyx mori.

The translationally controlled tumor protein (TCTP) is a highly conserved and multifunctional protein with activities ranging from cytoskeletal regulation to transcription regulation in numerous organisms. In insects, TCTP is essential for cell growth and proliferation. Recently, TCTP has been reported to affect the innate intestinal immune pathway in the Bombyx mori silkworm, a lepidopteran model insect. However, the comprehensive physiological roles of TCTP in the silkworm remain poorly understood. Here, we performed functional analysis of BmTCTP by using a binary transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/RNA-guided CRISPER-associated protein 9 nucleases) system. Disruption of BmTCTP led to developmental arrestment and subsequent lethality in third instar larvae. Histological analysis revealed that growth impairment originated from decreased cell size, and the proliferation and differentiation of intestinal epithelial cells were also affected. RNA-seq analysis revealed that genes involved in carbohydrate metabolism, lipid metabolism and digestive system pathways were significantly affected by BmTCTP depletion. Together, the results demonstrated that BmTCTP plays a key role in controlling larval growth and development.