Conventional immediate and high-temperature immediate acid treatment are crucial techniques for breaking the diapause state of silkworm eggs, but their molecular mechanisms remain unclear. This study prepared diapause eggs (CK), conventional immediate acid-treated eggs (46 °C, 5 min, and CG), and high-temperature immediate acid-treated eggs (47.5 °C, 7 min, and GW) and analyzed the transcriptome and metabolome to screen for key expressed genes and key metabolites. Transcriptome results showed that 688, 823, and 222 differentially expressed genes (DEGs) were obtained from CK vs. CG, CK vs. GW, and CG vs. GW, respectively, and 12 DEGs significantly upregulated in all three comparisons (CK vs. CG, CK vs. GW, and CG vs. GW), including glycine-N-methyltransferase, choline dehydrogenase, Hsp68, and Hsp70. The LC-MS analysis results showed that 854, 711, and 506 differential metabolites (DMs) were obtained from CK vs. CG, CK vs. GW, and CG vs. GW, respectively. A total of seven DMs upregulated in all three comparisons and with |log₂Fold Change| ≥ 0.5 in CG vs. GW, including tyrosine-isoleucine-histidine, phenylalanyl-tyrosine, tyrosine-phenylalanine-glutamate-lysine, and histidylleucine, as well as 12 downregulated DMs, were identified. Additionally, it was found that γ-linolenic acid and triglycerides were upregulated in CG vs. GW. The conjoint analysis results revealed that four small peptides, including tyrosine-isoleucine-histidine, phenylalanyl-tyrosine, tyrosine-phenylalanine-glutamate-lysine, and histidylleucine, exhibited a highly significant positive correlation with Hsp70 family genes such as Hsp68 and Hsp70. This suggests that these small peptides, along with γ-linolenic acid and triglycerides, may play a crucial role in the resistance of silkworm eggs to high-temperature stress and the associated oxidative stress.

The midgut of insects is a crucial organ that enables them to obtain nutrients and resist pathogen invasion. In this study, the midgut cell clusters of the silkworm (Bombyx mori), a model species of lepidopteran insects, were analyzed using single-nucleus RNA sequencing technology (snRNA-seq) (PRJNA1280782) to understand the functional diversity of midgut tissues, identify the key cell clusters involved in the response to pathogen infection, and screen molecular targets for disease resistance. Twenty cell clusters (C0-C19) were obtained and annotated. The known marker genes, KEGG and GO pathway enrichment analysis were used to identify five types of cells: enterocyte cells (ECs, 54.4%), goblet cells (GCs, 25.9%), enteroendocrine cells (EEs, 10.6%), intestinal stem cells (ISCs, 2.17%), and muscle cells (MCs, 7.0%). Additional representative cell marker gene Cel of GCs was identified. We conducted snRNA-seq on the midgut tissues of B. mori infected with the fungal and viral pathogens Nosema bombycis (N.b) and Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h (early stage of infection) and 96 h (later stage of infection) to determine the immune response patterns of different cell clusters. The immune-related genes of ECs C2 and C16 were significantly altered after pathogen infection, making them important cell clusters for screening disease resistance target genes. The common susceptible cell clusters C4 and C13 between different pathogens were successfully identified, and related genes that could simultaneously affect N.b and BmNPV proliferation were screened in C2, C13 and C16. The results provide a basis for exploring potential common molecular target genes of various pathogens.