Background: Artificial diet-reared silkworms (Bombyx mori) exhibit reduced gut microbial diversity and impaired growth performance compared to mulberry-fed counterparts. While Bacillus subtilis is widely used as a probiotic in livestock and aquaculture, its impact on silkworms remains unexplored. This study investigates whether dietary supplementation with B. subtilis enhances larval development and elucidates the underlying mechanisms involving gut microbiota and metabolic pathways. Results: Supplementing artificial diets with B. subtilis (6 × 10⁵ CFU/g) significantly increased larval body weight by 9.1–22.1% during instar stages and improved feed utilization efficiency (FUE) by 4.09%–6.80% compared to controls. Cocoon quality metrics, including cocoon shell weight (+ 9.77% in females) and cocoon shell ratio (+ 6.56%), also improved. Mechanistically, B. subtilis did not colonize the midgut but transiently modulated gut physiology: it elevated midgut fluid pH and enhanced α-amylase, trypsin, and lipase activities. 16 S rRNA sequencing revealed reduced gut microbial diversity (Shannon index, P < 0.01) and shifts in community structure, with decreased abundances of potential pathogens (e.g., Pseudomonas) and commensals (e.g., Lactobacillus). Targeted metabolomics identified a 3.1-fold increase in phenylalanine levels in hemolymph, linked to upregulated aromatic amino acid metabolism pathways (KEGG). Dietary phenylalanine supplementation (0.4%) replicated B. subtilis-induced growth promotion, confirming its pivotal role in host-microbe interactions. Conclusions: B. subtilis enhances silkworm growth and silk production through multi-faceted mechanisms: reshaping gut microbiota composition, improving digestive enzyme activity, and elevating phenylalanine biosynthesis. These findings establish B. subtilis as a promising probiotic for optimizing artificial diet systems in Lepidoptera and highlight the central role of amino acid metabolism in insect-microbiome symbiosis.

There remains a significant gap in production performance and disease resistance between silkworms reared on artificial diets and those reared on mulberry leaves. This study aims to identify key differential proteins through proteomic analysis of the midgut of silkworms fed artificial diets compared to those fed mulberry leaves. Utilizing molecular docking technology, three anti-nutritional factors that consistently bind to the UGT40B4, UGT340C2, and UGT40A1 proteins were selected, and the differential expression of these UGT genes in response to various anti-nutritional factors was examined. The findings indicate that variations in feed significantly influence the expression of digestive, metabolic, and immune-related proteins within the silkworm midgut. Notably, the expression levels of the UGT40B4, UGT340C2, and UGT40A1 genes vary across different silkworm organs and developmental stages, reflecting their respective physiological roles. Furthermore, the effects of soybean isoflavone, tannic acid, and arabinoxylan on silkworm growth and cocoon quality were found to differ when these substances were incorporated into semi-synthetic feed. This research is anticipated to provide valuable insights for future studies on the role of UGT genes in the silkworm midgut and the formulation of artificial diets for silkworms.

In recent years, the use of artificial diet to rear silkworm Bombyx mori (L.) (Lepidoptera: Bombycidae) has advanced rapidly in China. However, significant differences are found in the production and performance of silk from silkworms reared on artificial diet and mulberry leaves, thereby affecting the development of artificial diet usage in sericulture. To understand the reasons for these differences, we tested the following 3 rearing methods: all-instar mulberry leaf rearing (Mul), all-instar artificial diet rearing (Diet), and instars 1–3 reared on artificial diet followed by instars 4–5 reared on mulberry leaf (Mix). The results showed that the silk production was significantly lower under Diet than Mix and Mul. Electron microscopy images revealed that the protein synthesis and energy supply were decreased under Dier and Mix compared with Mul. Subsequent strength analysis indicated that the relative strength of silk was highest under Mix, followed by Mul, and weakest under Diet. However, no significant differences in elongation were observed among treatments. The β-sheet content of silk was significantly higher under Mix than Diet and Mul, and a similar trend was observed for the crystallinity. Furthermore, the elevated expression of BmChiNAG and the reduced expression of BmTpn genes may be a significant factor for the notable disparities in cocoon silk fineness and strength among the threes. These findings provide deep insights into the differences in silk produced by silkworms reared on mulberry leaves and artificial diet, as well as providing a reference for improving artificial diet for rearing silkworms.

As important silkworm varieties reared on artificial diet, Youshi No. 1 (YS) and Guangshi No. 1 (GS) showed remarkable differences in physiological characteristics. GS had significantly better performance than YS in body weight, cocooning ability, food intake, feed utilization efficiency, and digestive enzyme activities. We further performed metabolomics and 16S rRNA high-throughput sequencing to analyze their metabolic profiles and midgut microbiota. More than 40 differential metabolites were screened out, and four metabolic pathways related to feeding divergence were determined via KEGG enrichment, among which L-valine was enriched in multiple pathways. Significant structural differences were also observed in midgut microbiota, and Bacillus was positively correlated with pantothenic acid and valine metabolism. These correlational results disclosed that differential metabolites and gut microbiota might underlie the phenotypic variations between the two varieties. Integrated analysis combined with functional verification experiments demonstrated that supplementation of 1% L-valine or specific Bacillus strains in an artificial diet was associated with the improvement of the growth performance, cocoon quality, and feed utilization efficiency of the YS silkworm variety.