P4’ peptide-bearing Bacillus subtilis enhances the growth of Chinese mitten crab (Eriocheir sinensis) by enhancing muscle growth and molting performance

The addition of appropriate amounts of plant protein hydrolysates to feeds can improve the growth performance of aquatic animals, and this effect is closely linked to the bioactive peptides generated by hydrolyzing plant proteins. In a previous study, our laboratory developed a recombinant Bacillus subtilis capable of stably expressing the cmP4’ peptide (isolated from cottonseed meal hydrolysate) and demonstrated that supplementing feed with 10 9 CFU/kg of this recombinant strain promoted growth in the Chinese mitten crab ( Eriocheir sinensis ). However, the mechanisms underlying this growth-promoting effect remain unclear. The present study aimed to evaluate the effects of dietary supplementation with recombinant B. subtilis carrying the cmP4’ peptide on muscle growth and molting performance in E. sinensis . A total of 240 crabs (mean body weight:0.85 ± 0.01 g) were randomly divided into three groups and fed one of three diets for 8 weeks: a control diet without B. subtilis (CON), a diet with 10 9 CFU/kg of unmodified B. subtilis (BS9), and a diet with recombinant B. subtilis (RBS9). Each treatment was tested in four replicate tanks (20 crabs per tank). Compared with the CON group, both BS9 and RBS9 groups showed significant increases in final weight (FW), weight gain rate (WGR), body protein gain (BPG), crude ash content, pepsin, aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) activities, meat yield (MY), methyl farnesoate (MF) content, and expression of the cuticle protein gene ( cp cbm ). Feed conversion ratio (FCR), body moisture content, urea nitrogen (UN) levels, and expression of myostatin ( mstn ) and transforming growth factor-β type I receptor ( estgfbri ) were significantly reduced. Furthermore, the RBS9 group, compared with BS9, exhibited additional significant increases in FW, mortality rate (MR), feed intake (FI), crude protein content, trypsin activity, GOT and GPT activities, MY, MF content, molting rate, retinoid X receptor, cryptocyanin, and cp cbm expression, while body moisture, UN content, mstn , and estgfbri expression showed the opposite trend. These results suggest that both unmodified and recombinant B. subtilis can promote the growth of E. sinensis , and the superior performance of the recombinant strain is mechanistically linked to enhanced protein metabolism, suppression of muscle growth inhibitors, and activation of the molting signaling pathway. • Recombinant B. subtilis (RBS) expressing the bioactive peptide cmP4’ significantly promotes growth in Eriocheir sinensis . • Dietary supplementation with RBS enhanced weight gain, protein deposition, meat yield, and digestive enzyme activity. • Supplementation boosts molting via higher molting rate and upregulating cuticle formation ( cp cbm ) and retinoid signaling. • Recombinant strain outperforms wild-type in growth/metabolism, reduced anti-growth factors mstn/estgfbri and urea nitrogen. • RBS offer a strategy to enhance muscle growth and molting in crustaceans via bioactive peptide delivery.