Gut microbiome succession and cultivation-based functional screening during intestinal regeneration in Stichopus...

Stress-induced evisceration impairs sea cucumber survival and growth, affecting both their ecological roles and sustainable production in marine environments. Given the critical role of gut microbiome in maintaining host health, microbiome modulation emerges as a novel strategy to improve post-evisceration recovery. This study investigated the gut microbiome during intestinal regeneration in the tropical sea cucumber Stichopus monotuberculatus by combining 16S rRNA gene sequencing with culture-dependent isolation. We (i) tracked microbial community succession, and (ii) established a collection of indigenous gut isolates with digestive enzyme activities. The results demonstrated distinct gut microbial communities across regeneration stages, reflecting the combined influence of deterministic host selection and stochastic colonization. Dominant genera Delftia and Ruegeria aligned with taxa prevalent in native coral reef environments. These genera were nearly eradicated after evisceration but rebounded to baseline levels by late regeneration, suggesting host-selected retention of taxa with potential functional importance. Cultivation efforts under varied media and oxygen conditions yielded 163 bacterial isolates (78 species, four putatively novel), substantially expanding the known cultivable diversity of the holothurian gut microbiome. Enzymatic screening identified several multienzyme-producing isolates, including Priestia megaterium, Bacillus stratosphericus and Lysinibacillus fusiformis. These isolates exhibited amylase, lipase, and cellulase activities, supporting their potential as targeted digestive probiotics. Our integrated approach deciphers gut microbiome dynamics during regeneration, providing ecological insights into host-microbe-environment interactions and microbial resources for sustainable management of sea cucumber populations.