Abstract
This study comprehensively evaluated the physiological, metabolic, and microbial responses of Litopenaeus vannamei to graded levels of Clostridium autoethanogenum protein (CAP) used as the primary dietary protein source. The shrimp were randomly assigned to five iso-lipidic experimental diets containing 32%–48% crude protein. The fish meal content was fixed at 15% for each diet, and the protein level of the diet was adjusted by varying the amount of CAP added. The results indicated that: growth performance exhibited a distinct biphasic response, the CAP40 group achieved highest specific growth rate (SGR), whereas higher inclusions (CAP 44%-48%) significantly reduced SGR ( P < 0.05). Broken-line regression analysis identified 39.44% as the optimal dietary protein level for maximizing growth when CAP is the main protein source. Whole-body lipid content of shrimp increased significantly with higher dietary CAP inclusion ( P < 0.05), peaking in the CAP48 group. Physiological analyses showed that CAP40 enhanced intestinal digestion by upregulating the activity and gene expression of amylase, trypsin, and lipase. ( P < 0.05). Microbiota profiling revealed that CAP40 increased gut microbial alpha diversity ( P < 0.05), suggesting greater ecosystem stability. Functional metagenomic prediction using Tax4Fun further elucidated a critical trade-off: Moderate CAP inclusion (CAP40) enriched microbial pathways linked to lipid metabolism and host growth, but concurrently elevated the relative abundance of potential opportunistic pathogens such as Vibrio and Acinetobacter . In contrast, high CAP inclusion (CAP48) shifted the microbial community toward proteolytic amino acid catabolism and reduced pathogen load, yet this was accompanied by dysbiosis dominated by sulfate-reducing bacteria like Fusibacter . Collectively, these findings underscore CAP’s strong potential as a fishmeal replacer but highlight that its benefits are highly dose-dependent. Successful application in shrimp aquafeeds requires precise optimization of inclusion levels to balance growth promotion against the risks of metabolic imbalance and microbial instability.