Effects of L-theanine supplementation duration on antioxidant capacity, immunity, and gut microbiota in Chinese mitten crab (Eriocheir sinensis)

L-theanine (gamma-glutamylethylamine) is a unique non-protein amino acid found in tea and its benefits have been evaluated in various animals due to its various biological activities. However, the time-dependent effects of L-theanine supplementation on Chinese mitten crabs remain unknown. In the present study, we investigated the time-dependent effects of L-theanine supplementation on enzyme activities, transcriptome response, and intestinal flora composition of Chinese mitten crabs using the following experimental groups: non-L-theanine treatment (L0), 1.5 % L-theanine for four weeks (L4) and 1.5 % L-theanine for eight weeks (L8). Innate immune parameters (e.g., ACP and ALP) and activities of anti-oxidant enzymes namely SOD, T-AOC, and GSH-Px were positively correlated with L-theanine supplementation duration. Transcriptome results revealed that the number of differentially expressed genes (DEGs) increased from 222 (181 upregulated and 41 downregulated at four weeks) to 760 (724 upregulated and 36 downregulated at 8 weeks), and the number of DEGs was positively correlated with L-theanine supplementation duration. Furthermore, dietary L-theanine significantly affected intestinal microbiota composition, with the community abundance of Citrobacter , Lactovum , Pediococcus , Tyzzerella , and Exiguobacterium decreasing significantly with an increase L-theanine supplementation duration, whereas the abundance of Myroides , Kurthia , and Vagococcus bacteria increased significantly. In conclusion, L-theanine has a time correlation with the antioxidant enzyme activities and hepatopancreas metabolic function of E. sinensis , and a supplementation regimen of eight weeks is recommended for its optimal cultivation. • L-theanine exerts time-dependent effects on the growth and immunity of E. sinensis . • L-theanine exerts a time-dependent effect on the antioxidant enzyme activities. • L-theanine exerts time-dependent effects on hepatopancreas metabolic function. • Dietary L-theanine significantly affects intestinal microbiota composition. • We provide theoretical support for L-theanine use in aquaculture feed production.