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To investigate the effects of luteolin (LUT) on the growth, antioxidant capacity, non−specific immunity and intestinal microbiota of red swamp crayfish (Procambarus clarkii), 300 individual crayfish (4.17 ± 0.50 g average body weight and 50.19 ± 1.40 mm average body length) were randomly divided into five groups and fed with LUT doses of 0 mg/kg (CG), 50 mg/kg (LG50), 100 mg/kg (LG100), 300 mg/kg (LG300), and 500 mg/kg (LG500) for 8 weeks, respectively. Firstly, the results demonstrated that the survival and growth performance were significantly improved in all LUT added groups compared to the control (CG group), with no significant differences in hepatosomatic index, feed coefficient and muscle components, and the crayfish in LG100 group showed the maximal survival rate (SR) and specific growth rate (SGR) (P < 0.05). Secondly, histological observations displayed that the hepatopancreas and intestine tissues were not impacted in the LUT−supplemented groups, and the length of intestinal folds was significantly increased in LG100 and LG300 groups. Thirdly, LG50, LG100, and LG300 groups produced significantly increased hepatopancreatic antioxidant enzyme activities and hemolymph immune indicators compared with the control. In all experimental groups, malondialdehyde (MDA) contents were significantly reduced, as were aspartate aminotransferase and alanine aminotransferase activities. Lastly, dietary LUT specifically improved the structure and optimized the function of the gut microbiota. Compared to the control, the LG100 group displayed a significant increase in the index of Chao1 and Observed species, an increase in the relative abundance of Proteobacteria and Actinobacteria, a decrease in the ratio of Bacteroidetes to Firmicutes, and an increase in the abundance of Thermomonas , Turicibacter , Bdellovibrio and Enterococcus. PICRUSt analysis demonstrated a significant improvement in the KEGG pathway associated with isoflavone biosynthesis in the LG group. In conclusion, the present study confirms that dietary supplementation with 50−300 mg/kg LUT favors growth, non−specific immunity and antioxidant function in P. clarkii , with optimal additions ranging from 102−165 mg/kg. Furthermore, it optimized gut microbiota function, suggesting that LUT can be used as a potential feed additive for P. clarkii. These findings provide new insights for crayfish's healthy culture. • The optimal level of LUT addition to P. clarkii diet was 102−165 mg/kg. • Dietary addition of LUT improved the survival and growth performance of P. clarkii. • Dietary addition of LUT enhanced the antioxidant and immune capacity of P. clarkii. • Dietary addition of LUT improved intestinal microbiota structure of P. clarkii. [ABSTRACT FROM AUTHOR]

Thermal stress is a common experience suffered in rice-crayfish integrated systems (RCIS) by the red swamp crayfish, Procambarus clarkii. Based on our previous studies, the adaptability of this crayfish to non-hyperthermal stress over a specified period was explored within a short term by investigating the antioxidant capacity, immune function, HSP70 expression and intestinal microbial changes. Under the non-hyperthermal stress temperature (30 °C) determined by cumulative survivals, the malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), total super oxide dismutase (T-SOD) and catalase (CAT) activities in hepatopancreas significantly increased to the peak values during at 12 h or 24 h (P < 0.05), respectively, and then gradually recovered to the levels of the controls (24 °C) (P > 0.05); the immune-related enzymatic activities (alkaline phosphatase, AKP; acid phosphatase, ACP; lysozyme, LZM) and gene expressions (TNFAIP , TNF and Toll) rapidly increased and reach the peaks at 12 h and 48 h, respectively, and maintained subsequently the higher levels compared with the controls (P < 0.05) except for TNFAIP. The relative expressions of HSP70 in the hepatopancreas, intestine and gill all increased sharply and reached the peak at 6 h (P < 0.05), then decreased fast and roughly returned to the levels of the controls at 96 h (P > 0.05), while, there was still a relatively high expression during the later stages in the gill. High-throughput sequencing of 16S ribosomal DNA revealed the thermal stress altered the diversity and composition of intestinal microbiota. The increased abundance of Proteobacteria and decreased abundance of Bacteroidetes (mainly Bacteroides) and Firmicutes might be indicative of the elevated microbial instability and the unnecessity for high level of nutrient metabolism in the host. The abundance increase of relevant metabolic pathways (e.g., the biosynthesis and degradation of amino acids and lipids) predicted by MetaCyc might be caused by the abundance increase of Proteobacteria. The decreased abundance of two phyla (Actinobacteria and Fusobacteria) and genus Anaerorhabdus could adversely affect the crayfish's intestinal health status. While, the obvious abundance decrease of putatively opportunistic genera (Shewanella and Acinetobacter), combined with the abundance increase of beneficial phylum Tenericutes and genus Rhodobacter , suggested that there concurrently existed some adaptability to the non-thermal stress, which did not resulted in apparent risks of intestinal disorder. This study provides insights into the underling adaptive mechanisms associated with the anti-oxidation, immunity, intestinal microflora and nutrient metabolism of P. clarkii in response to the short-term non-hyperthermal stress. • Non-hyperthermal stress caused oxidant damage and induced the instability of intestinal microbiota. • The increased indices of oxidant damage, anti-oxidation and HSP70 gene expression could be gradually recovered in the short-term non-hyperthermal stress. • High levels of immune-related enzymatic activities and gene expression were sustained for the adaption to short-term non-hyperthermal stress. • The modulated gut microflora and decreased metabolic level could be beneficial to the adaption to short-term non-hyperthermal stress. [ABSTRACT FROM AUTHOR]