To test the performance of COI-based DNA barcoding in species delimitation, we amplified and sequenced the mitochondrial COI gene from six Schizothoracine fishes endemic to Xinjiang, China. We then characterized the COI gene dataset, quantified genetic divergence, and inferred phylogenetic relationships using distance-based approaches. Morphological examination supported clear phenotypic differentiation among taxa. In particular, Diptychus maculatus can be readily distinguished from the densely scaled Schizothorax species by having a single pair of barbels and relatively sparse scales. The COI gene sequences showed an evident AT bias (55.1%), consistent with patterns reported for teleost mitochondrial genomes. Across all samples, 11 haplotypes were identified. Pairwise comparisons indicated a maximum interspecific divergence of 15.296%, whereas the smallest interspecific distance (0.262%) occurred between Schizothorax barbatus and Schizothorax irregularis. Species-delimitation analyses and phylogenetic reconstruction supported D. maculatus and A. laticeps as distinct mitochondrial lineages, whereas S. biddulphi, S. eurystomus, S. irregularis, and S. barbatus were not fully resolved by COI gene. The shared haplotypes and low genetic distances among the four Schizothorax species may reflect recent divergence, incomplete lineage sorting, or historical gene flow. Overall, COI gene barcoding is effective for distinguishing the major lineages in this dataset, especially D. maculatus and A. laticeps.

Acute heat stress frequently causes mass mortality in farmed red swamp crayfish (Procambarus clarkii), yet the mechanisms underlying immune collapse remain poorly understood. We established an acute heat stress model (37 °C, 6 h) and performed an integrative analysis combining hemocyte profiling, redox and immune assays, RNA-seq, and qRT-PCR. Heat stress significantly increased mortality and disrupted the hemocyte system, with a ~25% reduction in total hemocyte count and a selective decline in granular cells. This was associated with severe redox imbalance, evidenced by ROS/H₂O₂ accumulation, suppressed SOD and CAT activities, and lipid peroxidation damage. Transcriptomic analysis revealed 1446 differentially expressed genes, indicating concurrent activation of ER stress and autophagy alongside suppression of energy metabolism. Key gene validation confirmed upregulation of pro-apoptotic factors (CASP3, P53) and ER stress markers (GRP78, XBP1), consistent with hemocyte depletion. These findings provide multi-level evidence that acute heat stress triggers a redox crisis (“oxidative burst–defense suppression”), which in turn activates ER stress and apoptosis, leading to selective loss of granular cells and systemic immune compromise. This study establishes a mechanistic framework for understanding heat-induced mortality in crustaceans and offers a theoretical basis for developing targeted interventions to enhance thermal resilience in crayfish aquaculture.

RhoA (Ras homolog A) is a prominent member of the Rho GTPase family, playing a key role in various cellular processes such as cytoskeletal dynamics, cell migration, and immune responses. However, its function in red swamp crayfish remains unclear. In this study, it is proposed that RhoA may regulate the innate immune response in P. clarkii. The gene was fully characterized as PcRhoA in P. clarkii. The results showed that the open reading frame (ORF) of PcRhoA is 663 bp, encoding a 220-amino acid protein with a conserved Rho domain of 174 amino acids. Phylogenetic analysis placed PcRhoA close to Cherax quadricarinatus RhoA. RT-qPCR analysis revealed high expression levels of the PcRhoA gene in the hepatopancreas, muscle, heart, ovary, and stomach, with lower expression in the blood, intestine, gills, and tentacle gland. Furthermore, PcRhoA mRNA transcript was significantly upregulated in the intestine following LPS and Poly I:C challenges. Knockdown of PcRhoA suppressed the expression of downstream genes in the immune signaling pathway. These results indicate that PcRhoA appears to play a pivotal role in regulating the immune response of crayfish.