Abstract:The red swamp crayfish, Procambarus clarkii, is one of the most economically important freshwater crustacean species in global aquaculture. As an ectothermic organism, its physiological homeostasis and immune function are highly vulnerable to environmental fluctuations, particularly sudden temperature changes and recurring pathogen outbreaks. Previous research has established that non-lethal heat shock (NLHS) acts as a physiological stimulus that can enhance resistance to subsequent pathogenic challenges in various invertebrate species. However, the molecular mechanisms linking environmental stress preconditioning to immune responses remain poorly understood. Integrins, which function as heterodimeric transmembrane receptors composed of non-covalently linked α and β subunits, occupy a central position in the regulation of cell-to-cell adhesion, cytoskeletal reorganization, and the activation of various immune cell lineages. Despite their recognized and indispensable roles in the innate immune architectures of both vertebrates and invertebrates, a truly comprehensive characterization of the integrin gene family within P. clarkii, alongside an analysis of its transcriptional dynamics under NLHS conditions, has remained an unexplored frontier in crustacean molecular biology. In the present study, we conducted a comprehensive, genome-wide identification of the integrin gene family members in P. clarkii. This involved the integration of BLAST homology searches against local genomic and with hidden Markov model (HMM)-based screening of conserved protein domains. All candidate sequences were rigorously validated through secondary conserved domain analyses to confirm the presence of canonical integrin α or β motifs. Our analysis identified a total of eight distinct integrin genes, which categorized into five α-type members, designated as PcITGA1 through PcITGA5, and three β-type members, labeled PcITGB1 through PcITGB3. Comprehensive physicochemical analyses demonstrated considerable diversity in the primary structures of these proteins. Their lengths ranged from 781 to 1,844 amino acids, molecular weights varied from 87.01 to 206.90 kDa, and their theoretical isoelectric points (pI) spanned 4.89 to 6.03. These extensive variations in biochemical properties strongly indicate a high level of structural and functional diversification within the family. The architectural analysis of conserved motifs and exon–intron organization further elucidated the evolutionary history of these genes. While the core functional domains essential for ligand binding and signal transduction were strictly preserved across all members, individual integrins exhibited differences in their specific motif arrangements and genomic structures. Chromosomal mapping showed that these eight genes are distributed across the genome without evidence of localized gene clustering, indicating the absence of recent tandem duplications. Additionally, comparative synteny analysis between P. clarkii and the Eriocheir sinensis, identified conserved collinear relationships for specific α-type integrins, underscoring a high level of evolutionary conservation among decapod crustaceans. Phylogenetic reconstruction successfully clustered the PcITGA and PcITGB sequences into two distinct clades corresponding to the α and β subfamilies, alongside their respective homologs from other arthropods, thereby confirming their taxonomic classification and evolutionary lineage. Three-dimensional structural modeling further indicated that while the global integrin fold is maintained, localized structural variations in the extracellular domains likely influence the specificity of ligand recognition. To explore the physiological roles of these genes, tissue-specific expression profiling was conducted using quantitative real-time PCR. The results showed that most identified integrin genes were highly expressed in hemocytes, gills, and the hepatopancreas, which are the primary tissues involved in immune surveillance, hematopoiesis, and pathogens clearance in crustaceans. In contrast, relatively lower expression levels were observed in the muscle and stomach. Notably, PcITGB3 displayed a distinct expression profile with elevated transcription in both the intestine and muscle, a finding that suggests potential functional specialization in these tissues. To further evaluate the transcriptional responsiveness of the integrin family to environmental and biological stressors, hemocytes from both NLHS-preconditioned and control crayfish were subjected to a challenge with the bacterial pathogen Vibrio parahaemolyticus. Expression dynamics were monitored across a series of temporal intervals. Our data revealed highly distinct temporal expression patterns among the different integrin members, indicating a complex regulatory network. Specifically, PcITGA4, PcITGA5, and PcITGB3 exhibited significant differential expression across multiple treatment groups and time points, highlighting their sensitivity to the synergistic effects of thermal stress and pathogenic stimuli. Conversely, PcITGA1, PcITGA2, and PcITGB1 were predominantly upregulated during the early phase of the challenge, particularly in the groups that had undergone NLHS preconditioning. This pattern suggests that these specific members are likely involved in the rapid onset of immune activation following stress-induced priming. In contrast, the expression of PcITGA3 and PcITGB2 was more markedly altered during the intermediate and late stages of infection, indicating their potential roles in sustained immune regulation or the resolution of inflammatory. Correlation analysis integrating data from previously characterized immune-related genes identified several significant positive associations, notably between PcITGA1 and COMMD8, PcITGA3 and MyD88, and PcITGB2 and NFκB. Subsequent network visualization provided further support for potential molecular interactions between these integrins and core components of the TLR/MyD88/NF?κB signaling axis. Although correlation in gene expression does not definitively establish direct regulatory interactions, these findings strongly imply that integrins serve as key coordinators in signal transduction and effector responses during pathogen invasion, particularly under the physiological conditions induced by NLHS immune preconditioning. Collectively, the findings of this study provide the first comprehensive overview of the integrin gene family in P. clarkii, offering detailed insights into their structural features, evolutionary relationships, tissue distribution, and stress-responsive expression patterns. The differential transcriptional profiles observed under the dual pressures of NLHS and bacterial infection support the hypothesis that integrins are instrumental in integrating environmental stress signals with innate immune activation. These results significantly expand the known molecular framework underlying NLHS-mediated immune enhancement in crustaceans and identify key candidate genes for future functional investigations. Such studies will be essential for developing targeted strategies to improve stress resilience and disease resistance in commercially farmed aquatic species.
