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In recent years, the red swamp crayfish (Procambarus clarkii, P. clarkii) farming industry has suffered huge economic losses due to the pathogenic bacterium Spiroplasma eriocheiris (S. eriocheiris). To elucidate the immune response mechanism and identify hub immune genes as well as their associated microRNAs that regulate the host response of P. clarkii against S. eriocheiris infection, we conducted a comprehensive analysis on P. clarkii hemocyte mRNA and microRNA (miRNA) transcriptomes at different infection stages using third- and second-generation sequencing technologies. In full-length transcriptome functional annotation, 8155 unigenes were annotated, and 1168 potential new transcripts were predicted. In the mRNA transcriptome, a total of 3168 differentially expressed genes were identified at different infection stages, including 1492 upregulated and 1676 downregulated genes (duplicate genes excluded). Transcriptome analysis revealed 880 differentially expressed genes involved in multiple pathways and processes such as endocytosis, autophagy, lysosome, mTOR signaling, phagosome, and the Fanconi anemia pathway. Mfuzz analysis was employed to integrate and cluster the differential expression trends of genes across the three infection stages. In the miRNA transcriptome, 234 miRNAs and 966 predicted target genes were identified, with 86 differentially expressed miRNAs identified across the three time periods. A significant difference (P < 0.05) was observed for miRNAs including pcl-miR-146-3p, pcl-miR-74-3p, pcl-miR-225-5p, and pcl-miR-68-5p. These miRNAs are involved in multiple immune and autophagy-related pathways and have regulatory effects on immune genes including Vps26 , lqf , and ERK-A. Based on the differentially expressed immune-related genes, we constructed a protein-protein interaction (PPI) network, which revealed the interactions among hub genes including Rac1 , Akt1 , Rho1 , and Egfr. We also constructed a miRNA-gene interaction network in immune and autophagy-related processes, highlighting the potential regulatory effects of miRNAs including pcl-miR-183-5p, pcl-miR-146-3p, pcl-miR-176-5p, and pcl-miR-225-5p on proteins including LST8, SNAP29, Rab-7A, and ERK-A. To conclude, this study has identified hub immune genes and corresponding regulatory miRNAs in P. clarkii hemocytes in response to S. eriocheiris infection and explored the roles of these genes in selected pathways and processes. These findings are expected to provide further insights into the molecular mechanisms that confer resistance to S. eriocheiris infection in P. clarkii. • 880 significant genes involved in multiple processes such as endocytosis, autophagy, lysosome, and phagosome. • A total of 234 miRNAs were identified, comprising 99 known miRNAs and 135 novel miRNAs. • Hub genes like Rac1, Akt1, Rho1, and Egfr emerged as important nodes that coordinate antibacterial immune processes. • Construction of immune and autophagy-related mRNA-miRNA interaction network involving 132 miRNAs and 966 mRNAs. • The key genes and corresponding regulatory miRNAs in hemocytes in response to an infection by S. eriocheiris. [ABSTRACT FROM AUTHOR]

Spiroplasma eriocheiris (S. eriocheiris) infection has caused heavy economic losses in Macrobrachium nipponense (M. nipponense) aquaculture industry. However, the interplay between immune responses of M. nipponense and S. eriocheiris infection has not yet been fully elucidated. Herein, full-length transcriptome profiling of M. nipponense hemocytes was performed with or without S. eriocheiris challenge. In total, 20,441 high quality unigenes were obtained, among which 733 differentially expressed genes (DEGs) were found, including 309 up-regulated and 424 down-regulated genes. Protein-protein interaction network (PPI) analysis of DEGs identified 4 hub immune-related genes, including MAP kinase-interacting serine/threonine-protein kinase 1 (MNK1), C-type lectin 4 (CTL4), gamma-interferon-inducible lysosomal thiol reductase (GILT), and anti-lipopolysaccharide factor (ALF). Tissue distribution and expression analysis of the key immune-associated genes demonstrated that they were widely expressed in 6 tissues, especially in gills, and were up-regulated by S. eriocheiris challenge. M. nipponense challenged by S. eriocheiris exhibited a significant reduction of bacterial clearance ability when the hub genes were silenced via RNA interference. Overall, our results indicated that the 4 hub immune-related genes play a vital role in anti- S. eriocheiris infection, thus providing insights into the immune defensive response mechanisms of M. nipponense against S. eriocheiris infection. • We identified 4 core genes in M. nipponense against S. eriocheiris infection. • The 4 core genes are mainly distributed in the gill of M. nipponense. • The 4 core genes are able to clear S. eriocheiris in M. nipponense. [ABSTRACT FROM AUTHOR]

In recent years, the industry in charge of the cultivation of Macrobrachium nipponense (M.nipponense) has suffered significant economic losses due to an infectious pathogen called Spiroplasma eriocheiris (S.eriocheiris). There has therefore been a need to identify the key immune and autophagy genes that respond to M.nipponense 's infection with S. eriocheiris to analyze its immune response mechanism and the regulation of related microRNAs (miRNAs). In this study, the mRNA and miRNA transcriptome of M.nipponense 's hemocytes were analyzed at different stages of infection. This analysis employed the second and third-generation sequencing technologies. In the mRNA transcriptome, 1656 genes were expressed in healthy and susceptible M.nipponense. 892 of these were significantly up-regulated, while 764 were down-regulated. 118 genes with significant differences in autophagy, endocytosis, lysosome, Toll, IMD, and VEGF pathways were obtained from the transcriptome. In the miRNA transcriptome, 312 miRNAs (Conserved: 112, PN-type: 18, PC-type: 182) were sequenced. 74 were significantly up-regulated, and 57 were down-regulated. There were 25 miRNAs involved in regulating the Toll and IMD pathways, 41 in endocytosis, 30 in lysosome, and 12 in the VEGF pathway. An integrated analysis of immune-related miRNAs and mRNAs showed that miRNAs with significant differences (P < 0.05) such as ame-miR-29b-3p, dpu-miR-1and PC-3p-945_4074, had corresponding regulatory relationships with 118 important immune genes such as Relish , Dorsal , Caspase-3, and NF-κB. This study obtained the key immune and autophagy-related genes and corresponding regulatory miRNAs in M. nipponense' s hemocytes in response to an infection by S.eriocheiris. The results can provide vital data that further reveals the defense mechanism of M.nipponense 's immune system against S.eriocheiris. It can also help further comprehension and interpretation of M.nipponense's resistance mechanism to the invading S.eriocheiris , and provide molecular research information for the realization of host-directed therapies (HDT) for M.nipponense. • Comprehensive mRNA and miRNA transcriptome profiling of M.nipponense challenged with S. eriocheiris. • 118 significant genes related to autophagy, endocytosis, lysosome, Toll, IMD, and VEGF pathways were excavated. • A total of 312 miRNAs, of which 74 were significantly up-regulated and 57 were significantly down-regulated. • The key immune and autophagy-related genes and miRNAs in hemocytes in response to an infection by S.eriocheiris. [ABSTRACT FROM AUTHOR]