Sepia esculenta is a popular economic cephalopod with high yield, delicious meat, and rich nutrition. With the rapid development of heavy industry and medical industry, a large amount of waste has been released into the ocean recklessly in recent years, inducing a significant increase in the content of heavy metals, especially cadmium (Cd) and copper (Cu), in the ocean. This phenomenon significantly affects the growth and development of S. esculenta, causing a serious blow to its artificial breeding. In this study, transcriptome analysis is used to initially explore immune response mechanisms of Cd and Cu co-exposed juvenile S. esculenta. The results show that 1,088 differentially expressed genes (DEGs) are identified. And DEGs functional enrichment analysis results suggests that co-exposure may promote inflammatory and innate immune responses in juvenile S. esculenta. Fifteen key genes that might regulate the immunity of S. esculenta are identified using protein-protein interaction (PPI) network and KEGG enrichment analyses, of which the three genes with the highest number of interactions or involve in more KEGG pathways are identified as hub genes that might significantly affect the immune response processes. Comprehensive analysis of PPI network and KEGG signaling pathway is used for the first time to explore co-exposed S. esculenta juvenile immune response processes. Our results preliminarily reveal immune response mechanisms of cephalopods exposed to heavy metals and provide a valuable resource for further understanding of mollusk immunity.

Immune defense systems are indispensable for living organisms. Within an immune network, problems with any given link can impact the normal life activities of an organism. Amphioctopus fangsiao is a cephalopod that exists widely throughout the world’s oceans. Because of its nervous system and locomotive organs, it has become increasingly studied in recent years. Vibrio anguillarum is one of the most common pathogenic bacteria in aquaculture organisms. It is highly infectious and can infect almost all aquaculture organisms. V. anguillarum infection can cause many adverse biological phenomena, including tissue bleeding. Study the immune response after V. anguillarum infection would help us to understand the molecular mechanisms of immune response in aquaculture organisms. In this research, we infected the primary incubation A. fangsiao with V. anguillarum for 24 h. We analyzed gene expression in A. fangsiao larvae via transcriptome profiles at 0, 4, 12, and 24 h after hatching, and 1,385, 734, and 6,109 differentially expressed genes (DEGs) were identified at these three time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify immune-related DEGs. Protein–protein interaction networks were constructed to examine interactions between immune-related genes. Twenty hub genes involved in multiple KEGG signaling pathways or with multiple protein–protein interaction relationships were identified, and their differential expression verified by quantitative RT-PCR. We first studied V. anguillarum infection of A. fangsiao larvae by means of protein–protein interaction networks. The results provide valuable genetic resources for understanding immunity in molluscan larvae. These data serve as a theoretical basis for the artificial breeding of A. fangsiao.

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Pathogen threats pose a significant limitation in the culture of marine organisms like cephalopods. Yet, there is a notable lack of immune information regarding cephalopods. Polyriboinosinic polyribocytidylic acid (Poly I:C), a synthetic virus-like molecule, can be recognized by the immune cells as pathogen-associated molecular patterns (PAMPs), and this process is often used to simulate the invasion of viruses. The white body is an immune and hematopoietic organ, and its RNA-seq information obtained from Amphioctopus fangsiao stimulated by Poly I:C is essential for understanding the antiviral immune response in this species. In this research, we performed transcriptome sequencing and bioinformatics analysis of A. fangsiao white body tissue within 24h stimulated by Poly I:C. A large number of differentially expressed genes (DEGs) were detected in this study, including 2,613 and 8 DEGs at 6h and 24h, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used for searching immune-related terms and genes. Afterwards, a protein–protein interaction (PPI) network was constructed to identify the relationship between immune genes. Finally, the 20 hub genes including RAC1, MAPK14, PIK3CA and other seventeen hub genes were identified based on the network and pathway analysis, and we validated the accuracy of 20 hub genes using qRT-PCR. These hub genes mainly participated in PI3K-Akt signaling pathway, Chemokine signaling pathway and other signaling pathways. These research results provided a valuable theoretical basis for researching A. fangsiao white body immunity and significantly improved our cognition about innate immunity of cephalopods.

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