Preliminary Study On The Toxicological Mechanism Of Sepia Esculenta Larvae Exposed To Cu And Cd

Industrial development promoted the discharge of heavy metal pollution into the ocean.Waste-water and materials often contained numerous heavy metals such as Cu,Cd,Zn,and Hg.Waste discharge has increased the concentration of heavy metals in the ocean,which seriously affected the growth and development of both wild and cultured organisms.Among these pollutants,Cu and Cd posed a significant threat to the survival of mollusks.They entered and accumulated in mollusks through multiple methods such as respiration,skin penetration,and ingestion and could induce a series of adverse reactions.Therefore,to prevent large-scale biological death caused by the increasing concentration of marine heavy metals,it was vital to comprehensively understand the toxicological mechanism of organisms exposed to Cu and Cd.In this study,an important economic cephalopod,Sepia esculenta(S.esculenta),was selected as the experimental subject.Through the determination of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione S-transferase(GST),and metallothionein(MTs)activities and RNA-Seq analysis,we explored the oxidative stress and metal toxicity of larvae of S.esculenta exposed to Cu and Cd.Additionally,we identified several key genes and signaling pathways,allowing scholars to further understand the toxicological mechanism induced by heavy metal exposure in cephalopods.In this study,0 h,4 h,and 24 h were selected as the experimental time points.After collecting samples from the 0 h control group,Cu,Cd,and co-exposure samples and control group samples were collected at 4 h and 24 h,respectively.RNA-Seq results showed that 1,995,607,306 raw reads were identified,which were then mapped to the reference genome.Through differential expression analysis,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment analyses,protein-protein interaction(PPI)network analysis,and weight gene co-expression network analysis(WGCNA),we identified the key genes,GO terms,and KEGG signaling pathways that regulating the toxicological process of S.esculenta larvae exposed to Cu and Cd.Analyzing these in depth would help to further understand the response mechanism of S.esculenta to heavy metals.The results of antioxidant enzymes and toxicity indicators suggested that Cu,Cd,and co-exposure induce significant oxidative stress and metal toxicity in S.esculenta larvae.The results of RNA-Seq indicated that 1,131 differentially expressed genes(DEGs)were screened after Cu exposure,and 12 key DEGs were identified based on the comprehensive analysis of protein-protein interaction network and KEGG signaling pathway.We found that Cu mainly induced metabolic toxicity such as disorder of protein digestion and absorption,purine metabolism,and cholesterol metabolism in the larvae of S.esculenta.Also,896 DEGs were screened from Cd-exposed S.esculenta larvae,and 20 key DEGs related to toxicology are identified and verified.The results reveal that the digestion and absorption,molecular function,especially the nervous system function of larvae were significantly inhibited.Among 572 DEGs identified after co-exposure,20 key DEGs were identified and verified.According to the expression of key genes,we speculated that the functions of cell connection and cell adhesion of S.esculenta larvae were damaged,and the structure of the extracellular matrix was destroyed.Furthermore,based on the WGCNA results,there were significant differences in the toxicological mechanisms induced by single exposure and co-exposure.Cu mainly inhibited the development of tissues and organs and promoted the decomposition of hydrogen peroxide;Cd inhibited a variety of metabolic processes and affected signal transduction between and within cells;and co-exposure induced the changes in the immune,metabolic,and nervous systems,as well as other processes in S.esculenta larvae.With the increase of exposure time,the neurotoxicity induced by Cu and Cd grew significantly,while the cell process and signal transduction process were further inhibited.Five hub genes based on the protein-protein interaction network were identified,including ADRA1 A,ARA1,JUN,KCNMA1,and SERPINB1,which were significantly related to three exposure methods and two exposure times,respectively.Analyzing the functions of the relevant modules and genes was conducive to further understanding the differences in the oxidation and toxicological mechanisms induced by Cu and Cd in S.esculenta.In this study,the comprehensive analysis of PPI network and KEGG signaling pathway and WGCNA methods were performed to analyze the toxicological mechanism of S.esculenta larvae exposed to Cu and Cd,and a large number of key genes that regulate oxidative stress and toxicological process were discovered.Our results laid a foundation for further understanding the toxicological mechanism of S.esculenta against heavy metals,which promoted the development of invertebrate environmental toxicology and provided theoretical assistance for S.esculenta artificial breeding.