Multi-omics Analysis Of The Effects Of Seawater Acidification On The Survival Of Four-armed Larvae Of Strongylocentrotus Intermedius

Seawater acidification(Ocean acidification or seawater acidification,OA)refers to a global environmental problem in which excess carbon dioxide gas(CO₂)dissolves in seawater,resulting in a decrease in seawater p H.Strongylocentrotus intermedius has the characteristics of short spines,good gonad quality and fast growth and development.It has become an important economic species in China's marine aquaculture industry.Our previous study found that the survival rate of S.intermedius four-armed larvae decreased significantly with the decrease of seawater p H.In order to further analyze the molecular mechanism of the effect of seawater acidification on the survival of S.intermedius four-armed larvae,according to the predictions of the United Nations Intergovernmental Panel on Climate Change,the current natural seawater p H(Control:p H=7.99±0.01)and laboratory-controlled acidification In seawater(OA₁:?p H=-0.3;OA₂:?p H=-0.4;OA₃:?p H=-0.5),the S.intermedius were induced and cultivated to the four-armed larval stage.To investigate the molecular mechanism of seawater acidification induced survival rate decline in S.intermedius four-armed larvae,we used high-throughput sequencing technology and liquid chromatography-mass spectrometry to sequence and analyze m RNAs and micro RNA(miRNA)transcriptomes,and metabolomes of S.intermedius four-armed larvae under different seawater acidification conditions.The main findings obtained are as follows:(1)Survival rate determination results showed that compared with the control group,the survival rate of larvae of sea urchins in different seawater acidification groups showed a very significant downward trend(P<0.01),and there is a dose-dependent decreasing trend with the degree of seawater acidification.Among them,the survival rate of S.intermedius four-armed larvae in the OA₁ group was the highest(70.00±2.00)%,the survival rate of the S.intermedius four-armed larvae in the OA₃ group was the lowest(51.33±3.06)%,and the survival rate of the S.intermedius four-armed larvae in the OA₂ group was the middle(62.67±4.16)%.(2)Analysis of m RNAs expression of S.intermedius four-armed larvae in response to seawater acidification,it was found that compared with the control group,there were 18differential expression genes(DEGs)in OA₁ group,798 DEGs in OA₂ group and 2033DEGs in OA₃ group,and the number of DEGs showed a linear upward trend with the deepening of seawater acidification.The results of KEGG enrichment showed that phospholipase D signal pathway,endocytosis,insulin signal pathway,glucagon signal pathway,adhesion,DNA replication,glutathione metabolism and were the main signal pathways affected by seawater acidification.At the same time,compared with the control group,with the deepening of seawater acidification,the number of KEGG signal pathways also showed a significant increase trend.The results showed that seawater acidification could affect the pathways in vivo by changing the expression levels of m RNAs in the S.intermedius four-armed larvae,and with the decrease of seawater p H,the number of pathways affected gradually increased and the types of pathways were complicated;in the OA₁ group,seawater acidification reduced the survival rate of S.intermedius four-armed larvae by affecting physiological processes such as cytoskeleton rearrangement,protein transport,and cell signal transduction;in the OA₂ group,seawater acidification affected the normal secretion of insulin and glucagon in S.intermedius four-armed larvae,resulting in an imbalance of blood glucose homeostasis in S.intermedius four-armed larvae,thereby reducing the survival of S.intermedius four-armed larvae;in the OA₃ group,seawater acidification not only affected physiological processes such as cytoskeleton reorganization and cell polarity in S.intermedius four-armed larvae,but also affected biological inheritance and immune mechanisms in S.intermedius four-armed larvae,which lead to the survival rate of S.intermedius four-armed larvae decreased sharply;with the continuous decrease of seawater p H,the effect of seawater acidification on the physiological mechanism types of S.intermedius four-armed larvae gradually increased,the additive effect of the different physiological mechanisms affected ultimately led to a decrease in the survival rate of S.intermedius four-armed larvae with the deepening of seawater acidification.(3)Analysis of miRNAs expression of S.intermedius four-armed larvae in response to seawater acidification,it was found that compared with the control group,there were 1differential expression miRNAs(DEMs)in OA₁ group,2 DEMs in OA₂ group and 2DEMs in OA₃ group,among them,miR-184 was identified in OA₁,OA₂ and OA₃groups,and miR-1 was identified in OA₂ and OA₃ groups.KEGG enrichment results showed that the KEGG signal pathways enriched by Dems target genes were mainly divided into aminoacyl t RNA biosynthesis,pyrimidine metabolism,glycine,serine and threonine metabolism,glycolysis/gluconeogenesis,pyruvate metabolism,pentose phosphate pathway,fructose and mannose metabolism,fat digestion and absorption,arginine and proline metabolism.The results showed that the number of DEMs in the larvae of S.intermedius four-armed larvae increased with the decrease of seawater p H,and the increase of the KEGG signaling pathway affected by the target genes of DEMs also meant that the diversity of pathway functions was increased;miR-184 was identified in all seawater acidification groups,and its effect on energy metabolism and apoptosis was one of the main molecular mechanisms for the decreased survival rate of S.intermedius four-armed larvae in all seawater acidification groups;with the decrease of seawater p H value,miR-1 was identified in OA₂ and OA₃ groups which effect of target gene(Myo D1)on the early bone formation of S.intermedius four-armed larvae may be one of the main reasons why the survival rate of OA₂ and OA₃ groups is lower than that of OA₁ group;the results of the KEGG pathway showed that seawater acidification in the OA₁ group only reduced the survival rate of S.intermedius four-armed larvae from the perspective of energy metabolism,while in the OA₂ and OA₃ groups,seawater acidification not only affected the energy metabolism of S.intermedius four-armed larvae,and also had various effects on the survival rate of S.intermedius four-armed larvae from the aspects of cell proliferation and immune regulation,and finally the survival rate of S.intermedius four-armed larvae decreased with the decrease of seawater p H.(4)Untargeted metabolomics analysis of S.intermedius four-armed larvae in response to seawater acidification,it was found that comparative metabolome analysis identified a total of 87 significantly differentially expressed metabolites(SDMs,UP:57,DOWN:30)in OA-treated groups compared with the control group.Twenty-threesignificantly differentially expressed metabolites,including carnitine,lysophosphatidylcholine(LPC)18:3,lysophosphatidyl ethanolamine(LPE)16:1,glutathione(GSH)and L-ascorbate,exhibited a linear increasing trend with decreasing seawater p H.Nine significantly differentially expressed metabolites exhibited a linear decreasing trend as the seawater p H declined,including hypoxanthine,guanine and thymidine.Among all significantly differentially expressed metabolites,we further mined 48 potential metabolite biomarkers responding to seawater acidification in four-armed larvae of S.intermedius.These potential metabolite biomarkers were mainly enriched in five pathways:glycerophospholipid metabolism,glutathione metabolism,purine metabolism,pyrimidine metabolism and the tricarboxylic acid cycle.The results showed that with the deepening of seawater acidification,the number of significantly differentially expressed metabolites gradually increased in the S.intermedius four-armed larvae,and the metabolic pathways enriched by potential biomarkers gradually increased,in OA₁ group,14 biomarkers can be divided into lipid and lipid-like molecules,biotoxic substance,antioxidants substance;in OA₂ group,34biomarkers can be divided into lipid and lipid-like molecules,genetic information,biotoxic substance and antioxidants substance;in OA₃ group,35 biomarkers can be divided into lipid and lipid-like molecules,genetic information and antioxidants substance;with the decrease of seawater p H,seawater acidification may eventually reduce the survival rate of S.intermedius four-armed larvae through Change lipid distribution,increase the content of biotoxic substance in the larvae,and reduce the synthesis of genetic information.To sum up,this study obtained the data of m RNA,miRNA and small molecule metabolites of S.intermedius four-armed larvae under different seawater acidification conditions,and explored the molecular expression regulation mechanism of sea urchin in response to seawater acidification from multiple angles,so as to provide theoretical basis and reference for comprehensively mastering the molecular mechanism of sea urchin in response to seawater acidification.