| The outbreak of Ulva prolifera in the South Yellow Sea area of China for 15 consecutive years had a great impact on coastal mariculture and ecological environment in Shandong Province.After the outbreak of U.prolifera,a large number of algae subsided to the seabed and then decayed,which is bound to affect marine benthic organisms,however,there are few relevant studies.Based on the above background,this study took Paralichthys olivaceus,a common benthic animal in the South Yellow Sea,as the experimental object.The juveniles of P.olivaceus was exposed to 2 g/L fresh U.prolifera algal body(representing the early stage of corruption,denoted by FU)and 2 g/L 7-day decomposing algal effluent of U.prolifera(representing a period of time of corruption,denoted by DU).After short-term stress for 14 days,the transcriptome analysis and weighted gene coexpression network analysis were used to study the expression of differential genes in the mixed immune tissues(liver,spleen,head kidney and blood),and the real-time fluorescent quantitative PCR technology was used to verify several significantly differentially expressed genes.Finally,the histopathological changes of the liver,spleen and head kidney of P.olivaceus caused by U.prolifera decay were analyzed by histopathological means.The experimental results obtained in this study are as follows:1.The results of transcriptome analysis showed that both FU group and DU group had a large number of down-regulated genes after exposure experiment,and there were more differentially down-regulated genes in DU group;After 7 days of exposure,the differentially expressed genes in DU and FU groups were mostly enriched to the biological processes related to maintaining cell homeostasis and metabolism.After 14 days of exposure,the differentially expressed genes in DU and FU groups were mostly enriched to the processes of oxidoreductase activity,immune response,organic acid metabolism and carboxylic acid metabolism.The results showed that U.prolifera had a greater impact on the immune tissue of P.olivaceus juveniles at the late stage of decay;U.prolifera decay affected multiple immune and metabolic related pathways in P.olivaceus juveniles.Among them,the steroid biosynthesis pathway was significantly enriched after 7 and 14 days of exposure in DU and FU groups,while PI3K-Akt signaling pathway was significantly enriched only after 14 days of exposure in DU group;After 14 days in DU group,several genes related to the immunity of P.olivaceus were significantly up-regulated or downregulated,such as heat shock protein 90,complement C1 q C-like,toll like receptor 2/4.The results suggested that U.prolifera decay might lead to immunosuppression of P.olivaceus juveniles in the late stage of corruption.2.Weighted gene coexpression network analysis preliminarily identified 64 hub genes,which were closely related to the changes of juvenile P.olivaceus after 14 days’ exposure to decomposing algal effluent of U.prolifera,such as interleukin 6,dual specificity phosphatase 1,dual specificity phosphatase 16,toll like receptor 8,toll like receptor 1,Heat shock protein 90 and serotransferrin,etc.In addition,through real-time quantitative PCR detection,it was found that the expression of several immune related genes in P.olivaceus was significantly down-regulated,such as complement C1 q sub component subunit C,solute carrier family 8,member 1,choline phosphate cytidyltransferase B-like,serotransferrin,cd44 antigen,glycerol-3-phosphate dehydrogenase 1-like protein,etc.3.Results of histopathological sections showed that the liver,spleen and head kidney of P.olivaceus in FU and DU groups were damaged to varying degrees.Among them,vacuoles appeared in the liver of both FU and DU groups,and the hepatic sinuses were significantly dilated.Only the liver of DU group had local aggregation of inflammatory cells.Melanin macrophage centers(MMCs)were found in the spleen of P.olivaceus in FU group,and macrophages increased significantly.Compared with DU group,there were more MMCs in the spleen of P.olivaceus.In addition,the head kidney of P.olivaceus in DU group also showed great changes.Compared with FU group,the head kidney structure was become loose,the number of MMCs was more,and there was obvious cavitation phenomenon.The analysis shows that the increase of MMCs in the spleen and head kidney may be caused by the destruction of blood cells,the release of iron by hemoglobin,and the increase of hemosiderin,and the destruction of blood cells may be related to the abnormal expression of key genes that exercise functions.This study investigated the ecological impact of U.prolifera on marine benthic fish from the molecular and tissue levels,and provided a theoretical basis and a new research perspective for further research on the molecular mechanism of U.prolifera decay causing harm to marine organisms in the future. |
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