Effects Of Nano-copper And Copper Sulfate Exposure On Histological Lesions And Endoplasmic Reticulum Stress In The Pufferfish

Takifugu fasciatus is a typical anadromous species,sensitive to environment changes,and its life cycle inevitably depends on the external ecological environment.In recent years,the increasingly scale of farming and the continuous development of farming technology,the pufferfish has a certain position in the economic fish market.In both aquaculture and wild,T.fasciatus is extremely susceptible to external influences.Previous studies have shown that the exposure of copper nano-copper(Cu-NPs)and copper sulfate(Cu SO₄;Cu²⁺for short)have adverse effects on T.fasciatus culture,but its mechanism is still unclear.A large number of studies have shown that endoplasmic reticulum stress(ERS),its unfolded protein response(UPR)and Ca²⁺homeostasis may play an important role in the process of toxic effects of pollutants.This study uses this point as a pointcut.Firstly,ERS-related genes were cloned from the liver of T.fasciatus.Secondly,the effects of two different forms of copper exposure on tissue lesions of T.fasciatus were compared.Responses of UPR signaling pathway and Ca²⁺homeostasis in T.fasciatus to Cu-NPs and Cu²⁺exposure.This study not only provides a theoretical basis for further revealing the regulatory mechanism of ERS-related genes in two different forms of copper,but also provides a valuable reference for further evaluation of the toxic effects of new pollutants Cu-NPs on aquatic organisms.The information also provides basic information for the healthy breeding of the T.fasciatus.The main results are shown as follows:1.Molecular characteristics and tissue expression analysis of endoplasmic reticulum stress-related genes in T.fasciatusThe full-length sequences of GRP78,CRT,PERK,ATF-6a gene and IRE-1?gene were amplified by RACE.The results showed that the total length of the c DNA of GRP78 gene was 2542bp,including 1963 bp open reading frame(ORF),encoding 653amino acids,and its 5'and 3'UTR regions were 140 bp and 439 bp,respectively.The total length of c DNA of CRT gene is 1583 bp(Gen Bank registration number:MN580497),including 1293 bp open reading frame(ORF),encoding 430 amino acids,and its 5'and 3'UTR regions are 131bp and 159 bp,respectively.The total length of c DNA of PERK gene was 5374 bp,including 3303 bp open reading frame(ORF),encoding1100 amino acids,and the 5'and 3'UTR regions were 151 bp and 1920 bp,respectively.The c DNA of the ATF-6?gene is 3505bp,including 1947 bp open reading frame(ORF),encoding 648 amino acids,and its 5'and 3'UTR regions are 144 bp and 1414 bp,respectively.The total length of c DNA of the IRE-1?gene is 3805bp,including 3228bp open reading frame(ORF),encoding 1075 amino acids,and its 5'and 3'UTR regions are 181 bp and 396 bp,respectively.Therefore,the liver may be the target organ for ER stress related genes to perform their functions.In this study,the quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the genes of GRP78,CRT,PERK,ATF-6?,and IRE-1?in seven tissues(brain,spleen,intestine,Muscle,gill,skin,and liver).The results showed that these five endoplasmic reticulum stress-related genes were highly expressed in liver tissues and the lowest in skin.Judging from the results,these five genes play an important role in the regulation of misfolded or unfolded proteins,so they all have higher expression levels in liver tissues with frequent protein synthesis.2.Pathological analysis of the liver and gill of T.fasciatus under Cu-NPs and Cu²⁺exposureTransmission Electron Microscope(TEM)was used to observe and analyze the characteristics of Cu-NPs in aquaculture water.The analysis results showed that Cu-NPs have accumulated in aquaculture water.Nanoparticles Tracking Analysis(NTA)was used to measure the inner diameter of Cu-NPs in cultured water,and the mean particle size was 187±56 nm,significantly larger than the initial particle size distribution(70±7.5 nm).Histopathological analysis showed that gill morphology was normal in all control animals not exposed to Cu²⁺or Cu-NPs.Exposure to concentrations of 20?g/L Cu²⁺or Cu-NPs showed edema of gill epithelial cells,lamellar fusion,stick tip and mucinous cell swelling.However,in gill tissue exposed to concentrations of 100?g/L Cu²⁺or Cu-NPs,more severe case pathology,such as branchial filaments deformities,aneurysms,and necrosis,was found.Similarly,the liver of the puffers in the control group showed normal histological morphology.The liver of individuals exposed to the 20?g/L Cu²⁺or Cu-NPs group showed edema and blood cells deposited in the vein,while the liver of individuals exposed to 100?g/L Cu²⁺or Cu-NPs showed more severe Lesions such as hepatitis-like injuries,necrosis,and pyknosis.From this result,Cu-NPs and Cu²⁺did cause toxic effects on the T.fasciatus,and the results of tissue lesions induced in the liver and gill of pufferfish were similar.3.Response of ERS-mediated UPR signaling pathway and Ca²⁺homeostasis under Cu-NPs and Cu²⁺exposure in the liver of T.fasciatusQuantitative Real-time Polymerase Chain Reaction(qRT-PCR)was used to detect the ERS marker gene(GRP78)and UPR signal pathway genes(PERK,e IF2?,ATF-6?,IRE-1?and XBP-1)and Ca²⁺homeostasis regulatory genes(CRT)in response to two forms of copper exposure.The results showed that after exposure to Cu-NPs(20?g/L and 100?g/L)and Cu²⁺(20?g/L and 100?g/L),m RNA expressions of PERK,e IF2?,ATF-6?,IRE-1?and XBP-1 all occurred significantly and the m RNA expression was significantly higher than that in the control group.Under the same form of copper exposure,the expression levels of the ERS marker gene,the UPR signaling pathway gene and the Ca²⁺homeostasis gene in the 100?g/L treatment group were higher than those in the 20?g/L treatment group.In addition,at the same concentration,m RNA expressions of most of the seven genes were not significantly different between the two forms of copper treatment groups.Similarly,GRP78,CRT,p-PERK,ATF-6?and p-IRE-1?were expressed at the protein level after exposure to Cu-NPs(20?g/L and 100?g/L)and Cu²⁺(20?g/L and 100?g/L),compared with the control group,a significant increase occurred.In addition,the expression of the vast majority of these five proteins was not significantly different between the two forms of copper-treated groups.These results indicate that exposure to Cu-NPs or Cu²⁺in the water body indeed induces ERS in the liver of T.fasciatus and activates its mediated UPR and signaling pathways.Cu-NPs and Cu²⁺exposure may have some similar mechanisms of toxic action on T.fasciatus.