Waterborne Copper Exposure Influencing Intestinal Microflora And Hepatic Lipid Metabolism In Cyprinus Carpio L.

In the current intensive farming model,many problems appeared such as lipid metabolism disorder of liver for fish farming.Cyprinus carpio L.is the main economic fish in north China.In recent years,farming carps appeared disorder such as accumulation of excess fat,due to many factors such as the deterioration of aquaculture water,high breeding density.In many environmental factors,copper was considered as an mineral nutrients and algae or pathogens inhibition of drugs,and was often used in the feed or water environment.When it reaches a certain concentration in the form of free metal ions,it will produce toxic on aquatic animals.The animal gut microbes of fish take important roles in the nutrition metabolism,especially the fat metabolism.Furthermore,gut microbes,the host and environmental factors exist complex interactions.In the present study,the changes of gut microbes of Cyprinus carpio L.was studied by using high-throughput sequencing technology,and the gene expression related to carp liver pancreas lipid metabolism and the binding protein genes in gut was detected by using fluorescence quantitative PCR.The relationship between changes of gut microbes and the lipid metabolism disorder induced by waterborne copper exposure was probed.The present research aimed to offer a new insight for regulation mechanism of lipid metabolism,and supply a scientific basis for healthy intestinal environment and healthy growth in fish.1.The effects on growth performance and excessive copper accumulation in the body tissue of Cyprinus carpio L.caused by waterborne copper exposureThe copper half lethal concentration?LC50 = 1.42 mg/L?was determined.A control group and three treatment groups?concentration were 0.00 mg/L,0.07 mg/L,0.14 mg/L,and 0.28 mg/L?were designed based on the copper half lethal concentration.The feeding experiment was carried out for 56 days.The initial weight was determined before feeding and the final weights was determined after feeding experiments,and then the weight gain rate?WGR?and specific growth rate?SGR?were calculated.The results showed that the growth performance,WGR and SGR of treatment groups were not significantly decreased?P > 0.05?compared to the control group.However,among the treatment groups,a significant decrease was detected in high concentration group than the low concentration group?P < 0.05?.Therefore,high concentration of copper effected on Cyprinus carpio L.may significantly reduced weight and induced metabolic disorders.The content of Cu²⁺ in liver pancreas and intestine were detected by atomic absorption spectrometry.The data demonstrated that the concentration of copper in liver pancreas and intestinal accumulated exponentially accompany with the increasing copper concentration treatment.The serum concentrations of total protein?TP?,high-density lipoprotein?HDL?,low-density lipoprotein?LDL?,triglyceride?TG?,immunoglobulin A?IgA?,immunoglobulin G?IgG?,and the activity of alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,alkaline phosphatase?ALP?were determined by using the automatic biochemical analyzer?AU-640,OLYMPUS?.The results showed that the abnormal significantly?P < 0.05?of ALT,LDL,TG,IgG were detected among the treatment groups,which indicated that liver may be suffered serious damage,especially in middle and high concentration group.2.Detection of lipid metabolism-related genes expression of liver and intestine in Cyprinus carpio L.by realtime fluorescence quantitative PCRThe expression of five kinds of lipid metabolism-related genes were detected using RT-PCR with 18 S rRNA as reference.The results showed that the expresion of lipiogenic factors?SREBP-1?and lipiogenic enzymes?ACC1,FAS?were significantly down-regulation?P < 0.05?,and the expression of lipolysis enzymes?LPL,CPT-1?were significantly up-regulation?P < 0.05?,which indicated that fat synthesis increased and fat decomposition reduced in the treatment groups.The expression of two kinds of intestinal mucosa tight binding protein genes?Zo-1,Occludin and Claudin-3?were also detected.Compared with the control group,Zo-1 gene expression quantity in treatment groups had no significant changes?P > 0.05?,while Occludin and Claudin-3 gene expression appeared down-regulation significantly?P < 0.05?.It indicated that intestinal wall permeability increased,so that intestinal metabolites may enter into the blood stream.It was reported that there is an intestine-liver axis in body.Under the normal circumstances,intestinal mucosa can allow few amount of LPS into portal vein to maintain the liver system active.When function of intestinal mucosa is lost,higher permeability is increased.With a large number of bacteria and LPS translocation,immune function of liver is impaired and cause liver injuried chronically.3.Analysis of intestinal microbial community caused by waterborne copper exposure by using high-throughput sequencing technology12 samples were sequencing using Illumina HiSeqTM 2500.838,774 original sequences have been obtained.And then 721,786 good quality sequences were selected after filtered out dirty quality sequences.Finally,29,951 OTU were got from all the samples and each OTU type represented a kind of system.The results showed good level of species diversity of the samples.The data of ACE value,Chaol,Shannon index,Simpson index had no significant changes after water copper exposure?P>0.05?.At the phylum level,all sequences were belong to 58 phylums,mainly included Proteobacteria?51.73%?,Fusobacteria?10.97%?.Bacteroidetes?5.24%?,Firmicutes?4.76%?,Cyanobacteria?4.24%?,Actinobacteria?3.02%?,Verrucomicrobia?2.25%?,Acidobacteria?2.69%?,TM6?2.16%?,Gemmatimonadete?1.11%?and others phylums?8.47%?.Among the microbes which were related to lipid metabolism,the proportion of Fusobacteria were increased in low concentration group compared to the control group.The proportion of Firmicutes and Bacteroidetes were decreased in high concentration groups compared to the control group.