| In the present study,experimental ecology,histology,physiology,omics,molecular microecology and bioinformatics were used to study the physiological response and adaptation characteristics of yellowtail kingfish to culture density,high temperature,low oxygen,land-sea relay transportation operation and growth.The effects of digestive tract microbial flora on growth and density adaptation of yellowtail kingfish in land-based greenhouse and deep-sea cage culture were revealed.The histological,microecological,omics and physiological adaptation mechanisms of yellowtail kingfish to hypoxia stress and reoxygenation were investigated and clarified,which provided basis for developing hypoxia adaptation regulation techniques.The microecological and physiological responses of yellowtail kingfish juveniles to high temperature stress were revealed,which provided important reference for the selection and regulation of suitable culture temperature during farming course.In addition,the histological,serum,physiological and omic effects of sea-land transportation operation stress on yellowtail kingfish were investigated,which provided theoretical support for the establishment of safe sea-land relay transportation technologies.The main research results are as follows:1.Microecological adaptation of yellowtail kingfish growth to the density of factory cultureWe studied the effects of low(5 kg /m3),medium(10 kg /m3)and high(15 kg /m3)densities on the growth of yellowtail kingfish(body weight 572.83±84.78 g)in factory culture,and analyzed the adaptation characteristics of yellowtail kingfish to density from the perspective of digestive tract flora.In order to explore the appropriate density of yellowtail kingfish culture and lay a theoretical foundation for the development of healthy culture technology.Illumina Mi Seq platform was used to perform 16 Sr RNA highthroughput sequencing of the digestive tract(stomach,intestinal tract,and pyloric blind sac)flora of yellowtail kingfish under different culture densities.The diversity,structural characteristics,and gene function prediction of the flora were analyzed.The results showed that the effects of density on the growth of yellowtail kingfish were mainly reflected in the inhibition of final body mass,specific growth rate,and serum stressrelated hormones at high density(P <0.05).At the phylum level,the microbial community of the digestive tract was mainly composed of Proteobacteria,Bacteroidetes,Firmicutes,and Actinobacteria,which did not change with the change of culture density.However,the proportion of bacterial flora structure changed.At the genus level,the abundance of Lactobacillus,Sphingomonas,Bifidobacterium,Faecalibaculum and Ruminococcaceae_UCG decreased in the medium and high density groups.The abundance of Klebsiella increased in the medium and high density groups,which were significantly different from that in the low density group(P < 0.05).Compared with the low density culture group,the composition and structure of gastrointestinal flora in the medium and high density culture groups showed a significant reduction in beneficial bacteria(Lactobacillus,etc.)and a significant increase in opportunistic pathogenic bacteria(Klebsiella)(P<0.05).In terms of KEGG functional annotation of flora,compared with the low density group,the total number of genes involved in functional pathways in the medium and high density groups increased significantly,especially the number of genes involved in the secondary functional pathways of metabolism,genetic and environmental information processing was significantly increased(P < 0.05).2.The microecological regulation of digestive tract microbiota on the growth of yellowtail kingfish larvae under two culture modesTo investigate the growth difference of yellowtail kingfish juveniles under indoor tank and deep sea cage culture modes,and its relationship with gastrointestinal flora as well as link to feed microbiota and culture water microbiota,six-month-old fish of the same size with average body length of(17.23±0.99)cm and body mass of(77.44±11.58)g were selected for a 30 day culture experiment under these two culture modes.The growth difference of yellowtail kingfish juveniles under these two culture modes was recorded,the structure and abundance of gastrointestinal microbiota(stomach,pyloric blind sac,intestine)as well as feed and culture water microbiota were analyzed by 16 S r RNA high-throughput sequencing and bioinformatic analysis.The results showed that the growth of cage cultured juvenile yellowtail kingfish was significantly faster than that of indoor tank cultured fish.As for gastrointestinal microbiota of juveniles cultured in cage,the abundance of Bacteroidetes and Firmicutes at the phylum level,as well as the abundance of Alloprevotella and Bacteroides at the genus level were higher than indoor tank cultured fish,wherein the abundance of Bacteroides was significantly higher.The composition of the gastrointestinal microbiota of indoor tank cultured fish is different from that of the feed microbiota,and significantly different from that of culture water microbiota,whereas the composition of the gastrointestinal microbiota of fish from cage culture was more similar with that of the feed microbiota,but also significantly different from that of the culture water microbiota.Beta diversity analysis showed that the gastrointestinal microbiota of juvenile yellowtail kingfish was more influenced by feed microbiota and less influenced by the culture water bacterial community.KEGG annotation analysis showed that the main functional pathways involved in the gastrointestinal microbiota of juvenile yellowtail kingfish in the cage culture were Phosphotransferase system(PTS)and NOD-like receptor signaling pathway,whereas carbohydrate metabolism and the carotenoid biosynthesis pathway was found in the indoor tank cultured fish.The present results showed that the gastrointestinal microbiota was involved in regulation of growth performance difference under indoor tank and cage cultured yellowtail kingfish juveniles by the way of changing structure and abundance and further functional pathways.Moreover,the feed microbiota has more influence on gastrointestinal microbiota than culture water microbiota.According to the results of our study,the faster growth of cage cultured fish may be due to the production of more SCFA(short-chain fatty acid)by the gastrointestinal microbiota(Example: Alloprevotella and Bacteroides)to induce the production of insulin-like growth factor 1(IGF-1).Furthermore,the results could provide micro-ecological support for high-efficient commercial feed and healthy culture technology for yellowtail kingfish.3.Physiological and microecological responses of yellowtail kingfish to sea-land relay transportation operationIn order to develop stress-reduction technology for yellowtail kingfish during sealand relay culture transportation,we studied the activities of serum antioxidant enzymes,liver transcriptome levels and the response characteristics of intestinal microbial flora of yellowtail kingfish,the timing points including ship transportation,land vehicle transportation,and 6h,12 h,1d(D1 group),3d(D3 group)and 7d(D7 group)after transportation.The results showed that: Serum cortisol(Cortisol),glucose(Glu),superoxide dismutase(SOD),catalase(CAT)and other anti-oxidative stress indicators were significantly increased during transportation.The level of yellowtail kingfish in D1 group returned to the control level,which was not significantly different from D3 and D7 groups.The change trend of yellowtail kingfish was contrary to MDA,GOT and GPT.The abundance of gut microbiota changed significantly at the genus level.NMDS analysis showed that compared with the control group,the shipborne transport group and the land vehicle transport group had a lower similarity of gut microbiota,while the D1 group had the highest similarity with the control group,but the D3 and D7 groups had a lower similarity with the control group.KEGG annotation results showed that the number of OTUs in the lipid metabolism pathway decreased first and then increased,with the lowest value in D1 group and significantly higher than that in the control group at D7.At the same time,the number of OTUs in the transcription and cell growth and death pathways showed the same changing trend.The abundance of intestinal flora was affected by transportation stress,but could quickly return to a steady state after 1 day.Furthermore,microbiota annotation to metabolic and transcriptional functions indicated active involvement in body repair during the recovery phase.The results of liver transcriptome analysis showed that the total number of DEGs enriched in the protein processing pathway of the endoplasmic reticulum was the most in each experimental group.After the end of transport(T2 group),the protein degradation system of endoplasmic reticulum(ERAD)was activated,in which hsp40,hsp70,hsp90α and other genes were significantly up-regulated.After 1 day of recovery,unfolded protein response(UPR)mediated ER stress,PEPK and Calmin genes were significantly down-regulated,and CRT was significantly up-regulated.The number of significantly up-regulated genes in this pathway increased after 3 days of recovery.After 7 days of recovery,ER stress-related genes returned to the level of the control group.The results indicate that during the landsea relay transport of yellowtail kingfish,nutritional fortification and addition of probiotics can be used to alleviate the transport stress according to the changes in microorganisms,genes and enzyme activity indicators to accelerate the recovery of the body and reduce the transport stress of yellowtail kingfish.4.Physiological responses of yellowtail kingfish to different cultured temperature-change patternsIn order to explore the response mechanism of yellowtail kingfish to temperaturechange patterns,two temperature levels(28℃ and 32℃)were set up,and further the acute temperature change mode and chronic temperature change mode in which were designed.The response characteristics of yellowtail kingfish juveniels to these two temperature-change patterns and the physiological changes after recovery from normal temperature(24℃)were studied.The results showed that under 28℃ condition,SOD,CAT and other antioxidant stress indexes of the two warming modes were significantly higher than those of the control group(P < 0.05),but there was no significant difference between the control group and the recovery group,and the trend of change was opposite to MDA,GOT,GPT and other enzyme activities.At the temperature of 32℃,the experimental fish died on the 4th day,and the levels of SOD,CAT,and MDA on the 3rd day were significantly higher than those of the control group.The levels of SOD,CAT and MDA in the chronic temperature change model were significantly higher than those in the control group.After recovery,there were no significant differences in the enzyme activities between the experimental groups and the control group.Heat stress changed the diversity and relative abundance structure of gut microbiota.Among the three experimental groups after recovery,the differences in Alpha diversity,relative abundance structure and KEGG function annotation between the 32℃ chronic temperature change model and the recovery group were the most significant,and the OTUs in the digestive tract were mainly annotated to the lipid metabolism pathway.The results of liver transcriptome showed that compared with the control group,there were 943 DEGs in the32℃ acute warming mode,of which 516 were up-regulated and 427 were down-regulated.The up-regulated genes PRKCSH,PERK,GRD94,hsp90α,hsp70 and hsp40 were mainly enriched in protein processing pathway of endoplasmic reticulum,lysosome,phagosome and oxidative phosphorylation pathway.The down-regulated genes FABP,SCD-1,ANGPTL4 and IGF1 were mainly enriched in lipid metabolism pathways such as cholesterol metabolism,fat digestion and absorption,PPAR signaling pathway and AMPK signaling pathway,indicating that yellowtail kingfish was activated by endoplasmic reticulum stress and lipid metabolism and growth were inhibited under this temperature change mode.There were 1468 DEGs in 32℃ chronic temperature change mode,of which 819 were up-regulated and 649 were down-regulated.Up-regulated genes were mainly enriched in protein processing pathways of endoplasmic reticulum and proteasome pathways,and down-regulated genes were mainly enriched in antigen processing and immune response pathways such as cysteine and methionine metabolism.This indicates that chronic warming stress only activates part of the protein repair mechanism,but the down-regulation of immune response related pathways indicates that yellowtail kingfish is more susceptible to disease outbreak under this condition.5.Physiological and microecological adaptation mechanism of yellowtail kingfish to hypoxia stress-reoxygenationYellowtail kingfish has emerged as one of the most promising marine fishes for aquaculture in China because it is tasty,fast growing,and has high economic value.Hypoxia is a frequently encountered phenomenon during fish culture,which can cause serious physiological stress and even death of farmed fish.To investigate the tolerance and adaptability to hypoxia of farmed yellowtail kingfish,juveniles were exposed to hypoxia for 5 days and then returned to normoxia for another 5 days.The results showed that hypoxia increased the gill lamellae length and spacing,which were reversible postreoxygenation.At the genus level,the relative abundances of Prevotella,Bacteroides,Roseburia,and Blautia in the gastrointestinal tract increased under hypoxia and were maintained post-reoxygenation.The liver transcriptome revealed that,compared with normoxia group,the different expression genes(DEGs)were mainly enriched in Steroid biosynthesis and PPAR signaling pathways in hypoxia group.Compared with normoxia group,the DEGs were mainly enriched in Ribosome biogenesis in eukaryotes,Steroid biosynthesis,Fatty acid biosynthesis,and PPAR signaling pathways in reoxygenation groups.Furthermore,compared with hypoxia group,the DEGs were mainly enriched in Ribosome biogenesis in eukaryotes and Ribosome pathways in reoxygenation groups.In contrast to normoxia,of the key genes of the PPAR signaling pathway,FABP4 was significantly downregulated,and SCD-1 and FATP were significantly upregulated.These findings indicated reduced lipid deposition and increased lipid decomposition in liver under hypoxia.The genes including PPARα,SCD-1,ANGPTL4,and FASN were significantly upregulated in lipid metabolism-related pathways,which indicated that lipid metabolism activity was more vigorous during reoxygenation.In contrast to the hypoxia group,almost all of the genes involved in Ribosome biogenesis in eukaryotes and Ribosome pathways for protein processing were significantly upregulated during reoxygenation;this is probably related to the clearance of misfolded proteins and the folding of the new proteins repairing there is damage to the body.The present results shed light on the possible synergetic function of lipid metabolism,protein repairment and synthesis,and gastrointestinal microbiota in resistance and homeostasis maintenance of yellowtail kingfish coping with hypoxic stress in aquaculture. |
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