Comparative Analysis Of Heat Tolerance Of Acclimatized Haliotis Discus Hannai Juvenile Under High And Low Temperature By Metabonomics And Transcriptionomics Technology

Pacific abalone Haliotis discus hannai Ino is one of the cultured vetigastropoda with high economic value in China.In recent years,frequently occurred massive summer mortality has restricted the industry development,while few is known on the causes.Pacific abalone,with the preference temperature range 15-22°C,naturally inhabit along northern Yellow Sea to the coast of Korea and northern east Japan.Since the 21 st century,with the rapid development of artificial nursery and aquaculture technology,Fujian and Guangdong have become major production areas of Pacific abalone.Most aquaculture companies applied "north-south relay" strategy to culture Pacific abalone.In early November,abalones with shell length of 2-3cm are transported to the south for overwintering,and then returned to the north in May of next year for estivating.Some abalones are retained at south in summer and thus experience warm acclimation during this process.It is still unknown whetehr warm acclimation benefit for high temperature tolerance.At the same time,understanding the mechanism of abalone thermal stress response is urgently need,especially from the perspectives of metabolism and molecular level,to provide theoretical support for new aquaculture mode development and genetic improving of abalones.In this study,we use H.discus hannai juvenile to analyze the thermal response and tolerance mechanism from four aspects: phenotype,physiological characteristics,metabolites and m RNA level.Main results are as follows:1.Phenotypic adaptability of temperature in Haliotis discus hannai juvenileAt the end of the 62-day culture process,there was no death in the lowtemperature acclimated population(group L),which maintained a 100% survival rate,while the high-temperature acclimated population(group H)had a survival rate of nearly 80%.We calculated average oxygen consumption rate(OCR)via 24-hour continuously monitoring the dissolved oxygen change.Rusult showed that OCR of individuls from group H was 1.98-fold as many as that of ones from group L(t =-5.912,P = 0.000).High OCR means high aerobic metabolic rate and more ATP output of oxidative phosphorylation(OXPHOS).Nevertheless,this increased energy flux didn't be used for the accumulation of body composition because of lower values of the average shell growth rate,shell width and bady weight growth rates of abalone from group H(0.196,0.040 and 0.006 of corresponding values of ones frome group L,respectively)indicating environmental temperature had a great influence on the growth performance of juvenile abalone,namely,excessive high temperature inhibits abalone's growth and weight gain.Although individuals in group H sacrificed theirs rapid growth performance,they had higher heat tolerance.After being matained at same temperature(20°C)for one week,45 abalones from each group were exposed to31°C immediately,the mortality rate of L group was 100% while the number of H group was only 2.2% during 48-hour observation period.2.Metabolic adaptability of temperature in Haliotis discus hannai juvenileThe hemolymph glucose(GLU)content of juvenile abalone in H group was significantly higher than that in L group(t =-2.615,P = 0.031),indicating that abalone under high temperature environment had higher demand for energy substance.Higher activity of ALT and AST together with lower content of hemocyanin was observed in hemolymph of ones from group H,but there were no significant difference between the two groups in these three parameters(P > 0.05).The physiological and biochemical analysis results of the foot muscle,gill,mantle and hepatopancreas showed that GLU concentrations and the activities of two major anaerobic metabolic enzymes of abalone--lactate dehydrogenase(LDH)and tauropine dehydrogenase(TDH)in these four tissues from H group were increased compared with those from L group indicating that anaerobic metabolism took part in fueling the cellular energy demand.Otherwise,the SOD activities in four tissues from H group were generally higher than that from group L whereas the CAT activities in gill,mantle and hepatopancreas were lower than that of L group.The content of lipid peroxidation biomarker--malonaldehyde(MDA)in gill and mantle were significantly higher in group H,this is one of the reason why organisms require more energy under heat stress.3.Analysis of heat stress response and heat resistance of Haliotis discus hannai based on metabolomeAbalones from two groups were kept at 20°C for one week in order to homogenization then put into 31°C immediately and incubated for 3 hours,labeled as LH and HH respectively.The hepatopancreas from group L,H,LH and HH were sampled for metabolome analysis.In total,1815 and 1314 significantly changed metabolites were found in LH and HH group compared with L and H group,respectively.Most of these metabolites in L vs LH were carboxylic acids and derivatives(17.65%),diazines(8.82%)and fatty acyls(8.82%),while the top three classes of differential metabolites in H vs HH were organooxygen compounds(18.92%),carboxylic acids and derivatives(16.22%)and diazines(5.41%)indicating the differential metabolites undergoing heat shock between the two groups were obviously unlike.A number of 42 and 100 signifcantly different metabolite biomarkers(MBs)were identifed in L vs LH and H vs HH,respectively,with parameter setting is FC >1.5 or < 0.6 and VIP value > 1.5.Among the 28 same MBs of these two group,associated with partly break up of amino acids in mitochondria were significantly accumulated.Such as 2-methylbutyroylcarnitine(P < 0.05),usually not detected in normal individuals,the elevation of 2-methylbutyrylcarnitine suggests a deficiency of isobutyryl-Co A dehydrogenase,which helps break down the amino acid valine.The L-kynurenine and DL-Indole-3-lactic acid which are partly break down metabolites of the amino acid tryptophan accumulated significantly(P < 0.05)in cells after heat stress.Kynurenine-3-monooxygenase(KMO)deficiency contributes to an accumulation of kynurenine and high level of this metabolite is proved to be associated with neurotransmitter metabolic disorders.DL-Indole-3-lactic acid induces caspase-8 and caspase-9,which results in caspase-3 activation and then apoptosis.Heat stress also led to a significant increase in the content of N-Acetyl-L-tyrosine(P< 0.05),a intermediate product of tyrosine metabolism and associated with aromatic L-amino acid decarboxylase deficiency.Significant high glutaric acid only be detected in LH group compared with group L(P < 0.05).Chronically high levels of glutaric acid are associated with at least three enzymes dysfunction including glutarylCo A dehydrogenase,electron transfer flavoprotein(ETF)and electron transfer flavoprotein dehydrogenase(ETF-QO).The glutaryl-Co A dehydrogenase deficiency in which the cell is unable to completely break down the amino acids lysine,hydroxylysine,and tryptophan.When one of ETF and ETF-QO is defective or missing,the mitochondria cannot function normally,partially broken-down proteins and fats accumulate in the cells and damage them.Parallelly,cellular contents of MBs associated with fatty acid oxidation disorder(FAOD)were observed of a large increment in ones expoesed to heat stress(P < 0.05).Such as stearoylcarnitine and L-palmitoylcarnitine which are found in significantly greater amounts of cells with carnitine palmitoyltransferase(CPT)II deficiency that is a metabolic disorder characterized by this enzymatic defect that prevents long-chain fatty acids from being transported into the mitochondria for utilization as an energy source.The long chain acylcarnitines have cytotoxicity,which can affect the molecular dynamics of cell membrane and induce apoptosis.Large amount change of suberic acid was detected in both LH and HH group(P < 0.05).Elevated levels of this unstaruated dicarboxylic acid are found in individuals with medium-chain acyl-Co A dehydrogenase deficiency(MCAD).Suberic acid is also found to be associated with carnitine-acylcarnitine translocase(CACT)deficiency,malonyl-Co A decarboxylase deficiency.Other metabolic biomarker,the decanoy-L-carnitine was detected significantly accumulated in LH group(P < 0.05).In a word,high temperature can result in incomplete decomposition of amino acids and fatty acids in hepatopancreas cells of juvenile abalone,illustrating that the pathway of oxidativing these two energy substances to yield energy was dysfunction.This phenomenon is first observed in marine mollusc.When organisms suffering heat stress,demand of energy greatly increased.Due to the fact that mitochondria are energy factories of cells,the stability of mitochondrial structure and function plays an important role in heat stress tolerance of Haliotis discus hannai.Some metabolic toxic substances such as glutaric acid,decanoy-L-carnitine,L-gulonic gamma-lactone and beta-alanine were found significantly increased only in the LH group(P < 0.05).While some beneficial substances such as D-glucose-6-phosphate,4-hydroxycinnamic acid,trans-2-hydroxycinnamic acid,N-acetyl-D-glucosamine and tiopronin were able to accumulate rapidly only in the individual of group HH(P < 0.05).In addition,metabolites related to promoting and stabilizing metabolism such as riboflavin,pantothenate,choline as well as CDP-choline which is related to stabilizing and repairing biomembrane and neuroprotection in hepatopancreas of group HH was significantly higher than that of group LH(P < 0.05).In a word,individuals of group H had obvious advantages in promoting,stabilizing metabolism,repairing molecular damage and protecting nerve,which can help explain the high heat tolerance of abalones from this group.4.Analysis of heat stress response and heat resistance of Haliotis discus hannai based on transcriptomeA total of 12 m RNA samples form L,LH,H and HH group(4 group and 3repetitions in every group)were sequenced then the clean data were mapped to the reference genome.Based on the mapped results,24,464 of novel genes were functionally annotated.Gene expression analysis was performed and differentially expressed genes were identified based on their expression levels in different samples.There were 478 differential expression genes in group L vs LH including 315 upregulated genes and 163 down-regulated genes while 709 genes in group H vs HH among which 562 up-regulated and 147 down-regulated.KEGG pathway enrichment analysis of shared differentially expressed genes of L vs LH and H vs HH was executed and results showed that the most significant pathway is protein processing in endoplasmic reticulum and genes enriched in this pathway were mostly molecular chaperone.The gene expression of 17 genes including molecular chaperone,ubiquitination-related proteins together with proteins involved in autophagy and apoptosis were estimated by q PCR assay.The gene expression pattern were consistent with the transcriptome results.Data showed that m RNA levels of HYOU1 and SIL1,Dna JC 3(belonging to HSP 40 family)and CCT7 these genes involved in protein recognition and processing were incresased significantly in the hepatopancreas of two populations after heat shock(P < 0.05).Although the transcriptional level of gene encoding HSPA5(also known as Bip or GRP 78)increased after heat stress,there was no significant change(P > 0.05).The m RNA contents of five chaperone proteins(HSPA1S?HSP110?Htp G?CRYAB and BAG3)in ER-related protein degradation pathway as well as Ubiqutin,were significantly up-regulated after heat shock in both groups(P < 0.05).It is noteworth that the transcription difference of CRYAB coding gene was more than 10,000 times pointing that alpha-crystallin protein may play an important role in the regulation of heat stress response in juvenile abalone Haliotis discus hannai.The expression levels of E1 and E2 protein gene did not change significantly before and after heat shock(P > 0.05).The expression level of E3 protein encoding gene,which regulates cell cycle,apoptosis,transcription,protein quantity control and signal transduction,increased significantly only in group HH(P< 0.05).In addition,the m RNA concentrations of the CRYAA protein which is involved in protein refolding and degradation,the p62 protein which is associated with toxic aggregate clearance were only significantly increased in the HH(P < 0.05).The gene expression of apoptosis inhibitor c IAP1 was increased significantly in LH and HH compared with L and H,respectively(P < 0.05)and value of HH was even higher than that of LH(P < 0.05).This indicates that from the aspect of regulation of cell fate,group H has a certain degree of advantage,which is one of the reasons for its strong heat resistance.