Tolerance Differentiation And Acclimation Of Abalone Under Hypoxia Stress

Since the middle of the 20th century,one of the most important changes in the global ocean has been the continuous decline in dissolved oxygen concentration in the open ocean and coastal waters due to global warming and excessive discharge of nutrient-rich wastewater caused by human activities.As an economically important shellfish,abalone has high requirements for water quality and has suffered a lot from hypoxia events in coastal waters.Therefore,it is of great significance to study the environmental hypoxia responses of abalone,as the abalone aquaculture system provides an important model for how economic species in the aquaculture system respond and adapt to global climate change.In this study,physiological methods and metabolomics were used to compare the metabolic response characteristics of different abalone species to acute hypoxia and reoxygenation conditions and to explore the effects of long-term thermal acclimation on the hypoxia responses of abalone.Population genetics,quantitative genetics,and transcriptomics were used to analyze the genetic basis of hypoxia tolerance in abalone and to explore the feasibility and mechanism of inducing phenotype differentiation of hypoxia tolerance in abalone by early hypoxia exposure.This study could be helpful for the breeding of hypoxia-tolerant abalone species,the establishment of scientific management strategy in aquaculture,the selection of suitable areas for abalone aquaculture,and evaluating and predicting the impacts of global climate change on mariculture systems.The results are as follows:1.Physiological and metabolic responses of two abalone species under acute hypoxia and reoxygenation conditionsAcute hypoxia events that happened in abalone aquaculture areas were simulated,and physiological and metabolomic methods were used to compare the metabolic responses of Pacific abalone Haliotis hannai discus（DD）and the hybrid H.discus hannai♀×H.fulgens ♂（DF）to acute hypoxia（～0.5 mg/L,12 h）and reoxygenation（～6.6 mg/L,10-20 h）.Compared with DF,DD abalones were less tolerant to hypoxia,indicating by higher mortality,more individuals leaving the shelter and losing adhesion.Under the conditions of hypoxia and reoxygenation,physiological indexes including the hemocyte concentration and phenol oxidase（PO）activity in the hemolymph differed a lot or even changed in distinct way in two abalone species.The differentially expressed metabolites in DF abalone were more involved in amino acid metabolism,carbohydrate metabolism,signal transduction,and excretion system under hypoxia and reoxygenation conditions.In addition,L-glutamate,2-hydroxy-butanoic acid,and 2methyl-3-hydroxybutyric acid were determined as potential biomarkers for hypoxia and reoxygenation response in abalone.2.Metabolic coordination in H.discus hannai,H.fulgens,and their hybrid abalone under acute hypoxia after long-term thermal acclimationA combination of physiological and metabolomic methods were used to compare the metabolic responses of the northern population of H.discus hannai（DL）,southern population of H.discus hannai（JJ）,hybrid H.discus hannai ♀×H.fulgens ♂（DF）,and H.fulgens（FF）to acute hypoxia（-0.5 mg/L）after long-term thermal acclimation at two temperatures（20℃ and 28℃）.Long-term thermal acclimation would increase the sensitivity of abalone to hypoxia（called "cross-tolerance"）,as indicated by the phenomenon that more abalones left the shelter,lost adhesion,and died.Long-term thermal acclimation shaped the hypoxia responses of abalone by reducing phosphorylated metabolites,which are important substrates involved in carbohydrate and amino acid metabolism.The metabolic dynamics vary among different abalone populations/species,possibly reflecting the stronger environmental resistance of JJ relative to DL and the hybrid DF relative to its parent populations.3.A genome-wide association study of hypoxia tolerance in H.discus hannai based on genome resequencingA multi-family mixed population of Pacific abalone was constructed.The hypoxia tolerance of 1,147 individuals in the population was determined based on the breakpoint of dissolved oxygen（BPDO）,and a total of 1,381,543 high-quality SNPs were identified by whole-genome resequencing of 585 individuals.A total of 21 SNP loci and 19 candidate genes were identified by the genome-wide association study（GWAS）.Some candidate genes were related to hypoxia regulation,including gria2 and lrp6 involved in signal transduction,tyms involved in DNA synthesis and replication,and rft2 and casp8 involved in apoptosis,indicating that hypoxia tolerance of abalone may be a quantitative trait controlled by multiple genes and affected by different biological processes.These results provide a basis for the analysis of hypoxia tolerance in abalone.4.Phenotype differentiation and plasticity of hypoxia tolerance of H.discus hannai at the early developmental stagesThe Pacific abalone was exposed to moderate hypoxia（about 4 mg/L）during the early development stages（from zygote to trochophora）.Assessments of hypoxia tolerance in the lab and at sea showed that the juvenile abalones that have been exposed to moderate hypoxia at the early developmental stages were more hypoxia-tolerant but with slower weight growth,probably because they utilize more energy to survive and less for growth,which is called trade-off between the growth and stress-tolerance.In this study,quantitative genetics and transcriptomic methods were used to illustrate the genetic differentiation and transcriptional regulation of larval abalones in the hypoxia group（H）and normoxia group（N）.（1）There was genetic differentiation between the two groups,and genes that were subjected to strong selection pressure,including pygm,map4k5,and tadh,were mainly involved in signal transduction,autophagy,apoptosis,and hormone regulation.（2）LncRNA regulation played an important role,and 3,831 significantly differential expressed genes（SDEmRNAs）and SDEmRNAs that targeted by 202 significantly differential expressed lncRNAs（SDElncRNAs）were enriched in DNA replication and repair,RNA transcription and degradation,signal transduction,myocardial activity,and hormone regulation.（3）A total of 54 genes that were coexpressed with SDElncRNAs were enriched in the HIF-1 signaling pathway,including mknk1,glut1,tlr2,pfk,vhl,and egln1,most of which were differentially expressed under hypoxia exposure.（4）Seven SDElncRNAs,including TCONS₀0105238,had the most co-expression targeting relationships with mRNAs and might be the core regulatory lncRNAs in abalone under hypoxia exposure.