| The Pacific abalone Haliotis discus hannai,the dominant aquaculture abalone species in China,is a commercially important shellfish providing "blue food" that meets the modern pursuit of "high nutrition,high-quality protein,and low fat".Fujian Province is the main abalone production area in China and yields 155,000 tons in 2020,accounting for 76.18%of the total national output.However,the Pacific abalone,which is naturally distributed in temperate waters,is suffering summer mortality in Fujian Province,causing huge economic losses.With the global warming and frequent heatwave events,the abalone summer mortality in the south of China has been intensifying,seriously restricted the further development of the abalone aquaculture industry.Thus,the subjects of this study are:to develop indicators of thermal tolerance of abalone;to understand the response mechanism of the northern and southern populations of the Pacific abalone to acute and long-term thermal stress and deeply understand the "summer mortality";to investigate the genetic basis of thermal tolerance in abalone,guiding the breeding of new abalone species(strains)with thermal tolerance.1.The development of a high-throughput thermal tolerance assessment method based on the adhesion capacity of abaloneA high-throughput evaluation method(heat adhesion duration,HAD)was developed based on the adhesion capacity of abalone.The optimum temperature for assessing the thermal tolerance of Pacific abalone through HAD is 32℃.The HAD values significantly increased with acclimation temperature(20℃,24℃;P<0.05)but were not affected by abalone size,age,or sex.The evaluation method is high-throughput and can be used to assess the thermal tolerance of 500 individuals in a single test with high survival of tested individuals.The stability and accuracy of the new method were verified by comparisons of survival time,Arrhenius breakpoint temperatures(ABT)of cardiac performance,and Kaplan-Meier survival curves.The assessments of HAD,ABT,and Kaplan-Meier survival curves indicated stronger thermal tolerance of the southern population(JJ)of Pacific abalone compared to the northern population(DL),suggesting the plasticity of abalone in thermal tolerance.In addition,the thermal resistance of five abalone species from two hybridization systems,H.discus hannai(DD),H.gigantea(GG),H.fulgens(FF),hybrid H.gigantea ♀× H.discus hannai ♂(GD),and hybrid H.discus hannai♀× H.fulgens ♂(DF),was evaluated by HAD and ABT,confirming the existence of heterosis for thermal tolerance.In all,HAD is an accurate,reliable,and high-throughput assessment method suitable for genetic selection for thermal tolerance in abalone.2.Annual domestication of southern and northern populations of the Pacific abalone on a farm in Fujian ProvinceThe year domestication experiment indicated that DL abalone grew faster in low-temperature seasons and JJ abalone grew faster in high-temperature seasons.The "summer mortality" occurred from late May to November,with mortality peak from the August to October.The cumulative mortality of DL abalone(85.21%)was significantly higher than that of JJ abalone(52.99%).With the extension of aquaculture duration in the south of China,the ABT values of DL abalone were increased but lower than that of JJ abalone.The ABT values of JJ abalone were stable between 30.11℃ and 30.63℃,indicating that the upper limit of thermal tolerance might be the main factor for the higher summer mortality.Transcriptomic analysis indicated that DL abalone adopted a relatively direct and "intense" strategy in response to thermal stress,in which more genes were triggered with higher expression levels and more modulation pathways were initiated;whilst the JJ abalone was more like an "energy saver" with a much milder response to temperature changes,leaving more energy to survive.The study suggested that efficient energy conservation may be the key for the abalone to cope with the persistent thermal stress in summer.3.Metabolomic responses of southern and northern populations of the Pacific abalone to long-term temperature acclimationThere was divergence in the growth and survival rates of DL and JJ abalone under six-month thermostatic acclimation(10～30℃).The appropriate growing temperature ranges of DL and JJ abalone were 13～20℃ and 18～24℃,with the optimum growing temperature of 16℃ and 20℃,respectively;the overall survival rates of abalone decreased with temperature increasing but the survival rates of JJ abalone were significantly higher than that of DL abalone at 28℃ and 30℃.The results of metabolomic analysis and enzyme measurement indicated that most metabolic pathways in DL abalone were down-regulated under thermal stress,whilst JJ abalone adopted a more flexible and efficient strategy for energy conservation in response to persistent thermal stress.Energy regulation and metabolic inhibition may be strategies for coping with prolonged thermal stress in Pacific abalone.4.Studies on transgenerational inheritance of heat tolerance in the Pacific abaloneThe assessment methods of thermal tolerance for abalone larvae including larval survival rate(LSR)under persistent thermal stress and larval survival level(LSL)under acute thermal stress,were developed,improving the thermal tolerance evaluation system of larvae-juvenile-adult abalone.The thermal tolerance assessments of 27 nest-designed abalone families were conducted.The heritability of LSR was 0.33,with 19.72%,31.01%,and 10.11%of the explainable variation for paternal,maternal,and parental interactions,suggesting that the maternal parent influences the thermal tolerance of the juvenile abalone more.According to the correlations between the thermal tolerance and gene expression of 27 families,a total of 432 heat tolerance associated genes(HTAGs)were identified.KEGG enrichment pathway analysis showed a network relationship between these genes significantly enriched pathways and apoptosis.While combining the transcriptome results of adult abalone and larvae under acute thermal stress,a total of 351 heat stress associated genes(HSAGs)were identified.Adult abalone responded more rapidly and strongly to acute thermal stress compared to larvae and JJ abalone compared to DL abalone.Similar key pathways and common HSAGs suggest that the responses to acute thermal stress can be inherited from parents to offspring.Combining the results of HTAGs,HSAGs,and long-term temperature acclimation transcriptome,protein processing in endoplasmic reticulum(ko04141)and MAPK signaling pathway(ko04010)were identified as potential key pathways in response to thermal stress in Pacific abalone.5.Genome-wide association study(GWAS)and genomic selection(GS)for thermal tolerance in the Pacific abaloneA total of 630 individuals randomly selected from 210 mixed families were measured for thermal tolerance using a modified ABT method and genotyped using whole-genome sequencing.Aquaculture model and shell color significantly affected ABT values,while there was no significant difference between that of female and male abalone.A total of 23 SNP loci were found significantly associated with thermal tolerance,10 of which were distributed in clusters on chromosome 13,10 of which were located within eight genes,including apoptosis-related genes tm117 and anxa6,proteostasis-related genes nktr and ubxn4,and immune-related genes ddx58,qvr,vwa7 and lin41.The heritability of the ABT value estimated by the six methods ranged from 0.12 to 0.43,with ssGBLUP estimating heritability of 0.20±0.09.The effects of statistic model,marker density,individual number,and mark source on the predictive ability of GS were assessed-GBLUP and BayesB showed the best performance for the predictive ability of GS;models with～5,000 markers provided predictive ability equivalent to that by using all available markers;the predictive ability of～300 individuals was similar to that of 630 individuals;the markers obtained by GWAS could effectively enhance the predictive ability. |
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