Hypersalinity Adaptation And Underlying Mechanisms Of The Estuarine Oyster (Crassostrea Ariakensis)

In the context of global climate change,environmental changes,such as salinity changes,have compounded the challenges to the survival and reproduction of marine organisms,particularly those in intertidal and estuarine areas that are more susceptible to external changes.Oysters,as a keystone sessile species living in intertidal and estuarine areas with complex and variable environmental conditions,have evolved various adaptabilities or responses to highly variable environments,making them an ideal model for exploring the environmental adaptation mechanisms of marine organisms under climate change,such as salinity adaptation.The Estuarine Oyster(Crassostrea ariakensis)is a typical large estuarine species with weak gene flow between different geographical populations,which makes it an excellent material for studying salinity adaptive divergence.In the early half of the last century,C.ariakensis was abundant and formed oyster reefs offshore of China.They played an important role in the ecological stability and the development of subsistence fisheries in the estuarine areas,with obvious economic and ecological significance.However,due to climate change,estuary diversion,and overfishing,the resources of C.ariakensis was in sharp decline in recent years.Especially in northern China,the small population size cannot match fishery demand,some of which are even functionally extinct.Therefore,it is of great practical significance for the resource restoration of C.ariakensis.In addition,C.ariakensis has a large commercial application potential.Given the current situation of mass mortality of C.hongkongensis,a dominant mariculture oyster species distributed in the same areas as C.ariakensis populations in southern China,developing complementary new strains of C.ariakensis can be valuable for the mariculture industry,and may even develop a new form of “large oyster” industry.Based on the different positioning of C.ariakensis in the southern and northern oyster industries,this study analyzed the salinity-adaptive divergences and underlying mechanisms among different germplasm,to provide a theoretical foundation of the selective breeding of the hypersalinity-tolerant strains in southern China and the development of hybrid strains in northern China.The main contents of this study are as follows:1.Salinity adaptive divergence and molecular mechanisms between sympatric closely related speciesComparing the responses of closely related species to environmental changes is an efficient method to explore adaptive divergence.As typical estuarine oysters,C.ariakensis and its closely related species,C.hongkongensis,are of sympatric distribution in southern China.The divergence in phenotypes and gene expression of C.hongkongensis and C.ariakensis in response to different salinity environments,and the relative contribution of species effect,environment effect,and their interaction to the divergence were explored.After a 2-month outplanting at high-and low-salinity areas in the same estuary,the high growth rate,percent survival,and high tolerance indicated by physiological responses in hypersalinity environment(21.0)suggested that C.ariakensis was more adaptive to higher salinity condition.While the high growth rate,percent survival,and high physiological tolerance in hyposalinity environment(13.6)indicated that C.hongkongensis was more adaptive to low salinity condition.Moreover,a transcriptome analysis revealed that the percentage and fold changes of differentially expressed genes between two species were significantly higher than those between salinity gradients,indicating that the expression divergence was mainly caused by the species effect.Several of the important pathways enriched in divergent genes between species were also salinity-responsive pathways,including transport,amino acid metabolism and energy metabolism pathways.Specifically,the pyruvate and taurine metabolism pathways and several SLC may contribute to the hypersalinity adaptation of C.ariakensis,and other SLC may contribute to hyposalinity adaptation of C.hongkongensis.Our findings provide insights into the phenotypic and molecular mechanisms underlying salinity adaptation in marine mollusks,which will facilitate the assessment of the adaptive capacity of marine species in the context of climate change,and will also provide practical information for marine resource conservation and aquaculture.2.Evolutionary and functional analyses of SLC superfamily in bivalves with different salinity adaptationConsidering that many different SLCs were found to play a role in the interspecific divergence of salinity response in the above results,this study further investigated the evolutionary patterns of the SLC family in bivalves with different salinity adaptation.In this study,the whole SLC superfamily of eight typical species of bivalves with different adaptation from freshwater to high salinity seawater were identified,including C.ariakensis in the genus Crassostrea.And we found that the variation of gene copy numbers in the following 5 SLC orthogroups(OG)was closely related to the salinity adaptation of these shellfish,from the perspectives of molecular evolution,gene expression response,and functional validation.The copy numbers of monocarboxylate transporter orthogroup I(OGMCTI)and taurine transporter orthogroup(OGTAUT)expand from freshwater species to high salinity species;while those of the anion transporter orthogroup(OGSLC26A5)contract from high salinity species to freshwater species.The monocarboxylate transporter orthogroup II(OGMCTII)is found only in oysters with low salinity adaptation.And the glycine transporter orthogroup(OGSLC6A5)is unique to Crassostrea species,with expanding copy numbers in with low salinity adaptation species.These orthologous proteins may be involved in bivalves adaptation to high or low salinity by transporting different substrates.This study revealed evolutionary and structural diversity within and between these orthogroups,as well as differential transcriptional expression in response to salinity stress.In addition,the pyruvate transport function of OGMCTI was verified in C.ariakensis,and the transport efficiency differed between copies from different evolutionary branches.The study on the SLC family of bivalve,including C.ariakensis,will provide new insights into the diversity of salinity responses among species with different salinity evolutionary adaptation,and lay the basis for future research on salinity-adaptive evolutionary mechanisms and functional regulatory mechanisms.3.Heterosis and underlying mechanisms of hybrids from intraspecific populations with salinity adaptive divergenceHybridization is a feasible way to breed new germplasm with genetic improvement and high environmental adaptability.C.ariakensis is broadly distributed in the estuaries of China and has evolved genetic divergence related to salinity adaptation and phenotypical divergences in salinity tolerance between populations;therefore,it is an ideal species to explore environmental resistance and its molecular mechanisms in intraspecific hybrids.In this study,we performed reciprocal hybridization between northern and southern C.ariakensis populations with salinity adaptive divergence,and determined fitness-related traits after culturing in both northern and southern habitats.The growth and survival of reciprocal hybrids were higher than those in inbred populations in both habitats,indicating heterosis in intraspecific hybrids of C.ariakensis.Moreover,hybrids reared in maternal habitats exhibited higher growth,field survival,and aerobic metabolism than alternative hybrid oysters under the same conditions,indicating maternal effect.We identified gene modules correlated with heterosis and maternal effect in reciprocal hybrids by Weighted Gene Co-expression Network Analysis,and these module genes were enriched in pathways of energy metabolism,protein modification,transport,and amino acid metabolism.Among them,energy metabolic pathways such as galactose metabolism,glycolysis/gluconeogenesis,and tricarboxylic acid cycle play a role in both heterosis and maternal effect.The expression level and expression plasticity of 29 candidate genes involved in these energy metabolic pathways were higher in the hybrid population than in the inbred population,suggesting that accelerated aerobic energy metabolism,which may result from evolutionary mitonuclear coadaptation,underlies the heterosis and maternal effect in the hybridization of C.ariakensis.This study reveals that an appropriate environment(maternal habitat)is critical for heterosis,which can effectively improve environmental resistance and growth rate,and provides important insights for germplasm management of aquatic species.In conclusion,by integrating phenotypic determination,bioinformatics analysis,and functional validation experiments,this study systematically investigated the salinity adaptive divergence and molecular mechanisms under natural salinity environments between C.ariakensis and its sympatric C.hongkongensis;the evolutionary and functional diversity of the salinity-responsive SLC superfamily in bivalves with different salinity adaptation;and the heterosis and underlying mechanisms of intraspecific hybrids from populations with salinity adaptive divergence.The findings would help guide the breeding of germplasm with high environmental tolerance,lay a theoretical foundation for resource conservation and mariculture industry development of C.ariakensis,and contribute to the assessment of adaptation potential under climate change.