Effects Of Hypoxic Preconditioning And Vibrio Harveyi Challenge On Hypoxia Tolerance Of Scapharca Broughtonii

The dissolved oxygen（DO）content in water is one of the key factors for the survival of aquatic organisms,affecting their growth,metabolism,reproduction and other life processes.Hypoxia often poses a threat to the normal growth and survival of aquaculture organisms,and the stress factors in complex are often superimposed in changeable environments.Especially,the increased water temperature in summer will decrease the dissolved oxygen,the increase of the number of pathogens,and the change in p H.The synergy of multiple factors will accelerate the stress on aquatic organisms.Many aquatic organisms develop tolerance and adaptation to hypoxia and other stress during the evolution process,but this organism tolerance and adaptation will be affected by many other factors.Scapharca broughtonii is a large-scale economic clam,which has various modes in northern China,such as shallow sea raft culture,bottom sowing culture and pond culture.Due to its poor mobility,it is often subjected to hypoxia.There have been many reports on the effects of hypoxia stress on the survival,physiology,biochemistry and molecular level of S.broughtonii,but there are few studies on the effects of other factors on its hypoxia tolerance.In this study,S.broughtonii was used as the research object,and the effects of two treatments,namely hypoxia preconditioning and pathogen stimulation,on hypoxia tolerance of S.broughtonii were explored.The main contents include the following three aspects:（1）Hypoxia preconditioning was performed on S.broughtonii,and the changes of physiological and biochemical indexes under hypoxia stress were analyzed.The effects of hypoxia preconditioning on hypoxia tolerance of S.broughtonii were discussed.（2）Through comparative genomic analysis,the gene families with contraction and expansion and the positive selection genes were screened to explore their potential functions in hypoxia tolerance and adaptation.（3）The comparative transcriptome was used to analyze the response difference between the stimulation group and the non-stimulation group of Vibrio harveyi to hypoxia stress.Differential genes were screened and enriched,and the effect of V.harveyi stimulation on the hypoxia response of S.broughtonii was discussed.The main contents were as follows:1.Taking S.broughtonii without hypoxic preconditioning as the control group（group C）,the changes of feeding,respiratory metabolism and enzyme activity of S.broughtonii with two hypoxic preconditioning（group H₂）and four hypoxic preconditioning（group H₄）were analyzed within 48 h under hypoxic stress with a dissolved oxygen concentration of about 2.0 mg/L.The results showed that the feeding rate（IR）of S.broughtonii in the three groups decreased sharply in the early stage of stress,and then gradually recovered with the extension of time.The recovery degree of group H was significantly higher than that of group C at 48 h（P<0.05）.The oxygen consumption rate（OR）of group C,H₂ and H₄ increased gradually with time,and increased by 1.15,1.08 and 0.73 times at 48 h than at 0 h,respectively.The ammonia excretion rate（NR）of the three groups showed different trends,which was1.67,1.30 and 0.97 times that of 0 h at 48 h,respectively.The oxygen-nitrogen ratio（O/N）in group C was relatively stable,and the variation range in group H was relatively large.The cytochrome C oxidase（COX）in the three groups showed a decreasing trend with the prolongation of hypoxia stress time.The lactate dehydrogenase（LDH）activity and reduced glutathione enzyme（GSH）content showed an overall upward trend.Compared with the control group,the enzyme activity in the pre-hypoxia group changed relatively smoothly during hypoxia stress,and the stress response was small.2.Gene family clustering,phylogenetic tree,expansion and contraction of gene family,positive selection and other comparative genomic analysis were carried out using the genome information of S.broughtonii and other 13 species downloaded by our group.The phylogenetic tree showed that S.broughtonii first clustered with S.broughtonii,and the divergence time was～68.5 Mya.A total of 727 gene families expanded and 563 gene families contracted,of which 48 were significantly expanded（P<0.05）.Taking the target species（S.broughtonii,Tegillarca granosa）as the foreground branch and other species as the background branch,81 positive selection genes were obtained（P<0.05）.The enrichment results of KEGG and GO of 48 distinct expansion gene families showed that these genes were mostly enriched in the metabolic pathways of sugar,lipid and amino acid.The specific metabolic pathways included the mutual transformation of pentose and glucuronic acid,arachidonic acid metabolism,linoleic acid metabolism,citric acid cycle（TCA cycle）,pyruvate metabolism,ascorbic acid and aldose metabolism,biosynthesis of unsaturated fatty acids,ketone body synthesis and degradation,galactose metabolism,glycolysis/gluconeogenesis and lysine degradation.A large number of studies have shown that when animals are subjected to hypoxia stress,the energy metabolism pathway plays an important role in the body’s response to hypoxia stress.These significantly expanded gene families may be related to the hypoxia tolerance response of S.broughtonii.The analysis of 81positive selection genes showed that 9 genes were related to hypoxia tolerance in organisms,including nuclear receptor coactivator 4（Ncoa4）,MYC binding protein（Mycbp）,enolase（Eno4）,MED protein gene（Med1）,ribonuclease gene（Rnc）,and so on.3.S.broughtonii without any treatment was used as the blank control group（NH group）,50μL 1 M PBS was injected as the treatment control group（SH group）,and 50μL 2×10⁸ CFU/m L V.harveyi was injected as the treatment group（VH group）.Three groups of S.broughtonii were cultured under the same conditions for 24 h,and the hypoxia stress was unified with DO of 2.5 mg/L.The gill tissues at 0 h,8 h,16 h,24h and 48 h were used for transcriptome sequencing analysis.The results showed that a total of 319.80 Gb Clean Data was obtained,and the average value of each sample was5.97 Gb.A total of 13,683 genes were obtained by annotation;the NH group was analyzed alone,and compared with the 0 h group,it was found that the number of differential genes increased with the prolongation of hypoxia stress time,indicating that hypoxia stress had an impact on the body of S.broughtonii,and the maximum number of differential genes reached 2,392 at 48 h.The differentially expressed genes were mainly concentrated in the phagosome（ko04145）,glycolysis/gluconeogenesis signal（ko00010）,phosphatidylinositol signal transduction system（ko04070）,cytochrome CP450 metabolism（ko00980）,glutathione metabolism（ko00480）,ketone synthesis and degradation（ko00072）and other pathways;compared with the SH group and the NH group at the same time point,the number of differential genes was less than that of VH vs NH;At the same time point,compared with NH group,VH group had the highest number of differentially expressed genes at 0 h and 8 h,which were 1,433 and1,566,respectively.KEGG analysis showed that these differentially expressed genes were enriched in PI3K-AKT signaling（ko04151）,carbon metabolism（ko01200）,purine metabolism（ko00230）,fatty acid biosynthesis（ko00061）and MAPK（ko04010）pathways.The results showed that the injection of V.harveyi changed the response of S.broughtonii to hypoxia at the transcriptional level,and the differential genes were mainly enriched in the metabolic-related pathways.