| The Hilsa shad Tenualosa ilisha,belonging to the Clupeiformes,Clupeidae,Tenualosa,mainly distributed in the Persian Gulf in the western Pacific region of India.Due to its closer kinship with T.reevesii,and considering market demand and ecological impact on native aquatic life,T.ilisha seem to be more suitable for breeding and farming in China than A.sapidissima.Therefore,based on mitochondrial molecular markers,in this study,we analyzed the genetic diversity and genetic differentiation of different T.ilisha populations in order to evaluate the theoretical feasibility of introducing T.ilisha.The American shad Alosa sapidissima.belonging to the Clupeiformes,Clupeidae,Alosa.At present,As a substitution of Tenualosa reevesii for table,it has become one of the most popular and valuable species in the Chinese aquatic product market.The breeding process of A.sapidissima includes overwintering,while the A.sapidissima is a kind of warm water fish with suitable temperature of 20-26℃.Due to the severe drop in water temperature in late December,too low temperatures will cause large-scale descaling deaths.Therefore,the breeding areas of A.sapidissima in China are mainly concentrated in some southern provinces and cities,and no report has been reported in northern areas.Therefore,the same problem may occur after the introduction of T.ilisha.Due to limited conditions,live T.ilisha could not be obtained,so this study used A.sapidissima with similar kinship and biological characteristics as an alternative species to explore the temperature tolerance and the antioxidant status and the expression level of stress-related genes under low temperature.It provides a reference for the breeding situation after the introduction of T.ilisha.1.Assessment of and genetic structure between two Tenualosa ilisha populations based on mitochondrial control region sequencesIn this study,the genetic diversity and population structure of two T.ilisha populations were analyzed by the sequence of mitochondrial D-loop region,and then the phylogenetic analysis of the two T.ilisha populations was performed using the A.sapidissima as an outlier.The results showed that 18 haplotypes appeared in two populations,4 of which were shared haplotypes.The Burmese population is high Hd and πtype.and the Bangladeshi population is high Hd but low π type.The fixation index Fst of the two populations is 0.627,and the gene flow Nm value is 0.297.The main source of variation came from among populations by AMOVA analysis,and the phylogenetic tree and haplotype network show that the two populations are closer Kinship.The above results show that there is a clear genetic differentiation between the two T.ilisha populations.The Burmese population has a higher genetic diversity and a more stable genetic structure.2.Effects of temperature tolerance and low temperature stress on antioxidation status of Alosa sapidissimaIn this study,the natural water temperature was used as the starting temperature(19.0±0.5℃),and the temperature was increased or decreased by 2℃ every 3 hours until all individuals died.The results show that in the low temperature test,the"starting death temperature" is 7℃,and the "peak death temperature" and "total death temperature" are between 4-5℃;in the high temperature test,they are 27℃,31℃and 33℃.When the temperature reaches about 2℃ before its starting death temperature,the A.sapidissima will experience slow swimming,reduced stress,and deepening of its body color,and the fish will appear to swim,roll over,and dephosphorize before death.In general,the temperature tolerance range of A.sapidissima introduced to China has changed adaptively.we also analyzed the hepatic antioxidative status and gill ionase activity of A.sapidissima under low temperature stress.The results showed that low temperature reduced gill Na+/K+-ATP activity,hepatic SOD activity,and total antioxidant capacity(T-AOC).Under low temperature stress hepatic GSH-PX activity increased first(at 0 h of treatment)and then decreased(24 h and 48h).There was no significant change in CAT activity and MDA content.In summary,low temperature stress reduced the antioxidant capacity of A.sapidissima.which led to the loss of the original homeostasis of the internal environment.The antioxidant enzymes SOD and GSH-PX are the key members of A.sapidissima to oxidative damage at low temperature,and Na+/K+-ATP also plays an important role.3.Molecule cloning and sequence analysis of hsp90 and ybx1 genes in Alosa sapidissimaIn this study,full-length cDNA sequences of hsp90 and ybx1 were obtained by rapid-amplification of cDNA ends(RACE)in A.sapidissima.The complete cDNA sequence of hsp90 contained a 92 bp 5’-untranslated region(5’-UTR),a 563 bp 3’-untranslated region(3’-UTR)and a 2181 bp open reading frame(ORF)encoded 726 amino acids.The complete cDNA sequence of ybx1 contained a 70 bp 5’-untranslated region(5’-UTR),a 416 bp 3’-untranslated region(3’-UTR)and a 939 bp open reading frame(ORF)encoded 312 amino acids.The HSP90 amino acid sequence has five conserved regions,with a HATPase(histidine kinase-like ATPase)domain at positions 34-188.The YBX1 amino acid sequence has a cold shock CSD(Cold shock domain)domain,and contains RNP-1 and RNP-2 two RNA binding motifs.Homologous and phylogenetic analysis showed that the amino acid sequence of HSP90 was close to Clupea harengus(92.77%),while the amino acid sequence of YBX1 was closer to that of Characin and Cyprinidae.4.Effects of low temperature on stress-realted genes expression in Alosa sapidissimaRT-qPCR was used to detect the changes of hsp70,hsp90 and ybx1 genes expression in the muscle tissue of A.sapidissima during low temperature stress.The results showed that the muscular hsp70 mRNA expression increased after low temperature treatment(12h and 48h)as well as hsp90 mRNA increased first(0h)and then recovered to the initial level.In A.sapidissima,ybx1 mRNA expression decreased at 48h.The above results indicate that low temperature induces a large amount of HSP90 protein synthesis to exert cell defense function in the early stage of stress,while HSP70 protein runs through the entire stress process. |
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