Effects Of High Temperature Stress On Mitochondrial Function Of Gonad In Strongylocentrotus Intermedius

The sea urchin Strongylocentrotus intermedius has the characteristics of short spines,good gonad quality and rapid growth.It is considered as one of the edible sea urchin species with superior gonad quality.At present,this species is the main farmed sea urchin species in coastal of China.The optimum growth temperature of S.intermedius is about 20 ℃.When the water temperature exceeds 23 ℃,the survival,feeding,growth and development of S.intermedius will be seriously affected,even cause large-scale death.Recently,due to the influence of global seawater warming,the water temperature of S.intermedius aquaculture area in summer in the north is usually overtop 25 ℃,which consequently leads to frequent outbreaks of large-scale death of cultured S.intermedius and a relatively large economic loss of sea urchin industry.Mitochondria are a main place for intracellular metabolism and energy production,which play an important role in cellular physiological processes such as apoptosis and autophagy.Mitochondrial dysfunction refers to the structural or functional dysfunction of mitochondria.Mitochondrial structural dysfunction includes destroy of mitochondrial membrane system,mitochondrial volume change and mitochondrial DNA damage.Mitochondrial functional dysfunction includes mitochondrial inner membrane oxidative phosphorylation alteration,decoupling,dysproduction of reactive oxygen species,abnormal calcium ion regulation,permeability transition pore opening and apoptosis initiation.To investigate the effects of high temperature on mitochondrial swelling,total citrate synthase activity and citrate synthase gene expression in the gonads of sea urchin mesocephus.In this study,we compare and analyze the changes of mitochondrial swelling and total citrate synthase(SiCS)activity in gonads of S.intermedius under the acute heat stress and gradual heat stress conditions.Full length c DNA of the SiCS gene of S.intermedius was cloned and its sequence characteristics were analyzed.From the transcriptional level,the expression law of this gene in adult Sea urchin intermediates is tested and its response to acute heat stress and gradual heat stress conditions is analyzed.The data obtained in this study further enriched the basic data related to CS genes in echinoderms,and provided certain reference materials for in-depth analysis of the molecular expression and regulatory mechanism of sea urchins in response to high temperature.The main results obtained are as follows:1.Effects of heat stress on mitochondrial swelling in gonads of S.intermedius.Results showed that the degree of mitochondrial swelling in gonads of S.intermedius decreased first,then increased and then decreased after acute heat stress at 23 ℃ and 26 ℃ compared with that of the control group.Two peaks of mitochondrial swelling degree in gonads of S.intermedius were observed at 1.5 h and 3 h after 23 ℃ actue heat stress.Only one peak of mitochondrial swelling degree in gonads of S.intermedius were observed at 3 h after 26 ℃ actue heat stress.Results of gradual heat stress experiments showed that compared with the control group(20 ℃),the degree of mitochondrial swelling in gonads of S.intermedius increased first and then decreased.One peak of mitochondrial swelling degree in gonads of S.intermedius were observed at 1.5 h after 23 ℃ gradual heat stress which is similar to the observation obtained at 23 ℃ acute heat stress,wherase,significant deacreased mitochondrial swelling degree was observed across all examined time pont in gonads of S.intermedius under 26 ℃gradual heat stress conditions.2.Effects of heat stress on total enzyme activity of citrate synthase(SiCS)in gonads of S.intermedius.Results showed that after acute heat stress at 23 ℃,total enzyme activities of SiCS in gonads of S.intermedius decreased significantly at all examined time points after 23 ℃ acute heat stress compared with that of control.However,two peaks of total enzyme activities of SiCS were observed at 3 h and 48 h in gonads of S.intermedius after 26 ℃ acute heat stress.As for gradual heat stress,two peaks of total enzyme activities of SiCS were observed at 24 h and 96 h in gonads of S.intermedius after both 23 ℃ and 26 ℃ gradual heat stress compared with that of control.3.Sequence characteristics and expression pattern of SiCS.Results showed that the full length of SiCS cDNA was 2971 bp,containing a 5’ end non-coding region(5’-UTR)of 97 bp,a 3’-UTR of 1480 bp and an open reading frame of 1395 bp which encoded 464 amino acids.The predicted molecular weight and the theoretical isoelectric point of SiCS protein are 51.48 kDa and 6.09,respectively.SiCS was a nontransmembrane and mild hydrophilic protein without signal peptide.Percent of typical conformation unit in the secondary structure of SiCS protein are as 43.86 %(α-helix),7.02 %(β-sheeted)and 49.12 %(random coil).Three-dimensional structure of SiCS was similar to that from Sus scrofa.Phylogenetic analysis indicated that the SiCS protein had the most similarity with CS protein from Strongylocentrotus purpuratus(similarity: 92.90 %).Relative gene expression data showed that SiCS was expressed in all examined tissues and the expression level from high to low was as coelomcytes >interdental muscle > gonad > tube foot > intestine.Total enzyme activity assay revealed that the level of total SiCS enzyme activity in different tissues from high to low was as tube foot > intestine > interdental muscle > gonad > coelomcytes.Acute and gradual heat stress experimental data indicated fluctuation alterations of relative expression of SiCS gene and the total SiCS enzyme activities in the gonads of S.intermedius.