Effect Of Low Temperature Stress On Respiratory Performance And Antioxidant Capacity Of Litopenaeus Vannamei

Litopenaeus vannamei has been widely cultivated due to its fast growth,delicious meat,high meat production rate and wide range of salinity tolerance.Therefore,as an important economic species,L.vannamei has become an important pillar industry of aquaculture in China.However,any changes of the water temperature may have a serious effect on the growth and survival of L.vannamei during breeding,and resulting in a great economic lose to further affect sustainable development of L.vannamei.In this study,we cloned and analyzed the expression of L.vannamei Ngb and Cygb under different temperature conditions.Respiratory relative physiological indexes and activities of antioxidant enzymes indexes were also detected.Furthermore,the structure changes of gill tissue of L.vannamei were observed after low temperature treatment.The main results are as follow: 1)The changes of gill structure after low temperature treatmentThe gill tissues were observed based on HE staining after low temperature treatment,it showed that the gill filaments at 25 °C were neatly and clearly arranged.And blood cells in the haemocoele were also observed.At 20 °C,the gill filaments were swollen and arranged irregularly,and most of the blood cells were swollen.Additionally,as the water temperature dropped from 20 °C to 10 °C,the structural damage of gill tissue aggravated,and the stratum corneum and epithelial cells became necrotic and exfoliated,and the blood cells ruptured.2)Effect of low temperature on L.vannamei hemocyanin content,respiratory metabolism and antioxidant enzyme activitiesThe result of hemocyanin concentration revealed that hemocyanin was gradually reduced as the temperature decreased.The activity analysis of SDH and LDH showed that the SDH activities gradually decreased with the decrease of the temperature,and reached the lowest at 10 °C in the gill,muscle and hepatopancreas.The LDH activities in gill,muscle and hepatopancreas gradually increased with the decrease of temperature.The activity analysis of antioxidant enzyme showed that the T-AOC content in gill and muscle increased firstly and then decreased with the decrease of the temperature,and reached the maximum at 15 °C.While the T-AOC content in hepatopancreas decreased with the decrease of temperature.The CAT activities gradually increased at low temperature and showed the highest level at 10 °C.The changes of MDA in gill and muscle were similar,which increased first and then decreased with the decrease of temperature,and reached the maximum at 20 °C and 15 °C respectively.However,the content of MDA in hepatopancreas decreased firstly and then increased.3)Molecular cloning and sequence analysis of L.vannamei Ngb and CygbIn this study,the cDNA sequences of Cygb and Ngb of L.vannamei were obtained based on bioinformatics technology and multiplexed PCR methods.The full length cDNA sequence of Cygb consisted of 1059 bp,including a 173 bp 5'-UTR,a 313 bp 3'-UTR and a 573 bp open reading frame(ORF).The predicted protein was 190 amino acid residues.The partial cDNA sequence of Ngb included a 528 bp coding sequence and a 567 bp 3'-UTR.Multiple sequence alignment of Ngb and Cygb amino acid sequences of L.vannamei with other species revealed the presence of shared globin superfamily domain.The phylogenetic tree with the amino acid sequences of Mb,Cygb and Ngb in different species was constructed.The result showed that,compared to the Ngb,Cygb and Mb had a closer evolutionary relationship.4)Effect of different temperature on L.vannamei Ngb and Cygb expressionThe real-time PCR results showed that the Cygb and Ngb genes were constitutively expressed in the detected tissues of L.vannamei at 25 °C.The Ngb mRNA levels gradually increased and reached the highest at 15 °C and then decreased in the muscle,brain,stomach and heart.The Ngb gene also showed the similar expression trend in the hepatopancreas and gill,which the expression increased first and then decreased,but increased again at 10 °C.The Cygb expression was similar to Ngb in the muscle,brain,stomach and heart.In the hepatopancreas,there were no significant changes with the decrease of temperature,while in the gill,the expression gradually increased.