Effects Of Dry Exposure On The Respiratory Metabolism-related Enzymes And HIF-1 Of Exopalaemon Carinicauda

The ridgetail white prawn Exopalamon carincauda is a medium-sized shrimp in coastal waters of China which has a high yield, strong disease resistance, high economic value. This prawn has been cultured on a large scale. In the process of production and transportation, E. carincauda inevitably encountered with environmental change, and faced with transition from aquatic environment to terrestrial environment. Therefore, the response of E. carincauda to dry exposure is really interesting. In this paper the effects of dry exposure on the respiratory metabolism-related enzymes and hypoxia-inducible factor gene regulation of E. carincauda was studied to provide directive significance of E. carincauda culture and transportation. The results were shown below:The first part: The purpose of this part was carried out to investigate the effects of dry exposure on respiratory metabolism-related enzymes in different tissues of E. carinicauda. E. carinicauda were divided into two experimental groups of dry exposure, including normal temperature(22℃-24℃) group and low temperature(4℃) group. Meanwhile, shrimp cultured in normal seawater as control group. Activities of respiratory metabolism enzymes and content of lactate were measured under dry exposure challenges at 60 min, 90 min, 120 min and 180 min. The investigated enzymes included succinate dehydrogenase(SDH), cytochrome c oxidase(CCO), fumarate reductase(FRD) and lactate dehydrogenase(LDH). SDH and CCO are the key enzymes of aerobic metabolism in animals. FRD and LDH are the key enzymes of anaerobic metabolism in animals. And the content of lactate is a sign of the degree of anaerobic metabolism. The results showed that the activities of SDH and CCO in hepatopancreas, gills, and muscles in normal temperature group of dry exposure had decreasing trend with time.which were significantly lower than those in the control(P<0.05). Activities of FRD, LDH and content of lactate in the muscles increased at first and then decreased with time. which were significantly higher than those in the control(P<0.05). Activities of respiratory metabolism enzymes and content of lactate in low temperature group had no significant difference than those in the control group at 60 min. After that, it showed the same trend as the normal dry exposure. The enzymes showed tissue dependence. Activities of respiratory metabolic enzymes in hepatopancreas and gills were higher than those in muscles. The results suggested that anaerobic metabolism increased after dry exposure. E. carinicauda still could maintain the aerobic metabolism in the early stage of low temperature dry exposure. The hepatopancreas and gills might be more sensitive tissues to dry exposure. Low temperature could increase the survival rate of dry exposure.The second part: RACE method was used to clone two genes, which were respectively named HIF-1α and HIF-1β. The length of HIF-1α and HIF-1β were 4398 and 2190 bp, respectively. The ORF(open reading frame) were 3186 bp and 1971 bp, which encoded 1062 and 657 amino acids, respectively. The putative protein of HIF-1α contained HLH, PAS and C-TAD conserved domain. The putative protein of HIF-1β contained HLH and PAS conserved domain. As shown by the alignment result of HIF-1α and HIF-1β amino acid sequences from different species, the HIF-1α amino acids sequence of E. carincauda presented the highest consistency with that of Palaemon pugio, the HIF-1β amino acids sequence of E. carincauda presented the highest consistency with that of Macrobrachium nipponense. HIF-1α gene expression levels were up-regulated under dry exposure. However, in low temperature dry exposure, relative expression of HIF-1α in hepatopancreas had no significant change, while relative expression of HIF-1α in gills and muscles had significantly increased. Similarly, the relative expression of HIF-1β in all three tissues were up-regulated under dry exposure, and the relative expression of HIF-1β in gills up regulated later under low temperature dry exposure than that under normal temperature dry exposure. The results indicated that HIF-1α and HIF-1β both played important roles in the adaptive response to hypoxia stress when exposed to dry condition, but the regulatory mechanism maybe different.The third part: This part used RNA interference to study the response of hypoxia inducible factor under dry exposure by injection of long double-stranded RNA to E. carinicauda. The results showed that: gene expression of HIF-1α and HIF-1β were significantly inhibited(P <0.05), expression of HIF-1α or HIF-1β which were suppressed in E. carinicauda with dry exposure, its survival rate is low. HIF-1α and HIF-1β play the role of responsing to dry exposure.