The Molecular Mechanism Of Pelteobagrus Vachelli In Response To Hypoxia Stress

A sufficient oxygen concentration is essential for electron transport within the mitochondria of aerobic organisms. Generally, in aquatic environments the amount of dissolved oxygen present fluctuates greatly depending on various natural conditions (e.g.,season, geographic location, composition, and water flow). Aquatic ecosystems are being stressed by nutrient enrichment, pollutants, and global warming more frequently, which seriously depletes oxygen concentration. To maintain homeostasis and organism function in low oxygen environments,aquatic organisms including fishes may respond to hypoxia with varied biochemical, physiological, and behavioral adjustments (e.g., depression of the metabolic rate, shifting of blood flow mainly to the brain and heart, and efficient production of energy). Even though fishes can have an acute reaction to hypoxia to maintain normal physical activity, a sudden serious lack of oxygen will result in mortality.Unearthing the molecular mechanisms of hypoxia adaptation in fishes will not only help us to understand fish speciation and the evolution of the hypoxia- signaling pathway but will also guide us in the breeding of hypoxia-tolerant fish strains.Pelteobagrus vachelli has delicious taste with little bone in muscle and high nutritional value. Moreover,it is omnivorous and has a remarkable ability to adapt to environment. The relatively high yield of P. vachelli coupled with an affordable price for consumers thus make it a very popular commercial species in Asia. However, the species is only distributed in some of Asia's larger rivers, such as the Liaohe, Huaihe, Yangtze,Xiangjiang, Minjiang and Pearl. It is not suitable for high-density pond farming because of the relatively high oxygen-consumption rate and low oxygen threshold; a sudden lack of oxygen will result in mortality among the fish and will eventually lead to pond turnover.These special characteristics of P. vachelli suggest that it is not only a significant aquaculture species but also a potential model organism for study of the molecular mechanisms of acute hypoxia. Indeed, no genomic and transcriptomic resources from this species have previously been available, and until now only about 150 EST and 105 protein sequences have been deposited in the NCBI GenBank. This dearth of genetic resources hinders P. vachelli molecular breeding as well as further studies on the mechanisms of specific biological processes.In the present study,as such offers deeper insight into the molecular mechanisms of hypoxia adaptation in aquatic organisms, this is the first re search on comparative quantitative miRNA-seq, mRNA-seq and proteomics using high throughput next-generation sequencing technology. These results provide new and important information about acute hypoxia responses in fishes,a significant step forward toward a more complete elucidation of the relationship between aquatic organisms and environmental hypoxia. The concrete results are as follows:1. Integrated analysis of mRNA-seq and miRNA-seq in the liver of P. vachelli in response to hypoxiaTo the best of our knowledge, this study is the first exploration to simultaneously characterise the mRNA-seq and miRNA-seq of fish in response to hypoxia. In this part we found that 712 genes were significantly up-regulated, while 249 genes were significantly down-regulated in response to hypoxia using mRNA-seq. We identified 162 miRNA-mRNA pairs with negative correlation as the key for analysis with the involvement of 18 differentially expressed miRNAs and 107 differentially expressed mRNAs in total. A large number of mRNAs and miRNAs from P. vachelli involved in diverse biological pathways were identified. Furthermore, the comparison of several key pathways (e.g., HIF-1 signaling, Glycolysis/Gluconeogenesis, and AMPK signaling pathways) provided informative results which could help us articulate the different mechanisms involved in the hypoxia response of P. vachelli. According to our data of integrated analysis, in order to maintain normal physical activity, fishes can have an acute reaction to acute hypoxia, including regulated proliferation of red blood cells, inhibiting cell apoptosis and stimulating angiogenesis, a shift in aerobic and anaerobic contributions to total metabolism, and a reduction in energy-consuming biosynthetic pathways. We provide a good case study for analyzing mRNA/miRNA expression and profiling a non-model fish species using next-generation sequencing technology.2. Effects of acute hypoxia and reoxygenation on oxygen sensors, respiratory metabolism, and hematology indicesIn this part we used next-generation sequencing technology to characterize the transcriptomes of P. vachelli and identified 70 candidate genes in the HIF-1 signaling pathway that are important for the hypoxic response in all metazoan species. The present study reported the effects of acute hypoxia and reoxygenation on oxygen sensors,respiratory metabolism, and hematology indices in P. vachelli. The predicted physiological adjustments show that P. vachelli's blood oxygen carrying capacity was increased through increased RBC, HB, and SI after hypoxia exposure. Glycolysis-related enzyme activities(PFK,HK,and PK) and LDH in the brain and liver also increased,indicating a rise in anaerobic metabolism. The observed reduction in oxidative enzyme level (CS) in the liver during hypoxia suggests a concomitant depression in aerobic metabolism. There were significant increases in oxygen sensor mRNA expression and HIF-la protein expression during hypoxia and reoxygenation exposure, suggesting that the HIF-1 signaling pathway was activated in the liver and brain of P. vachelli in response to acute hypoxia and reoxygenation. Our findings suggest that oxygen sensors (e.g., HIF-la) of P. vachelli are potentially useful biomarkers of environmental hypoxic exposure. These data contribute to a better understanding of the molecular mechanisms of the hypoxia signaling pathway in fish under hypoxia and reoxygenation.3. iTRAQ-based quantitative proteomic analysis of P. vachelli liver reveals acute hypoxic responsive proteinsIn order to shed further light on the molecular mechanisms of hypoxia adaptation in fishes, we conducted comparative quantitative proteomics on P. vachelli livers using iTRAQ. 511 acute hypoxia-responsive proteins from P. vachelli were identified.Furthermore, comparison of several of the key diverse pathways studied (e.g., Peroxisome,PPAR signaling pathway, Lipid metabolism, Glycolysis/Gluconeogenesis, and Amino acid metabolism) helped us articulate the different mechanisms involved in the hypoxia response of P. vachelli. Data from proteome analysis showed that in order to maintain normal physical activity,fish can have an acute reaction to acute hypoxia, including detoxification of metabolic byproducts and oxidative stress in light of continued metabolic activity (e.g., peroxisome), activation of catabolism to get more energy (e.g., lipolyis andamino acid catabolism), inhibitions of biosynthesis to reduce energy consumption (e.g.,biosynthesis of amino acids and lipids), and a shift in the aerobic and anaerobic contributions to total metabolism.4. Modulated expression and enzymatic activities of Darkbarbel catfish, P.vachelli for oxidative stress induced by acute hypoxia and reoxygenationLarge changes in oxygen availability in aquatic environments, ranging from anoxia through to hyperoxia, can lead to corresponding wide variation in the production of reactive oxygen species (ROS) by fish with aquatic respiration. In order to evaluate the effects of hypoxia and reoxygenation on oxidative stress in fish, the mRNA and protein expression of SODs (Cu/Zn-SOD and Mn-SOD) as well as indices (CP, LPO and MDA)and enzymatic activities (SOD, CAT, GPx, GR and GST) were analyzed in liver and brain tissues of P. vachelli. Predominant expression of PvSOD2 was detected in heart,brain,and liver. In contrast, PvSOD1 was highly expressed in liver. Based on the expression patterns of above parameters, we inferred that brain tissue of P. vachelli under 0.7 mg/L degree of acute hypoxia condition could experience hypometabolic states or no suffering stress, but brain tissue has effective mechanisms to minimize or prevent oxidative stress during the transition from hypoxia to reoxygenation. Our results also demonstrated an increased expression of SODs and enzymatic activities for oxidative stress in liver under hypoxic conditions, which supports the hypothesis that anticipatory preparation takes place in order to deal with the encountered oxidative stress during the recovery from hypoxia as proposed by M. Hermes-Lima. Therefore, this study will provide a clue to better understand the action mode of antioxidant genes and enzymes under oxidative stress in fish.