Various molecular pathways were obtained by Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. A total of 9,621 unigenes were mapped onto 317 pathways, and the most enriched sequences were metabolic pathways, which was followed by the biosynthesis of secondary metabolites, spliceosome and RNA transport. The top 20 pathways with the greatest number of annotated sequences are shown in . A total of 47 significantly changed GO terms were obtained, and the most significant change was in molecular functions, which was followed by catalytic activity, histone H4 acetylation, structural constituents of cuticle and chitin binding. All of the significantly changed (P . A KEGG pathway enrichment analysis was performed for the gene expression between salinity treatments to identify the number of significantly changed samples along the pathway that were relevant to the background number. The most significantly changed KEGG pathways were the glycosphingolipid biosynthesis, lysine degradation, glycosaminoglycan biosynthesis and malaria pathways. The gene recorded as B3GNT1,2 was significantly up-regulated both in the glycosphingolipid biosynthesis pathway and glycosaminoglycan biosynthesis pathway when shrimp were exposed to salinity at 3 psu (Figs and ). In addition, fatty-acid biosynthesis () was significantly enhanced, especially in short-carbon-chain fatty acids (C8-C18). In addition, low-salinity conditions enhanced polyunsaturated fatty-acid (PUFA) biosynthesis, especially that of highly unsaturated fatty acids such as ARA, EPA and DHA (Figs and ). All of the significantly changed (P . These annotations provide valuable information for studying the specific biological and metabolic processes and functions and molecular mechanisms under salinity stress in L. vannamei.
Nile tilapia Oreochromis niloticus is a freshwater fish but can tolerate a wide range of salinities. The mechanism of salinity adaptation at the molecular level was studied using RNA-Seq to explore the molecular pathways in fish exposed to 0, 8, or 16 (practical salinity unit, psu). Based on the change of gene expressions, the differential genes unions from freshwater to saline water were classified into three categories. In the constant change category (1), steroid biosynthesis, steroid hormone biosynthesis, fat digestion and absorption, complement and coagulation cascades were significantly affected by salinity indicating the pivotal roles of sterol-related pathways in response to salinity stress. In the change-then-stable category (2), ribosomes, oxidative phosphorylation, signaling pathways for peroxisome proliferator activated receptors, and fat digestion and absorption changed significantly with increasing salinity, showing sensitivity to salinity variation in the environment and a responding threshold to salinity change. In the stable-then-change category (3), protein export, protein processing in endoplasmic reticulum, tight junction, thyroid hormone synthesis, antigen processing and presentation, glycolysis/gluconeogenesis and glycosaminoglycan biosynthesis—keratan sulfate were the significantly changed pathways, suggesting that these pathways were less sensitive to salinity variation. This study reveals fundamental mechanism of the molecular response to salinity adaptation in O. niloticus, and provides a general guidance to understand saline acclimation in O. niloticus.
Because of the complexity of the physiological response to low-salinity stress in L. vannamei, several pathways (in addition to lipid metabolism) show potential importance in the shrimp's ability to cope with salinity stress. However, clear evidence of the direct involvement of these pathways during salinity challenges has not been observed; thus, the putative functions of these pathways including lysine degradation, cholinergic synapse, drug metabolism pathway, steroid hormones metabolism pathway, phosphonate and phosphinate metabolism, are only briefly discussed.
Steroid metabolism-related pathways such as steroid biosynthesis (), ovarian steroidogenesis (), sulfur metabolism and steroid hormone biosynthesis () played pivotal roles in response to salinity stress and are inextricably linked to other pathways in aquatic animals [, ]. The steroid regulatory metabolism under salinity stress in O. niloticus is discussed below.
According to the survival and growth parameters of O. niloticus ( and Tables), it is feasible to rear O. niloticus at saline water. From the molecular perspective as shown in , during salinity acclimation, O. niloticus produced significant changes in amino acid metabolism and synthesis, oxidation, protein synthesis and degradation, energy material utilization, and signal transduction. Glycolysis and fatty acids are involved in the regulation of acetyl coenzyme A synthesis and metabolism to participate in the TCA cycle and produce ATP for energy supply. Acetyl coenzyme A also participates in cholesterol synthesis by adjusting the needs of ovarian steroids and steroid hormone synthesis. Ovarian steroidogenesis activates the cAMP signal pathway to regulate adenylate cyclase, downstream gene expression and arachidonic acid metabolites. Among these actions, adenylate cyclase catalyzes ATP into cAMP to support signal transmission and the downstream genes of cAMP signal pathway cover various physiological processes. Arachidonic acid metabolites play extensive roles in maintaining homeostasis. Steroid hormone biosynthesis produces cholesterol-containing DHEA and cholesterol sulfate, which in turn participate in the PPAR pathway, immune-related pathways, cell connections and the PI3K signal pathway. The synthesis and metabolism of some amino acids reflect the reaction of tilapia to maintain osmotic stability. Protein synthesis and the metabolism in organisms are a prerequisite before responding to an environmental salinity challenge. In short, the steroid hormones, osmoregulation, lipid metabolism and cell-connected components are critical measures for salinity domestication in aquatic animals.
A total of 101 differentially expressed genes composed the stable-then-change category, with 20 down-regulated genes and 81 up-regulated genes. The chief sub-networks which contained in this category were the signaling pathway of PI3K and p53, steroid hormone synthesis and oxidative stress, fat synthesis and glycerophospholipid metabolism, cytoskeleton, endoplasmic reticulum activity, sugar utilization and pyruvate metabolism ().
A total of 162 differentially expressed genes were involved in the constant change category, and they were used to build a gene-act network profile. A total of 72 genes were down-regulated, while 90 genes were up-regulated. The main subnetworks included fat digestion and absorption, glycolysis/gluconeogenesis, steroid biosynthesis, complement and coagulation cascades, endoplasmic reticulum activity, cell connection and signal transport ().
The Kyoto encyclopedia of genes and genomes database was used to obtain significantly changed pathways containing differentially expressed genes. We divided the 6 tendencies into 3 categories (Tables –): constant change (containing tendencies 0 and 7), change-then-stable (containing tendencies 1 and 6) and stable-then-change (containing tendencies 3 and 4). Because tendencies 2 and 5 had no difference between the freshwater control and 16 psu, these pathways were not further analyzed. In the constant-change category, the complement and coagulation cascades contained four genes; the steroid hormone biosynthesis, steroid biosynthesis, and ovarian steroidogenesis contained 12 genes; and fat digestion and absorption, vitamin digestion and absorption and retinol metabolism contained eight genes. The stable-then-change category contained 31 pathways such as the biosynthesis of unsaturated fatty acids, fatty acid elongation, protein processing in endoplasmic reticulum, glycolysis/gluconeogenesis, pyruvate metabolism and tight junction. The change-then-stable category involved 29 pathways related to lipid metabolism, cell cycle and oxidative phosphorylation.
Payne AH and Hales DB: Overview ofsteroidogenic enzymes in the pathway from cholesterol to activesteroid hormones. Endocr Rev. 25:947–970. 2004. : :
Hanukoglu I: Steroidogenic enzymes:Structure, function, and role in regulation of steroid hormonebiosynthesis. J Steroid Biochem Mol Biol. 43:779–804. 1992. : :
The steroid biosynthesis pathway is up-regulated in the constant-change category. The quadrilateral in red represents the up-regulated genes. The 18.104.22.168 represents the squalene synthase; the 22.214.171.124 represents the squalene monooxygenase; the 126.96.36.199 represents the delta24-sterol reductase and the 188.8.131.520 represents the 3-keto steroid reductase. According to this pathway, the production of cholesterol is up-regulated.
The Pacific white shrimp Litopenaeus vannamei is a euryhaline penaeid species that shows ontogenetic adaptations to salinity, with its larvae inhabiting oceanic environments and postlarvae and juveniles inhabiting estuaries and lagoons. Ontogenetic adaptations to salinity manifest in L. vannamei through strong hyper-osmoregulatory and hypo-osmoregulatory patterns and an ability to tolerate extremely low salinity levels. To understand this adaptive mechanism to salinity stress, RNA-seq was used to compare the transcriptomic response of L. vannamei to changes in salinity from 30 (control) to 3 practical salinity units (psu) for 8 weeks. In total, 26,034 genes were obtained from the hepatopancreas tissue of L. vannamei using the Illumina HiSeq 2000 system, and 855 genes showed significant changes in expression under salinity stress. Eighteen top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly involved in physiological responses, particularly in lipid metabolism, including fatty-acid biosynthesis, arachidonic acid metabolism and glycosphingolipid and glycosaminoglycan metabolism. Lipids or fatty acids can reduce osmotic stress in L. vannamei by providing additional energy or changing the membrane structure to allow osmoregulation in relevant organs, such as the gills. Steroid hormone biosynthesis and the phosphonate and phosphinate metabolism pathways were also involved in the adaptation of L. vannamei to low salinity, and the differential expression patterns of 20 randomly selected genes were validated by quantitative real-time PCR (qPCR). This study is the first report on the long-term adaptive transcriptomic response of L. vannamei to low salinity, and the results will further our understanding of the mechanisms underlying osmoregulation in euryhaline crustaceans.