Xiaodan Wang

Transcriptome sequencing revealed the genes and pathways involved in salinity stress of Chinese mitten crab, Eriocheir sinensis

A total of 276.9 million reads were obtained and assembled into 206, 371 contigs with an average length of 614 bp and N50 of 1,470 bp. Comparison of digital gene expression between treatment and control group reveals 1,151 and 941 genes were significantly differentially expressed in crab gill and muscle, respectively. In gill and muscle, protein ubiquitination, ubiquinone biosynthesis, oxidative phosphorylation, and mitochondria dysfunction pathways were the top pathways differentially expressed following the challenge. EIF 2 signaling pathway and IGF-1 signaling pathway were the top ones among the signal-related pathways. Most of the amino acid metabolism pathways were found to be involved in this process. The expression patterns of 15 differentially expressed genes were validated by quantitative real-time RT-PCR (average correlation coefficient 0.80). This is the first report of expression analysis of genes and pathways involved in osmoregulation of Eriocheir sinensis through transcriptome sequencing. The findings of this study will further promote the understanding of the underlying molecular mechanism of salinity stress adaptation for crustacean species.

Transcriptome and Molecular Pathway Analysis of the Hepatopancreas in the Pacific White Shrimp Litopenaeus vannamei under Chronic Low-Salinity Stress

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.

Transcriptome Profiling and Molecular Pathway Analysis of Genes in Association with Salinity Adaptation in Nile Tilapia Oreochromis niloticus.

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.

Characterization and Expression of Glutamate Dehydrogenase in Response to Acute Salinity Stress in the Chinese Mitten Crab, Eriocheir sinensis

Background Glutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species. Methodology/Principal Findings GDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5′- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3′- untranslated region. E. sinensis GDH showed 64–90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA. Conclusions E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions.

Growth and Lipid Metabolism of the Pacific White Shrimp Litopenaeus vannamei at Different Salinities

ABSTRACT Juvenile white shrimp Litopenaeus vannamei (1.98 ± 0.28 g) were fed a commercial diet for 8 wk in triplicate to investigate growth and lipid metabolism at 3 salinities (3, 17, and 30). Shrimp weight gain and survival at 3 were significantly less than that at 17 and 30. No differences were found in whole-body proximate composition. Linolenic acid (18:3[n-3]) and (n-3) long-chain unsaturated fatty acid levels in the hepatopancreas, and n-3 long-chain polyunsaturated fatty acid level, especially eicosapentaenoic acid (EPA; C20:5[n-3]) and docosahexaenoic acid (DHA; C22:6[n-3]) in muscle at 3 were significantly greater than at other salinities. Fatty acid synthase, hormone sensitive lipase, lipoprotein lipase, adipose triacylglycerol lipase, acyl-CoA, diacylglycerol acyltransferase 2, elongase of very long-chain fatty acid 6, and &Dgr;5 and &Dgr;6 fatty acid desaturase activity was detected and showed a negative trend with an increase of salinity, and no significant differences were found among salinity groups (P > 0.05). The results indicate that the low salinity of 3 decreases the growth of L. vannamei. Although L. vannamei could not synthesize either DHA or EPA de novo, it possibly has the potential ability to convert linolenic acid to DHA and EPA regardless of salinity. However, the factors influencing this ability remain unknown and need further study.

Growth, Body Composition, and Ammonia Tolerance of Juvenile White Shrimp Litopenaeus vannamei Fed Diets Containing Different Carbohydrate Levels at Low Salinity

ABSTRACT Effects of dietary carbohydrate (CBH) levels on growth, body composition, and ammonia tolerance of juvenile white shrimp Litopenaeus vannamei at a low salinity of three were evaluated. Six isonitrogenous and isolipid diets containing different CBH levels were formulated and fed to juvenile L. vannamei for 42 days in triplicate for each treatment. Weight gain and survival rate of shrimp fed 20% CBH were the greatest and differed from those fed 5% CBH and 30% CBH. Shrimp whole-body crude protein of the 20% CBH group was significantly greater than that of other treatments. Whole-body crude lipid levels increased with the increase of dietary CBH, and was significantly higher in the 20%, 25%, and 30% CBH groups than in the control. Hepatosomatic index, condition factor, whole-body moisture, and ash contents were not affected by dietary CBH levels. Hepatopancreas soluble protein peaked in shrimp fed 20% CBH, and was significantly greater than in other groups. Hepatopancreas and muscle glycogen showed a similar tendency, but peaked in shrimp fed 15% CBH. Malate dehydrogenase, pyruvate kinase, and glucose-6-phosphate dehydrogenase activities were not affected significantly by dietary CBH levels. An ammonia challenge of 96 h showed that the shrimp in the 20% CBH group had the greatest survival rate, although no significant differences were observed among treatments. This study indicates that 15%–20% dietary CBH is optimal for growth and can improve the ability of L. vannamei to handle stress at low salinities.

Low Salinity Decreases the Tolerance to Two Pesticides, Beta-cypermethrin and Acephate, of White-leg Shrimp, Litopenaeus vannamei

Acute toxic effects of two commonly used pesticides, beta-cypermethrin and Acephate, to the white-leg shrimp, Litopenaeus vannamei, were tested at ambient salinity 5.0% and 20.0%, by using a static renewal method. The results showed that the mean LC50 values of beta-cypermethrin at 24, 48, 72 and 96 h were 0.437, 0.317, 0.203, and 0.170 μg/L to the white shrimp at 5.0%, and were 0.767, 0.440, 0.383, and 0.383 μg/L at 20.0%. The mean LC50 values of Acephate at 24, 48, 72, and 96 h were 51.250, 38.007, 27.783, 18.247 mg/L at 5.0%, and were 59.853, 43.490, 34.220, 27.337 mg/L at 20.0%. L. vannamei is more sensitive to ambient beta-cypermethrin and Acephate toxicity at salinity 5.0% than at salinity 20.0%. Beta-cypermethrin is highly toxic to L. vannamei at either salinity, while acephate has low toxicity to L. vannamei.

Comparative proteome analysis of the hepatopancreas from the Pacific white shrimp Litopenaeus vannamei under long-term low salinity stress

Litopenaeus vannamei is a typical euryhaline decapod model to study the osmoregulation mechanism in crustaceans. The proteomic was undertaken using isobaric tags for relative and absolute quantification together with the reverse phase in high-performance liquid chromatography mass spectrometry to quantitatively identify the proteins differentially expressed in the hepatopancreas under low salinity stress (3psu) compared with the control salinity (25psu). 533 proteins and 84 differentially expressed proteins were identified including 58 proteins with the 1.2-fold cut-off value under chronically low salinity stress. Among these proteins, 26 were up-regulated while 32 were down-regulated. 48 out of 58 differentially expressed proteins were annotated in the Uniprot database and were mapped into 38 pathways by KEGG analysis. These proteins were categorized into the pathways for energy metabolism, signaling, immunization and detoxification, lipid and protein metabolism. A more active glycometabolism, positive response detoxification pathway, immunosuppression and positive osmoregulation were identified in L.vannamei under low salinity stress. This study suggests that under chronically low salinity stress, L. vannamei showed low immunity and high demand for energy especially from glycometabolism. Signaling transfer related pathways, especially the Wnt signaling pathways were involved in the process of salinity adaption, but the in-depth mechanism warrants further investigation. SIGNIFICANCE In this study, a comprehensive physiological response was studied using proteomics to reveal the underlying mechanism of adaptation to low salinity in L.vannamei, which was the first report on the proteomic response of crustacean to salinity stress. The extensive proteomic investigation on hepatopancreas under low salinity stress provides a new insight into the adaptive mechanism of this euryhaline crustacean species to low salinity.

Molecular characterization and expression of AMP-activated protein kinase in response to low-salinity stress in the Pacific white shrimp Litopenaeus vannamei.

AMP-activated protein kinase (AMPK) serves as a major regulator of cellular energy metabolism by activating ATP production pathways and blocking ATP consumption. However, information on AMPK genes in aquatic animals is limited. In this study, three subunits of AMPK were cloned from the Pacific white shrimp Litopenaeus vannamei. The full-length cDNAs of the α, β and γ subunits were 1617, 1243 and 3467bp long, respectively, with open reading frames of 1566, 873 and 2988bp encoding for 521, 290 and 996 amino acids, respectively. Amino acid sequence alignments of the three subunits showed that the functional domains in the L. vannamei proteins retained the highest similarity with those of other animals, at 89%, 58%, and 75%, respectively. The expression levels of the three subunits were higher in the muscle and gills than in the eyestalk and hepatopancreas. The mRNA levels of AMPK-α and AMPK-β were up-regulated in the hepatopancreas and muscle after acute low-salinity stress at 3psu for 6h compared with control salinity at 20psu. After 8-week salinity stress at 3psu, AMPK-α and AMPK-β mRNA levels in the hepatopancreas were significantly higher than those of the control at 30psu. However, in the muscle only AMPK-γ mRNA was significantly up-regulated at low salinity relative to controls. Muscle and hepatopancreas showed increases in AMPK protein after 6h exposure to low salinity, but there were no differences seen after long term acclimation. The change patterns of protein were slightly differing from the mRNA patterns due to the distinguishing function of individual subunits of AMPK. These findings confirm that three AMPK subunits are present in L. vannamei and that all encode proteins with conserved functional domains. The three AMPK subunits are all regulated at the transcriptional and protein levels to manage excess energy expenditure during salinity stress.

MOLECULAR PATHWAY AND GENE RESPONSES OF THE PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI TO ACUTE LOW SALINITY STRESS

ABSTRACT To understand the underlying mechanism of the Pacific white shrimp Litopenaeus vannamei responding to acute salinity stress. RNA-seq was used to determine the transcriptome response of shrimp muscle and gill after ambient salinity changed from salinity of 20 (control) to 3 in 24 h. A total of 281.4 million reads were obtained and assembled into 105.153 contigs with an average length of 984 bp. Comparison of gene expression between shrimp exposed to salinity of 3 and the salinity control revealed that 991 and 3.709 genes were differently expressed in the gill and muscle, respectively. Both in muscle and gill, the changes of pathway can be categorized into oxidative pathways, signal transduction pathways, and metabolism pathways. More pathways significantly responded in gill than in muscle in metabolism and signal transduction. The significant change of pathways revealed that under acute low salinity stress, the increase of energy derived from carbohydrate, amino acid, or lipid in gill could satisfy the extra energy requirement of shrimp under salinity stress, but also lead to an overproduction of reactive oxygen species. For the maintenance of homeostasis, protein ubiquitination and relevant pathways were activated to remove the excessive reactive oxygen species and metabolite waste.