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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.

KEY WORDS: shrimp. RNA-Seq, oxidative stress, signal transduction, energy metabolism, Litopenaeus vannamei

INTRODUCTION

Aquatic crustaceans inhabit a variety of environments with different salinities from freshwater to seawater (Pequeux 1995). As one of the most important environmental factor, salinity can directly cause physiological stress on aquatic animals by changing osmoregulation. Therefore, osmoregulation is an essential physiological process for the majority of aquatic crustaceans because it enables them to cope with the discrepancy of ion concentrations inside the body and the environment (Pequeux 1995). Nevertheless, due to the diverse habitats of crustacean species, the osmoregulation of crustacean is a quite complex process (Pequeux 1995, Augusto et al. 2007, Sokolova et al. 2012), and the underlying mechanism of osmoregulation of crustacean is limited especially at the molecular level.

The Pacific white shrimp Litopenaeus vannamei is a typical euryhaline species and can adapt to both coastal and oceanic environments, as a strong osmoregulator (Cheng et al. 2006). Salinity tolerance research has shown that L. vannamei can tolerate a wide range of salinity from 0.5 to 50 (Pante 1990). Due to the special ecological niche of L. vannamei and its importance in aquaculture, research on the effects of ambient salinity and osmoregulation of L. vannamei has been extensively conducted, but the understanding on this animal is limited to its isoosmotic point (Diaz et al. 2001), growth and survival (Li et al. 2007), immunity (Wang & Chen 2005, Lin et al. 2012,), stress resistance (Li et al. 2007, Li et al. 2008b, Wang et al. 2013a), and genes controlling osmoregulation of L. vannamei (Lago-Leston et al. 2007, Li et al. 2009, Wang et al. 2013b). Nevertheless, the integrative molecular pathways of L. vannamei in response to salinity stress have not been studied.

To understand the complex molecular biological process of stress physiology at the whole transcriptome level, RNA-seq (whole-transcriptome shotgun sequencing) is an emerging technology to identify the relatively complete genes and pathways involved in the physiological response (Morin et al. 2008, Chu & Corey 2012). The RNA-seq has been used in the analysis of stress-response pathways in various aquatic species (Scott & Johnston 2012, Liu et al. 2013, Smith et al. 2013, Xia et al. 2013, Xu et al. 2013, Li et al. 2014), and is an proven tool to capture the genes and metabolic pathways at a specified physiological condition for aquatic animals (Li & Li 2014). Suppression subtractive hybridization has been used to capture the genes and pathways in juvenile Litopenaeus vannamei under a long-term low salinity stress. The most common genes in these libraries are immunity-related proteins and enzymes (Gao et al. 2012), but other genes or metabolic pathways contributing to salinity adaptation may be also included. Similarly, RNS-seq analysis in hemocytes of the Pacific white shrimp reveals that many immune pathways are involved into the process coping with acute salinity stress (31-16 for 24 h), and some energy metabolisms are found in this study but are not fully discussed (Zhao et al. 2015). Despite the low salinity environment (<5) for L. vannamei farming, the knowledge on the metabolic pathways and genes regulation of this euryhaline crustacean at low salinity is still limited.

Previous literature has shown that various organs are involved in the osmoregulation of aquatic animals (Pequeux 1995). Relatively to other organs in crustacean species, such as hepatopancreas and eyes stalk, gill, and muscle (with skin) are directly exposed to ambient water, and water salinity change, especially, would directly affect the physiological status of these two organs. Furthermore, gill is the main site for breathing and ion exchanging, and has been proved significant in osmoregulation in both fish and shrimp (Welcomme & Devos 1991, Fiol et al. 2006, Kiilerich et al. 2007). Because osmoregulation is a high energy cost process (Tseng & Hwang 2008), muscle has also been found involved in this process as the largest energy and osmolyte pool (Li et al. 2009).

Therefore, the aim of this study is to identify the change of pathways and genes responding to acute salinity stress in gill and muscle of juvenile Litopenaeus vannamei. On the basis of the data analysis, putative hypotheses are formulated on how L. vannamei responds to acute salinity stress in an attempt to improve the understanding on the underlying molecular mechanism of salinity adaptation in crustacean.

MATERIALS AND METHODS

Experimental A nimals

Juvenile Litopenaeus vannamei (2.6 [+ or -] 0.4 g) were obtained from a shrimp farm close to the Jinshan District, Shanghai, China. Shrimps were acclimated in tanks for 2 wk with fully aerated water at a salinity of 20 and at 24.3-27.4[degrees]. Then, the shrimps were deprived of food for 24 h and then divided into two treatment groups with three tanks for each group, and there were 30 shrimps in each tank. The group remains at salinity of 20 was the control, and the other group of shrimp was directed exposed into water with a low salinity of 3. No shrimp died within the 24 h. Shrimp at the intermolt stage C were anesthetized with cold ice, and then muscle and gill were sampled for RNA extraction at 24 h after low salinity challenge. Fifteen shrimps from each treatment were pooled for RNA-seq analysis (five shrimp in each tank). During the experimental period, shrimp were all derived of feed to reduce waste production and maintain water quality. Gill and muscle were preserved in liquid nitrogen and then transferred to -80[degrees] for storage before the RNA extraction.

RNA Extraction, Library Construction, and Sequencing

Total RNA from gill and muscle were extracted using Unizol reagent kit (Biostar, Shanghai, China) according to the protocol, and then the extracted RNA was treated with DNAase I. The quality of total RNA was determined using bioanalyzer 2100 and Quibt meter. The RNA-seq library preparation and sequencing were conducted at the HudsonAlpha Genomic Services Laboratory (Huntsville, AL) as previously described (Li et al. 2014). Raw read are available at NCB1 SRA under accession number SRP013882.

De Novo Assembly

All reads with quality scores less than 20 and length less than 15 bp were trimmed by removing ambiguous nucleotides with CLC Genomics Workbench (version 4.9; CLC bio, Aarhus, Denmark) before assembly, and then were used for further subsequent assembly using de Brujin graph assemblers (Li et al. 2012) with ABySS (version 1.2.5) and TransABySS (version 1.2.2) (Simpson et al. 2009). The k-mer size was from 25 to 49 in ABySS, and assemblies from all k-mer lengths were merged into one assembly by TransABySS to generate the transcriptome assembly. CD-Hit (4.5.4) CAP3 was used to remove redundancy and contigs greater than 200 bp (Kal et al. 1999. Li & Godzik 2006). The identify threshold was set at equal to 1 in CD-Hit, and the minimal overlap length and identity was set at equal to 100 bp and 99% in CAP3, respectively.

Gene Annotation and Ontology

The databases of nonreluctant (NR) protein and UniProtKb/SwissProt were used to BLAST the assembly contigs. The cutoff E-value was le-5, and only the top gene ID and the name were assigned to each contig. The UniProtKb/SwissProt BLAST results from Blast2GO (version 2.5.0) were used for the gene ontology (GO) annotation analysis (Gotz et al. 2008). After the gene ID mapping, GO term assignment, annotation augmentation, and generic GO-slim process; the annotation result was categorized into biological process, molecular function, and cellular component.

Identification of Differentially Expressed Contigs

The high-quality reads from each sample were mapped onto the TransABySS reference assembly with CLC Genomics Workbench software. The total number of mapped reads for each transcript was first determined, and then normalized to determine the reads per kilobase of exon model per million mapped reads. To identify the differently expressed genes between the control and the treated samples, the proportions-based test was used with P value setting at less than 0.05 for significant difference (Kal et al. 1999). Transcripts with absolute fold change values greater than 2, total read number greater than 5, and corrected P value < 0.05 were used in the analysis as differently expressed contigs.

Gene Ontology and Pathway Analysis by IPA Program

The analysis of significantly expressed GO terms was performed on Ontologizer 2.0 using the parent-child-intersection method with a Benjammini-Hochberg multiple testing correction to identify the overrepresented GO annotations by comparing to the broader reference assembly (Grossmann et al. 2007, Bauer et al. 2008). Gene ontology terms for each gene were obtained using the UniProtKb/SwissProt annotation.

The GO term assignment in the differentially expressed genes sets were compared with the reference assembly. The threshold was set at the FDR value less than 0.1. The differentiation of significantly expressed genes was analyzed by the Ingenuity Pathway Analysis (IPA) program (https:// analysis.ingenuity.com). All the pathways with one or more genes overlapping the candidate genes were extracted. In IPA, each of these pathways was assigned to a P value via Fisher's exact test, but only pathways with P < 0.01 were selected.

RESULTS

RNA-Seq Sequencing and De Novo Assembly

Totally, 281.4 million reads were obtained, including 68.5 and 67.0 million reads from the control muscle and gill, respectively, and 74.2 and 71.7 million reads from the gill and muscle of Litopenaeus vannamei under low salinity stress, respectively. After filtering, 256.9 million reads accounting for 91.29% of the total reads were used for transcriptome assembly.

De novo assembly with TransABySS generated 557,740 contigs in total with an average length of 333.4 bp and N50 size of 1,198 bp, including 156,686 contigs greater than 200 bp and 49,162 contigs greater than 1,000 bp. After redundancy filtering, 105,153 contigs were generated with an average length of 591.2 bp (Table 1).

Gene Identification and Annotation

Totally, 24,676 out of 105,153 (23.47%) TransAbySS contigs showed significant BLAST hit against the UniProt database with 11,551 unigene matches (Table 2). In contrast, 29,158 out of the TransAbySS contigs (27.73%) had significant BLAST hit against the NR database with 16,765 unigene matches. A total of 7,546 and 10,764 genes from the UniProt and NR databases were identified, which matched protein sequences in the public databases with the stringent criteria of a BLAST score greater than or equal to 100 and E-value less than or equal to le-20.

Identification and Analysis of Differentially Expressed Genes

After annotation, 991 (468 up and 523 down) and 3,709 (316 up and 3,393 down) unigenes differentially expressed after the low salinity challenge for 24 h relative to the control in the muscle and gill, respectively. More than half of the differentially expressed genes with a fold change below two and five, only less than 10% of the differentially expressed genes have been regulated more than 20-fold. In detail, there are 340 out of 991 unigenes were significantly changed more than 5-fold, and only 47 out of 991 unigenes were significantly changed more than 20-fold in muscle; and there are 1,623 out of 3,709 unigenes were differentially expressed more than 5-fold, and only 345 out of 3,709 unigenes were differentially expressed more than 5-fold in gill. Read coverage (average contig size) within the differentially expressed contigs ranged from 407.7 reads/contig in gill to 509.1 reads/contig in muscle. In muscle, the putative aquaporin gene was the top upregulated gene with a fold change of 113 relative to the control, and much higher than other genes. Similarly, in shrimp muscle, the antilipolysaccharide factor isoform and molt-inhibiting hormone 1(MIH1, sinus gland peptide A precursor) were found the most upregulated genes with fold changes of 66.33 and 60.05, respectively.

Gene Ontology and Pathway Analysis

Totally, 2,587 GO terms for muscle including 1,043 biological process terms, 705 molecular function terms and 839 cellular component terms were assigned to 1,300 unique gene matches using Blast2GO. In shrimp gill, GO terms including 3708 biological process terms, 2,179 molecular function terms, and 2,742 cellular component terms were assigned to 3,709 unique gene matches by Blast2GO. The metabolic process (GO: 0,008,152), binding (GO: 0,005,488), and cell (GO: 0,005,623) were the most common annotation terms in the three GO categories for both muscle and gill of the shrimp.

In both muscle and gill, three pathway categories, including oxidative stress-related pathways, signal transduction-related pathways, and metabolism-related pathways (especially energy metabolism pathways), significantly changed after the low salinity stress relative to the salinity control at salinity of 20 revealed by the IPA (Tables 3 and 4). In both muscle and gill, the protein ubiquitination pathway, oxidative phosphorylation and mitochondrial dysfunction significantly changed after the low salinity challenge among the oxidative stress pathways. Relatively fewer pathways related to the signal transduction and metabolism were found in muscle than in gill. For signal transduction-related pathways, hypoxia signaling in the cardiovascular system, integrin-linked kinase signaling and PI3K/ AKT signaling pathways toped in muscle, whereas in gill, aryl hydrocarbon receptor signaling, xenobiotic metabolism signaling, and rac signaling were the top three pathways. Other three pathways, including glucocorticoid receptor signaling, integrin signaling, and protein kinase A signaling were common in both gill and muscle. Among the metabolism pathways, those related to energy metabolism are dominant. In muscle, citrate cycle, purine metabolism and aminosugar metabolism were the top three pathways. Whereas in gill, valine, leucine, and isoleucine degradation; lysine degradation; and propanoate metabolism were the top three pathways.

DISCUSSION

Osmoregulation in crustacean is a complex process where many organs and systems are involved and contribute to the body hemostasis (Pequeux 1995). Litopenaeus vannamei has been recognized as one of the most euryhaline penaeid species, with adults and juveniles exhibiting a hyper- and hypoosmoregulatory pattern and being able to tolerate a wide salinity range, shown by the stable hemolymph osmolality, ionic concentration, or gill Na/K ATPase at various salinities (Gong et al. 2004, Sowers et al. 2006, Huong et al. 2010, Jennyfers et al. 2014). In this study, both gill and muscle responded significantly to acute low salinity challenge with a similar pattern in juvenile L. vannamei. Much more unigenes were differentially expressed in gill than muscle, but most of the genes were regulated between 2-fold and 5-fold in both tissues. The modest fold changes may indicate the ability of L. vannamei to quickly adapt the acute low salinity challenge. After annotation, more pathways changed in the gill than in the muscle after salinity challenge, which is similar to the finding in Eriocheir sinensis after the challenge with an acute salinity for 24 h (Li et al. 2014). This may be because gills are directly exposed to the environment, and are the primary site for the balance of ions between excretion and absorption (Evans et al. 2005, Evans & Somero 2008, Li et al. 2014). When facing an acute salinity challenge, gills are more responsible than the muscle to keep the hemostatics of shrimp. Though the number of pathway changes is different between these two organs, the patterns of change in the signal transduction pathways, oxidative pathways, and metabolism pathways in the gill and muscle were similar, and these patterns are also found in E. sinensis exposed to an acute salinity challenge for 24 h (Li et al. 2014).

Response of Signal Transduction Pathways to Acute Salinity Challenge

Aquatic animals need to quickly activate appropriate signal transduction pathways and send a message to specific target molecules to affect related function when suffered in osmotic stress (Evans 2002, Evans et al. 2005, Fiol et al. 2006, Fiol & Kultz 2007, Evans & Somero 2008). In this study, seven pathways in muscle and 13 pathways in gills were significantly changed and these pathways included glucocorticoid receptor signaling, integrin signaling, and protein kinase A signaling pathways in both gill and muscle. The osmosensory signal transduction pathway is hypothesized to regulate glucocorticoid expression in rainbow trout, and salinity exposure can modulate glucocorticoid expression and glucocorticoid signaling (Singer et al. 2007). Besides, in zebrafish, the glucocorticoid receptor can mediate Cortisol regulation of epidermal ionocyte development and ion transport (Cruz et al. 2013). These finding indicated that the glucocorticoid receptor signaling pathway was included in fish under salinity stress. Similarly, the glucocorticoid signaling in crustacean may have similar function of mediating the Cortisol regulation and ion transportation in respond to acute salinity challenge as in fish.

The protein kinase-A signaling pathway also significantly changed in both gill and muscle of Eriocheir sinensis after acute salinity challenge (Li et al. 2014). Because the protein kinase-A signaling can activate the adenylyl cyclase enzymes for catalyzing the formation of cAMP from ATP, and can regulate the metabolism of glycogen, sugar, and lipid metabolism (Li et al. 2014), the significant change of this pathway may indicate the increase of energy metabolism. It is known that shrimp need more energy to keep homeostasis by ion regulation under acute salinity stress (Tseng & Hwang 2008).

Integrins are a transmembrane receptor for extracellular matrix components and can modulate the calcium channel of animals (Chao et al. 2011) and participate in chondrocyte transduction under osmotic stress (Jablonski et al. 2014). Therefore, the significant response of the integrin signaling pathway in this study indicates that under acute low salinity challenge, the integrins in Litopenaeus vannamei would play an important role in ion modulation during osmoregulation. But further research should be done to explore how integrins work in osmoregulation during salinity stress.

In conclusion, the glucocorticoid receptor signaling, integrin signaling, and protein kinase A signaling pathways played important roles in Litopenaeus vannamei under salinity stress. They may active the subsequent process of ion transportation or energy metabolism during osmoregulation.

Energy Metabolism during Acute Salinity Challenge

Once shrimp senses the ambient salinity change and the signal transduction network is activated, signal are sent to osmo-effectors, which are responsible for acclimation to changes in environmental salinity (Fiol & Kultz 2007). Among these effector mechanism, energy metabolism plays a critical role for animal survival and maintains normal physiological status of organisms under stressful conditions (Sokolova et al. 2012). During salinity acclimation, aquatic animals need additional energy to modulate and stimulate ion transport (Tseng & Hwang 2008).

In this study, in both gill and muscle of shrimp, amino acid metabolism pathways were significantly changed by low salinity challenge, such as glutamate metabolism and arginine and proline metabolism, which is similar to the findings in Eriocheir sinensis (Li et al. 2014). A total of 10 amino acid metabolism pathways in gill and six pathways in muscle participating in the process of osmoregulation in E. sinensis. It may be because that muscle is the main site of gluconeogenesis (Vinagre & Da Silva 2002) in crustaceans, and this process also appears to occur in the gill (Oliveira et al. 2004). In addition, muscle is also the main amino acid pool in crustaceans, which plays a role in storing and supplying amino acids to the other tissues during stress (Wang et al. 2012). The amount of free amino acids in the muscle of E. sinensis will decrease after 12 h under salinity stress (Wang et al. 2012). Moreover, free amino acid and glycerol will be used by Neoheiice granulata to maintain the adequate glucose in other tissues through gluconeogenesis under hypoosmotic stress (Nery & Santos, 1993, Lauer et al. 2012). The finding in this study further corroborates the notion that free amino acids are important intracellular osmotic effectors in crustacean (Edwards 1982, McNamara et al. 2004, Augusto et al. 2007, Augusto et al. 2009, Wang et al. 2012).

Carbohydrate metabolism pathways for starch and sucrose metabolism, galactose metabolism, glycolysis/glucongeogenesis, and glycosaminoglycan degradation in gill, and aminosugars metabolism in muscle were also involved into the physiological process related to the osmo/ion regulation of shrimp after acute low salinity change in this study. Previous studies have showed that carbohydrate can directly meet the high energy demand of aquatic animals in a stress condition, especially in salinity stress (Welcomme & Devos 1991, Tseng & Hwang 2008). In previous study, it was found that suitable dietary carbohydrate of Litopenaeus vannamei can improve growth performance at low salinity by providing direct extra energy for growth and osmoregulation (Wang et al. 2014a). Moreover, under the basic demand for protein, the addition of appropriate carbohydrate can improve the survival of L. vannamei at low salinity (Wang et al. 2014b). The changes of these carbohydrate metabolism pathways can also reveal that carbohydrate or glucose can alleviate the osmotic stress by providing extra energy for shrimp during low salinity challenge.

Lipids play significant roles in osmoregulation as reported in previous studies (Lemos et al. 2001, Luvizotto-Santos et al. 2003, Sang & Fotedar 2004). In this study, acute low salinity change led to a significant change in pathways of fatty acid elongation in mitochondria, fatty acid metabolism, sphingolipid metabolism, and propanoate metabolism in gill. No pathway related to lipid metabolism was found in shrimp muscle, which may be because this acute salinity change stopped after 24 h and there was not enough time to respond by muscle compared with shrimp gill. Previous literature shows that modification of fatty acids composition in the gill with a high level of (n-3) PUFA can result in a large gill area to enhance osmoregulatory capacity of shrimp at low salinity, and increase survival (Palacios et al. 2004). Because the (n-3) HUFAs, especially docosahexaenoic acid, are mainly incorporated in cell membranes, and can increase membranes permeability and hence their fluidity (Martins et al. 2006, Sui et al. 2007). Furthermore, sphingolipid is a key factor in gill cell membrane and plays important roles for salt transport in aquatic animals (el Babili et al. 1996).

In summary, energy metabolism including protein, carbohydrate, and lipid metabolism, plays a critical role in the osmoregulation process in Litopenaeus vannamei after acute salinity stress. Free amino acids are important intracellular osmotic effectors in L. vannamei. Carbohydrate may alleviate the osmotic stress by providing extra energy for shrimp during low salinity challenge. Meanwhile, lipids may play some role in by increasing the ion transport under salinity stress.

Reactive Oxygen Species Overproduction and Scavenging

Similar to the findings in Eriocheir sinensis (Li et al. 2014), this study identified significant changes of the protein ubiquitination. mitochondria dysfunction, and oxidative phosphorylation pathways in both gill and muscle of Litopenaeus vannamei. The activity of oxidative phosphorylation can be modulated by tissue metabolic stress to maintain energy metabolism homeostasis. Under acute salinity stress, the energy metabolism in both gill and muscle reached the maximum as measured by the process of oxidative phosphorylation (Phillips et al. 2012).

Under oxidative stress, the elevation of reactive oxygen species (ROS), which is an unenviable product in aerobic metabolism, can damage cellular constituents (Lushchak 2011). In aquatic animals, salinity change can lead to various physiological responses such as elevation of plasma hormone, high metabolism and electrolyte disequilibrium due to ROS generation by salinity stress (Liu et al. 2007). Uniquinone functions as an antioxidant and serves as a cofactor of mitochondrial uncoupling proteins, and modification of the ubiquinone pool in mice results in increased longevity and higher resistance to oxidative stress (Liu et al. 2005). Oxidative stress could increase the levels of ubiquitin conjugates in various cell types (Shang et al. 1997, Liu et al. 2005, Shang & Taylor 2011) to remove the overproduced ROS. Protein ubiquitination and ubiquinone biosynthesis pathways could be activated by the oxidative stress of acute salinity challenge and increased energy metabolism to help shrimp eliminate the high level of ROS or related metabolite waste products. Similar findings were found in Eriocheir sinensis under acute salinity stress (Li et al. 2014) and Leuciscus waleckii in an extremely alkaline saline environment (Xu et al. 2013).

In summary, Litopenaeus vannamei may get more energy through by the process of oxidative phosphorylation. The increased protein ubiquitination and ubiquinone biosynthesis pathways may prevent the shrimp from the high level of ROS or related hazardous substances produced by oxidative stress of acute salinity challenge.

Potential Genes Coping with Acute Salinity Challenge

In this study, 468 genes in muscle and 316 genes in gill were upregulated, but the putative aquaporin gene was the top upregulated gene with a fold change of 113 times more in muscle at 3 than in the control (20). Aquaporin is a family of water-specific channel proteins that allow the transport of water and other solutes such as glycerol or urea in the presence of osmotic gradients (Borgnia et al. 1999). Therefore, under osmotic stress, the aquaporin can possibly regulate water and ions movement across membranes (Giffard-Mena et al. 2007). Aquaporin 3 from Sparus aurata express in various tissues except for blood, and higher expression is found in S. aurata under a hyposaline than a hypersaline condition (Deane & Woo 2006). Aquaporin 1 shows a major osmoregulatory role in water transport in the kidney and gut in seawater acclimated seabass Dicentrarchus labrax, whereas aquaporin 3 has a role in water transport in the gill of D. labrax acclimated to freshwater (Giffard-Mena et al. 2007). Similarly, aquaporin 3 from Cyprinus carpio also shows direct relation with environmental salinity C. carpio (Salati et al. 2014). Though no information is reported in aquaporin in crustaceans, all the findings together with the upregulated expression of the aquaporin gene in this study indicate its importance in regulating water transport under osmotic stress, especially at a hypoosmotic condition in both fish and shrimp.

In the gill of shrimp after 24 h-low salinity challenge, the top upregulated genes are antilipolysaccharide factor isoform and MIH1 (sinus gland peptide A precursor) with fold changes more than 60 times. Molt-inhibiting hormone is usually responsible for maintaining animals in at an intermolt stage due to the effect of MIH on the synthesis of a steroid molecule secreted by Yorgan (ecdysterioid) (Lago-Leston et al. 2007), but also has more possible biological functions such as hyperglycemic activity (Cheng et al. 2006, Lago-Leston et al. 2007). Two alternative slicing variants of MIH present in the eyestalks of Litopenaeus vannamei, and MIH 1 is the dominated hormone in L. vannamei regardless of ambient salinities. The highest expression values for MIH 1 were observed at the lowest salinity (10) tested in the study (Lago-Leston et al. 2007), which is in certain degree similar to the result of this study. Therefore, as in the Pacific rock crab Cancer antennarius, the high MIH expression induced by stress can inhibit ecdysteroid synthesis in the Y-organ during the intermolt stage of the crustacean and therefore inhibit the process of molting (Spaziani et al. 1989).

Antilipopolysaccharide factor is an important shrimp immune gene (Tharntada et al. 2008), and was significantly downregulated in gill and gut tissues when compared with muscle tissues of Penaeus monodon acclimated from salinity of 28 to 3 for 2 wk (Shekhar et al. 2006). High expressions of lipopolysaccharide were found in Litopenaeus vannamei reared at 2.5 and 5 than that at 15,25, and 35 for a period of 24 wk (Lin et al. 2012). This finding reveals either acute or chronic salinity stress will stimulate the immune response of L. vannamei, which will decrease its resistance against pathogens due to the reduction of immune parameters when exposed to low salinity conditions (Wang & Chen 2005, Li et al. 2010). A recently RNA-seq study with hemocytes of L. vannamei also collaborate this finding, though shrimp was only transferred from 31 to 16 for 24 h (Zhao et al. 2015).

SUMMARY

After acute low salinity challenge, shrimp could respond to ambient salinity change using various signal transduction pathways as summarized in Figure 1. Under low salinity stress, the glucocorticoid receptor signaling, integrin signaling, and protein kinase A signaling pathways played important roles in Litopenaeus vannamei under salinity stress. They may active the subsequent process of ion transportation or energy metabolism during osmoregulation. The energy metabolism including protein, carbohydrate, and lipid metabolism, plays a critical role in the osmoregulation process in L. vannamei after acute salinity stress. Free amino acids are important intracellular osmotic effectors in L. vannamei. Carbohydrate may alleviate the osmotic stress by providing extra energy for shrimp during low salinity challenge. Meanwhile, lipids may play some role in by increasing the ion transport under salinity stress. Under acute salinity stress, L. vannamei may get more energy through by the process of oxidative phosphorylation. The increased protein ubiquitination and ubiquinone biosynthesis pathways may prevent the shrimp from the high level of ROS or related hazardous substances produced by oxidative stress of acute salinity challenge. Despite the identification of various pathways under salinity stress, many functional responses to the transcriptome analysis remains unclear. Due to the complex of osmoregulation in crustacean, the functional roles of many genes involved on osmoregulation at low salinity are not fully discussed in this study. Therefore, the detained and specific response of vitally important pathways and functional genes should be further studied in the future.

ACKNOWLEDGMENTS

This research was supported by grants from the National Natural Science Foundation of China (No. 31472291, 31172422), the Special Fund for Agro-scientific Research in the Public Interest (No. 201203065), National "Twelfth Five-Year" Plan for Science & Technology Support (2012BAD25B03), the National Basic Research Program (973Program, No. 2014CB138803), and partly by the E-Institute of Shanghai Municipal Education Commission (No. E03009) and ECNU innovation fund.

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XIAODAN WANG, (1) SHAOLIN WANG, (2) CHAO LI, (3) KE CHEN, (1) JIAN G. QIN, (4) LIQIAO CHEN (1) AND ERCHAO LI (1) *

(1) Laboratory of Aquaculture and Environmental Health, School of Life Sciences, East China Normal University, Shanghai, 200241, China; (2) Department of Psychiatry & Neurobiology Science, University of Virginia, Charlottesville, VA 22911; (3) Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, 266109, China; School of Biological Sciences, Flinders University, Adelaide, SA 5001, Australia

* Corresponding author. E-mail: ecli@bio.ecnu.edu.cn

DOI: 10.2983/035.034.0330

TABLE 1.
Summary of de novo assembly results of Illumina sequence
data of Litopenaeus vananmei.

                                                          Trans-ABySS

Contigs ([greater than or equal to] 100 bp)                 557,740
Large contigs ([greater than or equal to] 1,000 bp)         49,162
Maximum length (bp)                                         13,707
Average length (bp)                                          333.4
N50 (bp)                                                     1,198
Contigs after length filtering ([greater than or equal      156,686
  to] 200 bp)
Percentage contigs kept after length filtering              28.09%
Average contig length after length filtering (bp)            984.2
Contigs (After CD-HIT-EST + CAP3)                           105,153
Average length (bp) (After CD-HIT-EST + CAP3)                591.2
Reads mapped to final reference (%)                          70.62

TABLE 2.
Summary of gene identification and annotation of assembled contigs
based on BLAST homology searches against UniProt and NR.

                             Annotated          Annotated
          Contigs with    contigs [greater   contigs [greater
          putative gene     than or equa      than or equal
             matches         to] 500 bp        to] 1,000 bp

UniProt      24,676            12,306             8,229
NR           29,158            13,846             8,960

                    Hypothetical   Quality
          Unigene       gene       unigene
          matches     matches      matches

UniProt   11,551         0          7,546
NR        16.765       5,339       10,764

TABLE 3.
Significantly changed pathways in muscle of Litopenaeus vannamei at
salinity of three relative to the control group at 20.

                          Pathways        -log (P value)

Oxidative            Protein                 1.62E01
  stress-related       ubiquitination
  pathways             pathway
                     Oxidative               1.17E01
                       phosphorylation
                     Mitochondrial           7.68E00
                       dysfunction
                     Ubiquinone              3.94E00
                       biosynthesis
Signaling-related    Hypoxia signaling       2.87E00
  pathways             in the
                       cardiovascular
                       system
                     Integrin-linked          2.8E00
                       kinase
                       signaling
                     PI3K/AKT signaling      2.17E00
                     Actin cytoskeleton      2.17E00
                       signaling
                     Glucocorticoid          2.11E00
                       receptor
                       Signaling
                     Integrin signaling      2.11E00
                     Protein kinase A          2E00
                       signaling
Metabolism-related   Citrate cycle           5.43E00
  pathways           Purine metabolism       4.66E00
                     Aminosugars             3.78E00
                       metabolism
                     Pentose phosphate       3.36E00
                       pathway
                     Glutamate               2.74E00
                       metabolism
                     Arginine and             2.5E00
                       proline
                       metabolism
                     Urea cycle and          2.39E00
                       metabolism of
                       amino groups
                     D-glutamine and         2.18E00
                       D-glutamate
                       metabolism

                          Pathways                 Molecules

Oxidative            Protein              CRYAB, USP14, PSMA3, UBE2A,
  stress-related       ubiquitination     PSMD7, UBE2D2, USP2,
  pathways             pathway              HSPA5, TCEB1, PSMB6,
                                            HSP90B1, PSMC6, PSMD10,
                                            USP47, UCHL5, PSMA2,
                                            UCHL3, PSMA6, PSMB5,
                                            DNAJC19, HSPA9, PSMD6,
                                            PSMA1, THOP1, HSPD1,
                                            PSMD5, UBE3A, SKP1/
                                            SKP1P2, PSMD8, DNAJC21,
                                            PSMB7, PSMC1, PSMB2,
                                            UBE2G1, PSMD12, PSMA5,
                                            PSMB1, PSMA4, HSP90AA1,
                                            PSMD4, PSMC3
                     Oxidative            NDUFA10, UQCR11, NDUFB8,
                       phosphorylation      ATP6AP1, NDUFS1, NDUFA5,
                                            UQCRFS1, ATP5F1, NDUFS4,
                                            ATP6V0E2, ATP5A1, ATP5C1,
                                            UQCR10, Atp5h, NDUFV2,
                                            ATP6V1H, NDUFA11, NDUFA6,
                                            UQCRC2, NDUFB7, ATP6V0D1,
                                            COX5A, SDHD, CYCl, UQCRQ,
                                            NDUFA7
                     Mitochondrial        NDUFA10, ATP5A1, NDUFB8,
                       dysfunction          ATP5C1, NDUFS1, PRDX3,
                                            NDUFA5, PARK7, NDUFV2,
                                            NDUFA11, NDUFA6, NDUFB7,
                                            UQCRC2, COX5A, UQCRFS1,
                                            SDHD, CYCl, NDUFA7,
                                            PINK1, NDUFS4
                     Ubiquinone           NDUFA5, NDUFS1, NDUFA10,
                       biosynthesis         NDUFV2, NDUFA11, NDUFA6,
                                            NDUFB7, NDUFB8, NDUFA7,
                                            NDUFS4
Signaling-related    Hypoxia signaling    P4HB, HSP90B1, NFKB1A,
  pathways             in the               UBE2A, UBE2G1, UBE2D2,
                       cardiovascular       HSP90AA1, LDHA
                       system
                     Integrin-linked      ITGB1, nuILMYH10, PXN,
                       kinase               MYL6, RHOC, PIK3R1, MYH7,
                       signaling            PARVB, MTOR, FLNA,
                                            PPP2R3A, PPM1L, MYH9,
                                            RPS6KA5
                     PI3K/AKT signaling   ITGB1, MTOR, HSP90B1,
                                            NFKB1A, YWHAE, PPP2R3A,
                                            PIK3R1, PPM1L, HSP90AA1,
                                            CDKN1B
                     Actin cytoskeleton   1TGB1, MYH10, ARHGEF4, PXN,
                       signaling            MYL6, PIK3R1, ARPC5, CRK,
                                            MYH7, TTN, MYL12A, ACTR3,
                                            FLNA, MYH9, ARPC1A
                     Glucocorticoid       PRKAB1, PIK3Rl, HSPA9,
                       receptor             SLP1, PCK1, HSPA5, null,
                       Signaling            HSP90B1, NFKB1A, NFAT5,
                                            HMGB1, PCK2, POLR2H,
                                            HSP90AA1, A2M, PPP3CA,
                                            POLR2K
                     Integrin signaling   RAP1B, ITGB1, CAPN5, PXN,
                                            RHOC, PIK3R1, ARPC5, CRK,
                                            TTN, PARVB, MYL12A, ARF1,
                                            ACTR3, ARPC1A
                     Protein kinase A     RAP1B, MYH10, PXN, MYL6,
                       signaling            YWHAE, PPP1R3C, RYR2,
                                            TTN, null, PHKA2, MYL12A,
                                            PYGM, NFKB1A, NFAT5,
                                            FLNA, ADD1, PDE5A,
                                            PPP1CA, PPP3CA
Metabolism-related   Citrate cycle        SUCLA2, PCK2, SUCLG2, AC02,
  pathways                                  SDHSD, PCK1, IDH3A, ACLY
                     Purine metabolism    Pkm, ATP5A1, PAICS, MYH7,
                                            TYMP, HSPD1, HSPA5,
                                            AMPD2, null, POLR1C,
                                            ATP5C1, null, PSMC1,
                                            Atp5h, PSMC6, ADK, AK1,
                                            PNP, POLR2H, HSP90AA1,
                                            MYH9, PDE5A, ATP5F1,
                                            POLR2K
                     Aminosugars          HK1, NAGK, CHIT1, HEXB,
                       metabolism           PDE5A, PPM1F, UAP1, CHIA,
                                            null, GFPT2
                     Pentose phosphate    DERA, PGD, PGLS, PGM1, RGN,
                       pathway              PFKP
                     Glutamate            NAGK, SUCLG2, GLS, GLUD1,
                       metabolism           GOT2, GFPT2
                     Arginine and         P4HB, PRODH, OAT, GLUD1,
                       proline              GOT2, ODC1, ALDH7A1,
                       metabolism           PYCR1
                     Urea cycle and       OAT, GLUD1, ALDH18A1, ODC1,
                       metabolism of        PYCR1
                       amino groups
                     D-glutamine and      GLS, GLUD1
                       D-glutamate
                       metabolism

TABLE 4.
Significantly changed pathways in gill of Litopenaeus vannamei at
salinity of three relative to the control group at 20.

                          Pathways         log (P value)

Oxidative            Mitochondrial            2.93E00
  stress-related       dysfunction
  pathways           Oxidative                1.89E00
                       phosphorylation *
                     Protein                  1.83E00
                       ubiquitination
                       pathway *
Signaling-related    Aryl hydrocarbon         3.5E00
  pathways             receptor
                       signaling
                     Xenobiotic               3.07E00
                       metabolism
                       signaling
                     Rac signaling            2.85E00
                     Sertoli                  2.78E00
                       cell-sertoli
                       cell junction
                       signaling
                     Estrogen receptor        2.48E00
                       signaling
                     Glucocorticoid           2.38EOO
                       receptor
                       signaling
                     Huntington's             2.38E00
                       disease
                       signaling
                     Integrin signaling       2.36E00
                     Tight junction           2.07E00
                       signaling
                     Synaptic long-term       2.06E00
                       potentiation
                     BMP signaling            2.04E00
                       pathway
                     Protein kinase A         2.03E00
                       signaling
                     ERK/MAPK signaling        2E00
Metabolism-related   Valine, leucine,         1.02E01
  pathways             and isoleucine
                       degradation
                     Lysine degradation       7.83EOO
                     Propanoate               7.81E00
                       metabolism
                     Butanoate                7.22E00
                       metabolism
                     [beta]-alanine           5.74E00
                       metabolism
                     Citrate cycle            5.61E00
                     Ascorbate and            5.44E00
                       aldarate
                       metabolism
                     Glycine, serine,         4.38E00
                       and threonine
                       metabolism
                     Tryptophan               4.29E00
                       metabolism
                     Glutamate                4.09E00
                       metabolism
                     Pyruvate                 4.O8EOO
                       metabolism
                     Arginine and             3.33EOO
                       proline
                       metabolism
                     Purine metabolism        3.29E00
                     Calcium signaling        3.14E00
                     Nucleotide sugars        2.51E00
                       metabolism
                     Fatty acid               2.48E00
                       elongation in
                       mitochondria
                     Fatty acid               2.39E00
                       metabolism
                     Pantothenate and         2.35E00
                       CoA biosynthesis
                     Starch and sucrose       2.3IE00
                       metabolism
                     Galactose                2.25E00
                       metabolism
                     Sphingolipid             2.23E00
                       metabolism
                     Glyoxylate and           2.12E00
                       dicarboxylate
                       metabolism
                     Sulfur metabolism        2.12E00
                     Glycolysis/             2.09 E00
                       gluconeogenesis
                     Glycosaminoglycan        2.05E00
                       degradation
                     Pyrimidine               2.03EOO
                       metabolism
                     Assembly of RNA          2.99E00
                       polymerase II
                       complex

                          Pathways                  Molecules

Oxidative            Mitochondrial         COX1, NDUFV1, COX7A2,
  stress-related       dysfunction           CPT1A, NDUFA10, CASP3,
  pathways                                   PRDX5, XDH, PSENEN,
                                             NDUFB8, GPX7, App,
                                             ATP5C1, NDUFA5, ATP5B,
                                             CYB5R3, NDUFA12, SDHD,
                                             CYC1, OGDH, NDUFA7,
                                             PINK1, AIFM1
                     Oxidative             COX1, NDUFV1, COX7A2,
                       phosphorylation *     ATP6V1D, NDUFA10,
                                             UQCR11, NDUFB8, ATP6V1A,
                                             ATP6AP1, ATP6V0A1,
                                             ATP5C1, NDUFA5, Atp5h,
                                             ATP5B, ATP6V0D1,
                                             NDUFA12, SDHD, CYC1,
                                             ATP5F1, NDUFA7, ATP6V1B2
                     Protein               COX1, NDUFV1, COX7A2,
                       ubiquitination        ATP6V1D, NDUFA10,
                       pathway *             UQCR11, NDUFB8, ATP6V1A,
                                             ATP6AP1, ATP6V0A1,
                                             ATP5C1, NDUFA5, Atp5h,
                                             ATP5B, ATP6V0D1,
                                             NDUFA12, SDHD, CYC1,
                                             ATP5F1, NDUFA7, ATP6V1B2
Signaling-related    Aryl hydrocarbon      ALDH4A1, GSTM5, NFKB1,
  pathways             receptor              SMARCA4, EP300, ARNT,
                       signaling             GSTT1, NCOA7, ALDH1A1,
                                             NCOA2, ALDH5A1, CHEK2,
                                             GSTK1, ALDH7A1, ALDH1B1,
                                             SRC, MGST1, ALDH8A1,
                                             SLC35A2, ALDH9A1, GSTO1,
                                             A1P, CDKN1B, ALDHI6A1,
                                             MCM7
                     Xenobiotic            ALDH4A1, CHST4, SULT1C4,
                       metabolism            CAMK1, GSTM5, ABCC2,
                       signaling             PIK3R1, NFKB1, P1K3R4,
                                             ARNT, EP300, CUL3,
                                             GSTT1, CAMK2D, ALDH1A1,
                                             UGT2B17, MAP3K7, PPM1L,
                                             CHST11, SMOX, null,
                                             ALDH5A1, GSTK1, ALDH7A1,
                                             ALDH1B1, MGST1, ALDH8A1,
                                             GRIP1, UGT8, Sult1d1,
                                             ALDH9A1, GSTO1, AIP,
                                             PPP2R1A, PRK, CI,
                                             MAPK14, SULT1A1,
                                             ALDH16A1, ABCC3,
                                             EIF2AK3, DNAJC7
                     Rac signaling         ITGB1, ABI2, PAK2, PIK3R1,
                                             ARPC5, PIK3R4, NFKB1,
                                             ANK1, MCF2L, PIP5K1A,
                                             CYFIP2, ACTR3, PRKCI,
                                             CYFIP1, ARPC1A, PARD3,
                                             PI4KA, NCKAP1, ITGA4
                     Sertoli               PRKACB, SPTBN1, AXIN1,
                       cell-sertoli          MLLT4, MPP6, CTNNA2,
                       cell junction         SORBS1, MAP3K7, AKT3,
                       signaling             MTMR2, CTNNB1, ACTN1,
                                             ITGA4, 1TGB1, PLS1,
                                             DLG1, SRC, EPN1, TJP2,
                                             TUBG1, TUBA1B, ATF2,
                                             null, MAPK14, TUBA1A,
                                             PRKAG2, MAGI2, PRKAR1A
                     Estrogen receptor     MED12L, SRC, TAF11,
                       signaling             POLR2D, MED23, null,
                                             MED21, TAF10, ERCC2,
                                             SMARCA4, EP300, null,
                                             GTF2B, TAF1, POLR2A,
                                             TAF5, NCOA2, MED13L,
                                             NCOR1, TAF3, POLR21
                     Glucocorticoid        PRKACB, TAF11, POLR2D,
                       receptor              PRKAB1, PIK3R1, SMAD3,
                       signaling             SLP1, PBX1, TAF10,
                                             HSPA5, NFKB1, PIK3R4,
                                             SMARCA4, EP300, null,
                                             GTF2B, POLR2A, NFAT5,
                                             NCOA2, MAP3K7, AKT3,
                                             NCOR1, TAF3, TAB1,
                                             PPP3CA, null, HSPA9,
                                             CHP1, TAT, ERCC2, TRAF6,
                                             MAPK14, TAFl, TAF5,
                                             PRKAG2, POLR21
                     Huntington's          POLR2D, VTI1A, PIK3R1,
                       disease               GNB2L1, HSPA5, PIK3R4,
                       signaling             VTI1B, EP300, null,
                                             POLR2A, HDAC7, AKT3,
                                             PLCB1, TCERG1, NCOR1,
                                             GOSR1, CASP3, GLS,
                                             HSPA9, CLTC, ITPR1,
                                             STX1A, NAPG, ATF2, DNM1,
                                             DYNC112, PLCB4, PRKC1,
                                             ATP5B, CAPN9, POLR21
                     Integrin signaling    RAP1B, RAPGEF1, ARHGAP26,
                                             PIK3R1, ARPC5, CRK,
                                             TLN1, NCK1, PIK3R4,
                                             MYLK, BRAF, PARVB,
                                             ACTR3, ITGAV, AKT3,
                                             ARPC1A, ACTN1, ITGA4,
                                             ITGB1, SRC, PAK2, RHOC,
                                             TTN, RAC3, ARF1,
                                             PPP1R12B, CAPN9, ITGA7,
                                             CTTN
                     Tight junction        PRKACB, CPSF2, TJP2,
                       signaling             MARK2, MLLT4, MYH7,
                                             null, NFKB1, SMURF1,
                                             MYLK, PPP2R1A, PRKCI,
                                             LLGL1, MPP5, PPM1L,
                                             PRKAG2, AKT3, MYH9,
                                             INADL, MAGI2, CTNNB1,
                                             CSTF3, PRKAR1A
                     Synaptic long-term    RAP1B, PRKACB, PPP1R3C,
                       potentiation          CHP1, ITPR1, ATF2,
                                             GR1NA, EP300, PLCB4,
                                             CAMK2D, PRKC1, PPP1R7,
                                             ADCY1, PRKAG2, PLCB1,
                                             PPP3CA, PRKAR1A
                     BMP signaling         PRKACB, SMAD6, NFKB1,
                       pathway               SMURF1, CHRD, ATF2,
                                             BMPR1B, MAPK14, MAP3K7,
                                             PRKAG2, SMAD1, TAB1,
                                             PRKAR1A
                     Protein kinase A      RAP1B, PRKACB, FLNB,
                       signaling             SMAD3, PPP1R3C, PHKG2,
                                             GNB2L1, NFKB1, PHKA2,
                                             MYLK, R0CK2, BRAF,
                                             CAMK2D, NFAT5, PHKB,
                                             PLCE1, PPP1R7, FLNA,
                                             PLCB1, CTNNB1, APEX1,
                                             PPP3CA, YWHAE, CHP1,
                                             ITPR1, ANAPC7, TTN,
                                             ATF2, PDE8A, AKAP13,
                                             PLCB4, PYGM, PRKC1,
                                             ADCY1, PRKAG2, ADD1,
                                             AKAP9, TCF7L2, CDC27,
                                             PRKAR1A
                     ERK/MAPK signaling    RAP1B, PRKACB, RAPGEF1,
                                             PIK3R1, PPP1R3C, CRK,
                                             TLN1, RAPGEF4, PIK3R4,
                                             KSR1, BRAF, PPP1R7,
                                             PPM1L, ITGA4, ITGB1,
                                             SRC, MYCN, PAK2, MKNK2,
                                             RAC3, ATF2, PLA2G6,
                                             PPP2R1A, PRKC1, PRKAG2,
                                             PRKAR1A
Metabolism-related   Valine, leucine,      ALDH4A1, BCKDHB, HSD17B8,
  pathways             and isoleucine        ALDH1A1, BCAT2, BCKDHA,
                       degradation           OXCT1, MCCC1, ACADM,
                                             HSD17B4, HADHA, ALDH7A1,
                                             ALDH1B1, ECHS1, MUT,
                                             ALDH9A1, ELOVL6, ACADL,
                                             ACADVL, PCCA, HJBADH,
                                             AUH, AGXT2, HADH, MCCC2
                     Lysine degradation    ALDH4A1, ALDH1B1, WHSC1,
                                             ECHS1, AASS, PLOD1,
                                             GCDH, ALDH9A1, ELOVL6,
                                             EP300, HSD17B8, TMLHE,
                                             ALDH1A1, FAM213B, AUH,
                                             OGDH, HSD17B4, HADHA,
                                             SHMT2, HADH, ALDH7A1
                     Propanoate            ALDH4A1, ALDH1B1, ECHS1,
                       metabolism            SUCLG2, MUT, ALDH9A1,
                                             ACADL, SUCLA2, ALDH1A1,
                                             PCCA, ACADVL, SRD5A3,
                                             AUH, ACSS2, AGXT2,
                                             ACADM, LDHA, ACSL1,
                                             HADHA, ALDH7A1
                     Butanoate             ALDH4A1, ALDH1B1, ECHS1,
                       metabolism            SUCLG2, PRDX6, ALDH9A1,
                                             ELOVL6, HSD17B8, BDHl,
                                             ALDH1A1, FAM213B, AUH,
                                             OXCT1, SDHD, HSD17B4,
                                             ALDH5A1, HADHA, HADH,
                                             ILVBL, ALDH7A1
                     [beta]-alanine        ALDH4A1, ALDH1B1, SRM,
                       metabolism            DPYS, DPYD, ECHS1,
                                             ALDH9A1, ACADL, ALDH1A1,
                                             ACADVL, AUH, AGXT2,
                                             ACADM, HADHA, ALDH7A1
                     Citrate cycle         SUCLA2, PC, SUCLG2, AC02,
                                             SDHD, 1DH2, IDH3A, MDH1,
                                             OGDH, ACLY, AC01, IDH3B
                     Ascorbate and         ALDH4A1, ALDH1B1, ALDH1A1,
                       aldarate              FAM213B, BCKDHA, RGN,
                       metabolism            ALDH9A1, GST01, ALDH7A1,
                                               BCKDHB
                     Glycine, serine,      GNMT, GLYCTK, TARS,
                       and threonine         ELOVL6, SARDH, PLCB4,
                       metabolism            PLCE1, FAM213B, PHGDH,
                                             PLCB1, SMOX, GOT1,
                                             ALAS2, GLDC, SARS,
                                             AGXT2, CHKB, SHMT2
                     Tryptophan            ALDH4A1, BC02, GCDH,
                       metabolism            BCKDHB, ACR, HSD17B8,
                                             ACMSD, ALDH1A1, BCKDHA,
                                             SMOX, OGDH, HSD17B4,
                                             HADHA, ALDH7A1, ALDH1B1,
                                             ECHS1, HAAO, Nedd4,
                                             ALDH9A1, TMLHE, WARS,
                                             SRD5A3, AUH, Cyp2j9,
                                             KYNU, HADH
                     Glutamate             ALDH4A1, NAGK, GMPS,
                       metabolism            SUCLG2, NADSYN1, GLS,
                                             CCDC92, GOT1, CAD,
                                             ALDH5A1, GSTO1, GFPT2
                     Pyruvate              ALDH4A1, ALDH1B1, PC,
                       metabolism            ACOT9, MDH1, ALDH9A1,
                                             GLO1, BCKDHB, ALDH1A1,
                                             BCKDHA, ACSS2, PDHX,
                                             LDHA,  ACSL1, HAGH,
                                             HADHA, ALDH7A1
                     Arginine and          ALDH4A1, ALDH1B1, SRM,
                       proline             OAT, AMD1, ALDH9A1, ACR,
                       metabolism            BCKDHB, P4HA1, ALDH1A1,
                                             PRODH, BCKDHA, GOT1,
                                             SMOX, ASL, ALDH7A1
                     Purine metabolism     POLR2D, IMPAD1, XDH, POL1,
                                             HSPA5, POLRMT, SMARCA4,
                                             BCKDHB, MPP6, null,
                                             POLR2A, POLR3A, PRPS1,
                                             BCKDHA, ENTPD5, ADA,
                                             ATP5F1, DLG1, PEX6,
                                             TJP2, AK3, PAICS, MYH7,
                                             REV3L, NT5C2, PDE8A,
                                             ATP5C1, ENPP3, NT5C3,
                                             Atp5h, ATP5B, GMPS,
                                             MPP5, IMPDH1, ATF7IP,
                                             ADCY1, PNP, MYH9, GMPR2,
                                             POLR21, CANT1
                     Calcium signaling     RAPlB, PRKACB, CAMK1,
                                             CHRFAM7A, ATP2A1, null,
                                             EP300, GR1NA, CAMK2D,
                                             NFAT5, TNNT3, HDAC7,
                                             ASPH, PPP3CA, CHRNA4,
                                             LETM1, TP63, ATP2C1,
                                             CHP1, CHRNA9, MEF2A,
                                             MYH7, ITPR1, ATF2,
                                             MICU1, CAMKK1, PRKAG2,
                                             MYH9, PRKAR1A
                     Nucleotide sugars     UGDH, FAM213B, UGP2, GALE
                       metabolism
                     Fatty acid            ECHS1, AUH, HSD17B4,
                       elongation in         HADHA, HADH, HSDI7B8
                       mitochondria
                     Fatty acid            ALDH4A1, ALDH1B1, CPT1A,
                       metabolism            ECHS1, GCDH, ALDH9A1,
                                             HSD17B8, ACADL, ALDH1A1,
                                             ACADVL, AUH, ACSL4,
                                             Cyp2j9, HSD17B4, ADHFE1,
                                             ACADM, HADHA, ACSL1,
                                             HADH, ALDH7A1
                     Pantothenate and      ENPP3, DPYS, DPYD, BCAT2,
                       CoA biosynthesis      PPCDC, ILVBL
                     Starch and sucrose    PGM2, PGM1, HPSE, GUSB,
                       metabolism            GANAB, ENPP3, DDX6,
                                             UGDH, PYGM, UGT2B17,
                                             UGP2, GAA, GBE1
                     Galactose             B4GALT2, PGM2, FAM213B,
                       metabolism            GALK2, UGP2, GLB1, GALE,
                                             GAA, PGM1, GANAB
                     Sphingolipid          CERS6, ARSH, SPTLCl, PlGO,
                       metabolism            ARSD, PIGF, UGT8, SGMSl,
                                             ASAH1, PPAP2A, GLB1,
                                             SPTLC2, PPM1F, ARSB,
                                             KDSR
                     Glyoxylate and        APTX, GLYCTK, AC02, MDHl,
                       dicarboxylate         MTHFD1, ACO1
                       metabolism
                     Sulfur metabolism     SULT1A1, ETHE1, CHST1l,
                                             null, Sult1d1, SUOX
                     Glycolysis/           PGK1, ALDH4A1, ALDH1B1,
                       gluconeogenesis       PGM2, PGM1, Tpi1
                                             (includes others),
                                             ALDH9A1, GALM, ALDH1A1,
                                             PDHX, ACSS2, ADHFE1,
                                             LDHA, ACSL1, ALDH7A1
                     Glycosaminoglycan     MGEA5, GLB1, HPSE, GALNS,
                       degradation           ARSB, GNS, GUSB
                     Pyrimidine            DPYS, DPYD, POLR2D,
                       metabolism            IMPAD1, AK3, POL1,
                                             REV3L, DCTD, POLRMT,
                                             CTPS1, NT5C2, ENPP3,
                                             NT5C3, null, POLR2A,
                                             POLR3A, ENTPD5, PNP,
                                             CAD, POLR21, CANT1
                     Assembly of RNA       TAF11, GTF2B, null,
                       polymerase II         POLR2A, TAF1, POLR2D,
                       complex               TAF5, null, TAF3,
                                             TAF10, ERCC2, POLR21
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Author:Wang, Xiaodan; Wang, Shaolin; Li, Chao; Chen, Ke; Qin, Jian G.; Chen, Liqiao; Li, Erchao
Publication:Journal of Shellfish Research
Article Type:Report
Date:Dec 1, 2015
Words:9673
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