Gene 534 (2014) 298–306

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Identification of differentially expressed genes in hepatopancreas of oriental river prawn, Macrobrachium nipponense exposed to environmental hypoxia Shengming Sun a,1, Fujun Xuan b,1, Xianping Ge a,⁎, Hongtuo Fu a,⁎, Jian Zhu a, Shiyong Zhang c a Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China b Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers University, Yancheng 224051, PR China c Wuxi Fishery College, Nanjing Agricultural University, Wuxi 214081, PR China

a r t i c l e

i n f o

Article history: Accepted 15 October 2013 Available online 26 October 2013 Keywords: Macrobrachium nipponense Transcriptome Hemocyanin Hypoxia Hepatopancreas

a b s t r a c t Hypoxia represents a major physiological challenge for prawn culture, and the hepatopancreas plays an important role in these processes. Here, we applied high-throughput sequencing technology to detect the gene expression profile of the hepatopancreas in Macrobrachium nipponense in response to hypoxia for 3 h and hypoxia for 24 h. Gene expression profiling identified 1925 genes that were significantly up- or down-regulated by dissolved oxygen availability. Functional categorization of the differentially expressed genes revealed that oxygen transport, electron transport chain, reactive oxygen species generation/scavenging, and immune response were the differentially regulated processes occurring during environmental hypoxia. Finally, quantitative real-time polymerase chain reaction using six genes independently verified the tag-mapped results. Immunohistochemistry analysis revealed, for the first time, hemocyanin protein expression as significant hypoxia-specific signature in prawns, which opens the way for in depth molecular studies of hypoxia exposure. The analysis of changes in hepatic gene expression in oriental river prawn provides a preliminary basis for a better understanding of the molecular response to hypoxia exposures. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction Among many environmental factors, dissolved oxygen concentration is the major variable limiting water quality for the intensification of prawn aquaculture (Cheng et al., 2003), particularly in rearing ponds that do not use aerators in the hot summer weather. Hypoxic conditions cause stress and inhibit the optimal development of

Abbreviations: PCR, Polymerase chain reaction; RNA, Ribo nucleic acid; mRNA, Messenger ribo nucleic acid; cDNA, DNA complementary to RNA; EST, Expressed sequence tag; NCBI, National Center for Biotechnology Information; GO, Gene Ontology; RNA-Seq, High-throughput RNA-sequencing; TPM, Transcripts per million; FDR, False discovery rate; FPKM, Fragments Per Kilobase of transcript per Million mapped reads; DEGs, Differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; COX I, Cytochrome oxidase subunit I; CCO I, Cytochrome c oxidase subunit I; Cu/Zn-SOD, Copper/zinc superoxide dismutase; HSP70, Heat shock protein 70; GST, Glutathione-Stransferase; α2M, Alpha-2-macroglobulin; qRT-PCR, Quantitative real-time polymerase chain reaction; HIFs, Hypoxia-inducible factors; ATP, Adenosine triphosphate; NADH, Nicotinamide adenine dinucleotide plus hydrogen; ROS, Reactive oxygen species; DO, Dissolved oxygen; L, Light; D, Dark; HE, Hematoxylin and eosin; OD, Optical density; DAB, Diaminobenzidine; PBS, Phosphate-buffered saline. ⁎ Corresponding authors at: Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China. Tel./fax: +86 510 85557892. E-mail addresses: [email protected] (X. Ge), [email protected] (H. Fu). 1 These authors contributed equally to the work.

crustaceans, resulting in reduced frequency of molts, metabolic changes, avoidance behavior, slow growth and even death (Allan and Maguire, 1991; de Oliveira et al., 2005; Mangum, 1997; McMahon, 2001; Racotta et al., 2002; Wu, 2002). Although these responses are valuable indicators of low oxygen conditions, they are not effective as biomarkers of hypoxia because they involve in situ measurements of the organisms. The hepatopancreas of crustaceans has long been thought to function not only as a site for secretion of digestive enzymes, but also as a center for lipid and carbohydrate metabolism (Hill et al., 1991a,b). During exposure to hypoxia, some aquatic organisms rely on physiological mechanisms to extract as much oxygen as possible from the water and transport it to tissues, switch to anaerobic metabolic pathways to supply energy or both, to adapt to fluctuations of dissolved oxygen (Harper and Reiber, 2006; Qiu, 2011; Rosas et al., 1998, 1999). Alternative routes of anaerobic carbohydrate catabolism are less efficient in producing ATP and do not provide enough energy to maintain aerobic consumption, thus, the responses of crustaceans to hypoxia can lead to behavioral, physiological, cellular and molecular changes depending on the duration and severity of hypoxia, include behavioral response for some mobile species (Craig et al., 2005), growth and reproduction (Brown-Peterson et al., 2008; Ocampo et al., 2000), metabolism and immune response (McMahon, 2001; Paschke et al., 2009; Qiu, 2011), and molecular responses of differentially expressed genes in crustaceans

0378-1119/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.036

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(Brown-Peterson et al., 2008; Li and Brouwer, 2009, 2013). However, the molecular basis of these adaptations in Macrobrachium species has so far not been extensively investigated. The oriental river prawn Macrobrachium nipponense, a member of the Palaemonidae family of decapod crustaceans, is widely distributed in freshwater and low-salinity regions of estuaries (Ma et al., 2011), which are especially prone to eutrophication, pollution and hypoxia in China. This species is particularly sensitive to oxygen limitation (Li et al., 2004), and therefore, was considered as a good subject for ecotoxicological studies. We hypothesized that M. nipponense exposed to hypoxia will show different and possibly more complicated and dynamic changes in gene expression than previous studies in other crustaceans. Previous research in M. nipponense has focused on the effects of hypoxia on respiratory metabolism (Guan et al., 2010). There is growing evidence that changes in molecular indicators such as energy production, antioxidant enzymes and oxygen-carrying proteins can effectively imply chronic and acute hypoxia exposure in invertebrates (Brouwer et al., 2004, 2007; Choi et al., 2000), but changes in the gene expression profile of M. nipponense in response to environmental hypoxia remain unknown. As a result of this research interest, large-scale expressed sequence tag (EST) libraries of the M. nipponense have been sequenced (Ma et al., 2012; Qiao et al., 2012; Wu et al., 2009), which will allow more comprehensive molecular studies. Recently, high-throughput RNA-sequencing (RNA-seq) has emerged as a strategy for analyzing the functional complexity of transcriptomes (Marioni et al., 2008; Wang et al., 2009). Compared with conventional transcriptome analysis approaches, RNA-seq has a major part to play in deciphering the complexity of the transcriptome by speeding up the identification of isoforms, allelic expression, untranslated regions, splice junctions, antisense regulation and intragenic expression (Mane et al., 2009; Pan et al., 2008; Trapnell et al., 2010). Furthermore, unlike hybridizationbased approaches, RNA-seq is not limited to detecting transcripts that correspond to existing genomic sequence (Mortazavi et al., 2008), which permits the analysis of organisms that lack genomic information. In the present study, we used M. nipponense juveniles to examine the effects of environmental dissolved oxygen levels on gene expression in vitro using high-throughput sequencing technology. The study aimed to compare the expression data of the different libraries and, in addition to gene identification and functional annotation, to develop a better understanding of the genetic-level responses of M. nipponense when exposed to hypoxia.

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library ranged from 21.3 to 24.6 million. Among the clean reads, the number of sequences that could be mapped to unigenes ranged from 11.7 to 14.9 million, and the percentage of these clean reads ranged from 54.72 to 63.36% in the different libraries (Table 1). We concluded that these libraries were fully saturated and were large enough for gene expression analysis. 2.2. Differentially expressed genes and clustering To map reads to known genes, a reference M. nipponense unigene dataset containing 42,551 contigs and 38,860 singletons was used. The libraries were relatively uniform with respect to mapping efficiency. Among the 29,925 and 28,642 unique genes detected from hypoxia 3 h vs. normoxia 3 h and hypoxia 24 h vs. normoxia 24 h samples, the differentially expressed genes (DEGs) in each of the two comparisons were identified and uploaded to the website www.ffrc.cn/gene/list. asp. To avoid the possibility of noise signals from high-throughput sequencing, genes with an average TPM (transcripts per million) of less than 1 in these two comparisons were excluded. In this study, the absolute log2fold change ≥2 and a false discovery rate (FDR) b0.001 were used to define DEGs. According to this analysis, a total of 1925 genes were detected in at least one of the two comparisons in M. nipponense (Fig. 1), and there were 1139 and 1273 genes specifically regulated by hypoxia 3 h and hypoxia 24 h, respectively (Table A.1 and Table A.2). The magnitude distribution of the significantly changed genes was illustrated by MA plot analysis (Fig A.1). 2.3. Biological processes and pathways regulated by hypoxia Genes with altered expression responses spanned a wide variety of regulatory and metabolic processes. Approximately 39.5% of the differentially expressed genes were assigned to one or more of the three categories: biological process, cellular component, and molecular function (Fig. 2). The categories “metabolic process”, “binding”, “cellular process”, “catalytic activity”, “cell part”, and “response to stimulus” represented most of these genes. The differentially expressed genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic and regulatory pathways. Significant differential expression caused only by hypoxia affected a wide range of KEGG pathways, including the lysosome, focal adhesion, amoebiasis, peroxisome, metabolism of xenobiotics by cytochrome P450 and Glycolysis/ Gluconeogenesis (Fig A.2).

2. Results 2.4. Genes associated with major biological process were affected by hypoxia

2.1. RNA-seq of the transcriptome An immediate application of our transcriptome sequence data included expression profiling of groups held in different dissolved oxygen treatments. cDNA libraries were sequenced from the hepatopancreas of M. nipponense that had been subjected to normoxia for 3 h, hypoxia for 3 h, normoxia for 24 h and hypoxia for 24 h. The average number of reads produced for each treatment groups was approximately 25 million (Table 1). After filtering, the number of clean reads per Table 1 Summary of the RNA-seq data collected from hepatopancreas of Macrobrachium nipponense in response to environmental hypoxia. Category

Normoxia 3h

Hypoxia 3h

Normoxia 24 h

Hypoxia 24 h

Total sequence collected Low quality reads (%)

26,472,741 2,149,167 (8.12%) 24,323,574 (91.88%) 13,587,148 (55.86%)

23,175,718 1,865,777 (8.05%) 21,309,941 (91.95%) 11,660,800 (54.72%)

26,765,423 2,205,186 (8.24%) 24,560,237 (91.76) 14,145,563 (57.60%)

25,590,728 2,115,723 (8.27%) 23,457,005 (91.73%) 14,862,358 (63.36%)

Clean reads (%) All tag mapping to gene (%)

Partial DEGs involved in major processes associated with prawn hypoxia based on Gene Ontology categorization are listed in Table 2. Suppression of cytochrome oxidase subunit I (COX I) and cytochrome c oxidase subunit I (CCO I) mRNA expressions was observed during hypoxia 3 h and 24 h, and hypoxia-induced transcription of some glycolytic genes, such as 6-phosphofructokinase-like isoform 1, pyruvate kinase and alcohol dehydrogenase. Consistent with these changes in transcript levels, the lactic acid concentrations in the hemolymph also significantly increased during hypoxic stress, suggesting that anaerobic fermentative pathways replace aerobic ATP-producing mechanism (Fig. 3). The expression of hemocyanin decreased at hypoxia 3 h and then increasing at hypoxia 24 h. Some of the differentially expressed genes were associated with antioxidant ability and stress response. A gene encoding extracellular copper/zinc superoxide dismutase (Cu/Zn-SOD) showed an increase in expression in response to hypoxia 3 h, but the expression of Cu/Zn-SOD was not significantly affected by hypoxia 24 h. The increased expression of heat shock protein 70 (HSP70), peroxiredoxin and glutathione-S-transferase (GST) in hepatopancreas upon exposure to hypoxia 3 h and 24 h was also observed in prawns exposed hypoxia. In addition, immune response genes encoding alpha-2-macroglobulin

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Fig. 1. Venn diagram showing 1925 differential expression genes (DEGs). DEGs were filtered using a threshold of false discovery rate (FDR) ≤0.001 and an absolute log2fold change N2.

Fig. 2. Functional categorization of DEGs in response to environmental hypoxia in hepatopancreas of Macrobrachium nipponense based Gene Ontology distribution. GO-slim terms are on the y-axis. Percentage distribution of genes shown as GO terms for biological process, cellular components and molecular function.

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Table 2 List of fourteen DEGsa involved in major processes associated with prawn hypoxia based on Gene Ontology categorization. Go term

Gene identifier

Gene description

N3h vs. H3h log2 FCb

N24h vs. H24h log2 FCb

Oxidation–reduction process

Isogroup00421 HD75PE301BRU5M HD75PE302GS1VH Isogroup02452 Isogroup06858 Isogroup09357 Isogroup01128

Hemocyanin subunit Cytochrome oxidase subunit I Cytochrome c oxidase subunit I 6-Phosphofructokinase-like isoform 1 Pyruvate kinase Alcohol dehydrogenase Copper/zinc superoxide dismutase

−4.12 – −11.48 2.80 2.68 2.33 5.51

3.49 −11.59 −4.40 – 3.05 4.15 –

HD75PE301BDVSG Isogroup08368 Isogroup25249 Isogroup02265 Isogroup02887 Isogroup07342 Isogroup16157

Peroxiredoxin Glutathione-S-transferase Heat shock protein 70 Alpha-2-macroglobulin Plus agglutinin C-type lectin 4 Crustin

2.42 2.33 4.38 −4.51 −6.64 −7.26 −7.06

4.31 2.37 4.68 −2.40 −6.36 −4.27 −6.79

Glycolysis

Oxygen and reactive oxygen species

Response to stress Immune response

a b

DEGs were filtered using a threshold of false discovery rate (FDR) ≤0.001 and an absolute value of log2 ratio ≥2. Fold-change for genes higher or lower expressed in prawn in response to hypoxia. N3h, normoxia 3 h; H3h, hypoxia 3 h; N24h, normoxia 24 h; H24, hypoxia 24 h.

(α2M), plus agglutinin, C-type lectin 4 and crustin, were found to be downregulated in response to hypoxia (Table 2). 2.5. Verification of DEGs in response to hypoxia To confirm the results of differentially expressed genes in M. nipponense, six genes were selected for qRT-PCR analysis over the time-course of the dissolved oxygen treatment. RNA sampled from the hepatopancreases of M. nipponense from the different treatment groups provided the template for qRT-PCR validation of the sequencebased transcription profiles of six of the differentially expressed genes (Fig. 4). Representative genes associated with hypoxic stress for the analysis were those involved in the antioxidant system, respiratory proteins, and mitochondrial electron transport. The expressions of the six genes (hemocyanin, COX I, CCO I, Cu/Zn-SOD, peroxiredoxin, GST) by using qRT-PCR agreed well with the RNA-seq analysis pattern. 2.6. Localization of hemocyanin in the hepatopancreas from M. nipponense The hemocyanins of M. nipponense resulted from aggregation of ~75 kDa subunits as other crustaceans (Fig. 5A). Following continuous serial transverse sections, we also found that the structure of hepatopancreas consisted of numerous blind ending tubules and each tubule consisted of four main cell types: E-cells, R-cells, F-cells and B-cells (Fig. 5B). However, since our target was to test the genetic results from the transcriptional level, here we analyzed the hemocyanin

variations in each section of hepatopancreas in response to hypoxia entirely and without separating the distinct cell types (Figs. 5C–E). With the durative hypoxia, the highest intensity of hemocyanins was found in the group treated with hypoxia for 24 h compared with the normoxic group (P b 0.001), but weak signals were found in groups treated with hypoxia for 3 h compared with the normoxic group (P b 0.001, Fig. 5G). Thus, our immunohistochemical results were also consistent with the above gene expression analysis. Few labeled cells were observed when antibodies to hemocyanin were replaced with normal rabbit IgG as the primary antibody (Fig. 5F). 3. Discussion 3.1. Differential gene expression profiling between hypoxic and normoxic groups To better understand how organisms adapt to such dynamic changes in oxygen levels at the molecular level, a global analysis of transcriptome could facilitate the identification of systemic gene expression and regulatory mechanisms for the hypoxia tolerance of a prawn. In the present study, a transcriptome profiling of hepatopancreas was performed to identify genes that are differentially expressed in response to hypoxia. A sequencing depth of 23.2–26.8 million reads per library was reached (Table 1), and more than 36% of the distinct clean reads could not be mapped to the transcriptome reference database. The occurrence of unknown reads was most likely due to the lack of genome sequences, many reads matching noncoding RNAs and the usage of alternative polyadenylation and/or splicing sites (Mattick, 2009; Pan et al., 2008). Despite the facts that prawns physiology was influenced and gene expression was altered by hypoxia, we did not detect significant induction of HIF-1α genes. Similarly, early studies using custom cDNA macroarrays also showed that there was no significant difference among the expression levels of HIF-1α in grass shrimp (Palaemonetes pugio) under normoxia, and severe (DO 1.5 mg/L) and moderate (DO 2.5 mg/L) chronic hypoxia (Li and Brouwer, 2007). Since HIF exerts its action at the level of protein and not at mRNA expression level, the absence of HIF among the DEG is not surprising, in addition, HIF-1α protein and mRNA levels would decrease back to normal over the hypoxic course of several hours, which may be related to mRNA stability (Stroka et al., 2001). 3.2. Energy metabolism was affected by hypoxia

Fig. 3. The lactic acid concentration in hemolymphs of Macrobrachium nipponense when exposed to environmental hypoxia. All values are presented as the mean ± SE (n = 3). Error bars indicate standard error and asterisks indicate significant difference (P b 0.05; t-test).

A clear and striking result in this study was that mitochondria were identified as the most strongly suppressed organelles by hypoxia, M. nipponense's sensitivity to dissolved oxygen may be due to the fact that inhibition of complex III of the mitochondrial respiratory chain

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Fig. 4. qRT-PCR validation of DGE results (A — hypoxia 3 h vs. normoxia 3 h; B — hypoxia 24 h vs. normoxia 24 h). 4 up-regulated genes and 2 down-regulated genes have been identified by qRT-PCR, including hemocyanin, COX I, CCO I, Cu/Zn-SOD, peroxiredoxin, and GST.

leads to blockage of HIF-1 DNA binding activity and expression of HIF-1 target genes under hypoxic conditions (Chandel et al., 1998). Two differentially expressed genes encoding COX I and CCO I, both associated with mitochondria-related processes (coupling electron transfer) were downregulated in M. nipponense in response to hypoxia for 3 h and 24 h, suggesting that M. nipponense mitochondria were less efficient at electron transport during anaerobic respiration. Under hypoxic conditions, dissolved oxygen limits oxidative phosphorylation, and prawn cells depend on alternative metabolic pathways to produce ATP, which is not surprising since the major adaptive changes associated with hypoxia are due to enzyme-mediated alterations in the mitochondrial electron transport chain (Colleoni et al., 2013; Heerlein et al., 2005; Vijayasarathy et al., 2003). In the present study, gene expressions involved in glycolysis and fermentation increased quickly between hypoxia 3 and 24 h. This finding indicates that the major energy source is the glycolytic pathway, which produces two ATP and two pyruvate molecules per unit of hexose while concomitantly reducing NAD+ to NADH. 3.3. ROS generation/scavenging and immunity-related genes were affected by hypoxia Hypoxia increases ROS via the transfer of electrons from ubisemiquinone to molecular oxygen at the Qo site of complex III of the mitochondrial electron transport chain (Bell et al., 2007). In crustaceans, hypoxia and subsequent reoxygenation result in the generation of high levels of reactive oxygen species (ROS) (Zenteno-Savín et al., 2006). ROS production promotes oxidation of cellular components and oxidative stress (Halliwell and Gutteridge, 2006; Lushchak et al., 2001; Zenteno-Savín et al., 2006). The hepatopancreas is considered to be more susceptible to oxidative stress during hypoxia because of its high metabolic rate and its role in digestion, storage of reserve material, metabolism of lipids and carbohydrates, and absorption and

detoxification processes (Ruppert et al., 2003). In this study, a statistical analysis of the qRT-PCR results confirmed that there were significant differences in the expressions of Cu/Zn superoxide dismutase (Cu/Zn SOD), peroxiredoxin and glutathione-S-transferase (GST) of hepatopancreas in prawns during fluctuating dissolved oxygen levels. Similar changes in endogenous antioxidants in response to hypoxia and hypoxia-recovery have been reported in tissues of the cultured pacific white shrimp Litopenaeus vannamei (Parrilla-Taylor and ZentenoSavín, 2013), and the estuarine crab Chasmagnatus granulata (de Oliveira et al., 2006). An increase in HSP70 mRNA levels was observed under hypoxic conditions, and this increase could have resulted from cell injuries caused by hypoxia (Scumacher et al., 1996). Interestingly, hypoxia resulted in down-regulation of alpha-2-macroglobulin, plus agglutinin, C-type lectin 4 and crustin of prawns in response to hypoxia, which play an important role in virus resistance in innate immune response (Chaikeeratisak et al., 2012; García et al., 2009; Sun et al., 2007), suggests that the prawns' immune system may be compromised with exposure to hypoxia. An earlier study suggested that hypoxia results in a depression of the generalized innate immune response in grass shrimp Palaemonetes pugio and Penaeus vannamei based on measurements of circulating hemocytes and survival of shrimp exposed to Vibrio (Burnett and Burnett, 2000). Thus, prolonged hypoxia may have population consequences, as individuals that have already downregulated their aerobic metabolism also have decreased immune defenses, which might result in an increase in the prawn population's susceptibility to microbial infections. Namely, the immune system appears to respond to hypoxia, but the particular transcripts involved in the immune system responses vary with the length of exposure to hypoxia. Thus, several genes could potentially be used as molecular indicators of hypoxia in M. nipponense at specific time points. However, the changes in the expressions of these significant genes were too dynamic to serve as biomarkers of hypoxic stress in M. nipponense.

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Fig. 5. Localization of hemocyanins in hepatopancreas in response to hypoxia. (A) The specificity of the hemocyanin polyclonal antibody as determined by western blotting; (B) The histological transverse section of hepatopancreas with hematoxylin–eosin staining. Immunohistochemical detection of hemocyanin accumulation in the hepatopancreas of M. nipponense in response to three dissolved oxygen stages, (C) normoxia 24 h, (D) hypoxia 3 h and (E) hypoxia 24 h. (F) The negative control, the first antibody (anti-hemocyanin antibody) was replaced with normal rabbit IgG. B: B-cells; E: E-cells; F: F-cells; L: lumen; R: R-cells. Bar indicates 100 μm in length. (G) Mean optical density values used for quantification of the expression of hemocyanin in response to above three conditions. All values are presented as the mean ± SE (n = 3). Bars with different letters are significantly different (LSD, P b 0.001).

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3.4. Oxygen transport proteins were affected by hypoxia Studies have revealed that up-regulated gene expression occurs in hemocyanin of Cancer magister in response to hypoxia (Head, 2010), however, there is less evidence of its involvement in the functional adaptation to hypoxia exposure. Structure of the hemocyanin gene fits this mode of synthesis and secretion, as its encoded protein possesses a signal sequence (Sellos et al., 1997). Immunohistochemistry revealed that hemocyanin expression in M. nipponense was moderate, weak, and strong in response to normoxia, hypoxia for 3 h, and hypoxia for 24 h, respectively, which was consistent with the results obtained in the qRT-PCR analysis. The qRT-PCR analysis confirmed that hemocyanin gene expression was upregulated in M. nipponense in response to hypoxia 24 h, which was similar with a previous study in freshwater giant prawn Macrobrachium rosenbergii (de Man), where the hemocyanin concentration in the prawn showed marked elevations after exposure to hypoxic conditions for 24 h (Mauro and Malecha, 1984). During hypoxia, M. nipponense was able to turn on mechanisms that either enhanced the affinity of the oxyhemocyanin or increased the hemocyanin concentration to adapt to hypoxic stress (Bridges, 2001; Burnett, 1997). Further study is needed to clarify the relationship between hypoxia and the oxygen affinity of hemocyanin in this crustacean. In the present study, a significant reduction in hemocyanin expression was recorded in animals exposed to hypoxia for 3 h, but then significantly increased after 24 h of hypoxia, suggesting that the mechanisms involved in hemocyanin synthesis are markedly affected several hours after exposure to low DO levels (2 mg/L), probably first depending on increasing the affinity of the actual oxyhemocyanin to tolerate hypoxia, and then depending on increasing hemocyanin concentration. The lactic acid concentration in the treatment group was significantly elevated compared with the control group after exposure to hypoxia for 3 and 24 h. Similarly, anaerobic responses such as hyperglycemia and increased blood lactate concentration have been reported in lobsters exposed to hypoxia (Ocampo et al., 2003), suggesting that the juvenile M. nipponense would utilized anaerobic respiration as early as 3 h after exposure to hypoxia. In conclusion, we have generated a quantitative gene profile of the hepatopancreas of M. nipponense in response to environmental hypoxia using high throughput sequencing. Dissolved oxygen is mainly regulated in respiratory metabolism, especially in hemocyanin synthesis in the hepatopancreas, and is highly correlated with the energy consuming mechanisms, antioxidant ability and immune response. These quantitative results make a major contribution to understanding the mechanism of M. nipponense's response to environmental hypoxia. 4. Materials and methods 4.1. Experimental prawns Several healthy oriental river prawns with wet weights of 2.86–3.45 g were obtained from Tai Lake in Wuxi, China (120°13′44″ E, 31°28′22″N). All the samples were transferred to the laboratory of the Freshwater Fisheries Research Center and maintained in six 300-liter tanks with aerated freshwater for one week prior to experimentation. During acclimation periods, prawns were fed commercial flake food twice daily, the culture conditions were 22.6 ± 0.5 °C, pH 8.2 ± 0.08, DO 6.5 ± 0.2 mg/L, total ammonia-nitrogen 0.08– 0.09 mg L−1, under a photoperiod of 14L/10D. A control group was maintained under normoxic conditions (6.5 ± 0.2 mg O2 L−1). Hypoxic (DO 2 mg/L) conditions for 3 h and 24 h within the treatment tanks were maintained by bubbling with N2 gas until the desired O2 concentrations were reached; oxygen levels were maintained by adding N2 gas when needed. All exposures were conducted in triplicate for both controls and treatments. The hepatopancreases of six prawns in hypoxia 3 h and hypoxia 24 were removed as pooling samples in each tank, respectively. In parallel, six prawns that were held in air-saturated water (normoxia) compared to time-

matched animals were pooled as each negative control tank. Finally, these samples were immediately frozen in liquid nitrogen and stored at −80 °C until processed. Hemolymph was sampled randomly from control and treatment groups at 3 h and 24 h, after they were placed in the refrigerator at 4 °C for overnight. The samples were then centrifuged at 1000 ×g for 30 min and the supernatant was used for subsequent lactic acid assays. 4.2. Library preparation and sequencing Total RNA was extracted using a TRIzol® Reagent (Invitrogen, USA) according to the protocol of the manufacturer and treated with DNase I. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using a Nanodrop ND-1000 spectrophotometer (LabTech, Washington, DC, USA). RNA integrity was further verified by electrophoresis through a 1.5% (w/v) agarose gel. Three pooled RNA samples were obtained in each group. The RNA samples were used to isolate poly(A) mRNA and to prepare a nondirectional Illumina RNA-Seq library with an mRNASeq Sample Prep Kit (Illumina). The libraries were loaded onto flow cell channels for sequencing using an Illumina Genome Analyzer at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) in 2012. Library quality control and quantification were performed with a Bioanalyzer Chip DNA 1000 series II (Agilent). Each library had an insert size of 200 bp; 42- to 50-bp sequences were generated via an Illumina HiSeq™ 2000 sequencing machine. 4.3. Transcriptome analysis The raw data was filtered to remove low quality sequences, including ambiguous nucleotides and adaptor sequences. To link the expressed signatures to known genes from M. nipponense, the unigene dataset (http://www.ncbi.nlm.nih.gov/sra/?term=SRA051767.2) from the transcriptome of M. nipponense was used as a reference database (Ma et al., 2012). The transcription level of each gene was deduced by determining the total number of reads mapped to each gene using Picard tools (http://picard.sourceforge.net/). To provide a relative assessment of transcript-abundance, the numbers of raw reads that were mapped to individual contigs were normalized for sequence length (i.e., Fragments Per Kilobase of transcript per Million mapped reads, FPKM). DEGs were identified by the DESeq package in the R software (Anders and Huber, 2010), using two-fold change (log2 (fold-change) ≥2 or ≤−2) and p-value b0.05 (cut-off at 5% FDR) as the threshold. After data normalization by the p-value and FDR calculation, the resulting expression intensity values were analyzed by the MA plot-based method, as previously described (Wang et al., 2010). For pathway enrichment analysis, all differentially expressed genes were mapped to the terms in the KEGG database and searched for significantly enriched KEGG terms compared to the whole transcriptome background. Functional enrichment analyses including GO and KEGG were performed using the following ultra-geometric test to identify which DEGs were significantly enriched in GO terms (P ≤ 0.05) and metabolic pathways (q-value ≤ 0.05) compared with the whole transcriptome background (Kanehisa et al., 2006, 2008). 4.4. Real-time quantitative RT-PCR Real-time quantitative RT-PCR (qRT-PCR) was used to validate the expression of a selection of the genes identified as being differentially expressed. The sequences of the primer pairs (designed using Primer Express 3.0) are listed in Table A.3. Real-time quantitative PCR reactions were carried out using the Bio-Rad iCycler iQ5 Real Time System (Biorad Inc., Berkeley, CA, USA) using the β-actin gene as a internal control (Zhang et al., 2013). The PCR temperature profile and PCR reaction condition analyses were performed according to the instructions of the SYBR Premix Ex Taq kit (TaKaRa, Dalian, China). For the negative

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control, DEPC-water replaced the template. A relative standard curve was constructed using a 10-fold serially diluted cDNA. Each sample was run in triplicate along with the internal control gene. To ensure that only one PCR product was amplified and detected, a dissociation curve analysis of the amplification products was performed at the end of each PCR reaction. The relative copy number of gene mRNAs was calculated according to the 2−ΔΔCT comparative CT method (Livak and Schmittgen, 2012).

Acknowledgments

4.5. Immunohistochemistry

References

To better understand the genetic results from the transcriptional level, another three individuals of each condition were used to further test the protein variations in response to hypoxia. Hepatopancreases were dissected out from M. nipponense grown under hypoxic (3 h, 24 h) or normoxic conditions and fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (0.1 M PBS; pH 7.4) for 24 h at room temperature. After dehydration in a series of graded alcohols and clearing in xylene, hepatopancreases were embedded in paraffin, mounted on a microtome and cut into 5 μm transverse sections. The primary antibodies to hemocyanin were ordered and produced in Shanghai Sangon Biotech Co., Ltd. The specificity of this antibody for prawn antigens was confirmed by western blotting. Following normal immunohistochemistrical procedures, the sections were incubated with this affinity-purified polyclonal antibody against hemocyanin (dilution 1:20000 in 0.1 M PBS) overnight at 4 °C and treated with a ChemMate™ EnVision™/HRP complex with diaminobenzidine (DAB) as a substrate (GK500705, Gene Tech). All sections were treated in the same way and the presence of hemocyanin signals was seen as a brown reaction. No or few labeled cells were observed when antibodies to hemocyanin were omitted or replaced with normal rabbit IgG as the primary antibody (provided by Shanghai Sangon Biotech Co., Ltd., dilution 1:20000 in 0.1 M PBS). Some sections were stained with hematoxylin and eosin (HE) to observe and ensure adequate tissue preservation. The images were captured at 200× magnification and digitized by means of a CCD camera adapted to a Leica DMR microscope, linked to a Dell computer and analyzed with the software image pro plus 6.0. For each individual, ten transverse sections ranged from the whole hepatopancreases were quantified to analyze the expression of hemocyanin using mean optical density (OD) values. The background obtained from adjacent sections but in the negative control group (using normal rabbit IgG as the primary antibody) was subtracted from each section. These values from the same condition were then averaged to yield mean OD levels for each condition. Statistical significance was determined by one-way ANOVA. 4.6. Analysis of lactic acid concentrations in M. nipponense Lactic acid concentrations in the hemolymph were determined via a previously described method. The absorbance of the sample was measured at 340 nm on a spectrophotometer (UV-2600) and converted to L-lactate concentrations using a calibration curve constructed with standards of known lactic acid concentrations (Gutmann and Wahlefeld, 1974). The results were expressed in mmol lactate per liter. Three replicates were conducted for each sample. When the overall treatment effect was significantly different, t-test was conducted to compare the means between the dissolved oxygen treatment and control. The level of significant difference was set at P b 0.05. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.10.036. Conflict of interest statement The authors declare they have no conflict of interest.

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We acknowledge Yan Wang and Yi Ren (Shanghai Majorbio Biopharm Biotechnology Co., Ltd.) for their kind help in sequencing and in the bioinformatics analysis. This work was supported by the National “Twelfth Five-Year” Plan for Science & Technology Support (2012 BAD25B07) and the National Natural Science Foundation of China (31272654).

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