Recently, the farming of Portunus trituberculatus has prospered and its dependence on fresh trash fish and feed has increased. Also, the ammonization of organic compounds by microorganisms in the water has increased the likelihood of exposure to high concentration of ammonia-N (Ren et al. 2015). Among water deterioration factors, a high concentration of ammonia-N is the most common environmental limiting factor for crustaceans. Ammonia-N is normally present in water in the ionized (NH4+) and unionized (NH3) states. The relationship between the concentrations of NH3 and NH4+ may be estimated by the Henderson-Hasselbalch equation: pH=pK+log [NH3]/[NH4+]. Physiological solutions act as weak bases (pK of 9.3-9.4), which are mainly present in the protonated form, NH4+. However, NH3 has higher lipid solubility, which makes it easier to diffuse through the phospholipid bilayers (Cameron and Heisler 1983). Ammonia-N has been reported to cause a series of physiological reactions in crustaceans, including mainly behavioral reactions (Zimmer and Wood 2017), antioxidant response (Liang et al. 2016; Pinto et al. 2016), immune stress (Yue et al. 2010), ion regulation (Romano and Zeng 2007), and ammonia excretion process (Wang et al. 2003). Many studies have suggested also that a high concentration of ammonia-N can influence physiological response processes, such as moulting, growth and reproduction, and even lead to death (Chen and Kou 1992; Dutra et al. 2016). Therefore, ammonia-N is now being increasingly considered as a main threat to crustaceans. However, there are only a few studies on the transcriptome level under ammonia-N stress.
A high concentration of ammonia-N has been reported to severely damage the hepatopancreas of crustaceans and even induce the apoptosis of hepatopancreatic cells (Liang et al. 2016). In addition to performing the function of the digestive gland, the hepatopancreas of crustaceans is known as a metabolic factory, which is the center of lipid and carbohydrate metabolism. Furthermore, it exerts an important role in ammonia detoxification, including the conversion of ammonia to urea and uric acid through the ornithine-urea cycle (OUC) (Chen and Chen 1997) and purine nucleotide anabolism (Bernasconi and Uglow 2011). Moreover, amino acid metabolism is carried out and glutamine (Gln) is synthesized mainly through the combined action of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) (Pan et al. 2018). Moreover, the hepatopancreas may serve also as a site for the consumption and storage of organic substances to support a variety of important life activities, such as moulting (Gaxiola et al. 2005), vitellogenin synthesis (Tseng et al. 2001) and ovarian maturation (Chen et al. 1998). The hepatopancreas is one of the immune organs that may serve as a major site for the synthesis and secretion of immune molecules, such as antibacterial peptide (AMP) (Ried et al. 1996), beta-1, 3-glucan binding protein (LGBP) (Roux et al. 2002), and lectin or lectin-related proteins (Gross et al. 2001). However, the underlying mechanism of the hepatopancreas in crabs on ammonia-N stress is still unclear and many more genes may be involved in this metabolic process, which needs to be explored. Therefore, large-scale identification of functional genes from hepatopancreas tissue is of great significance and is a necessary condition for studying the metabolic mechanism of crabs.
Next-generation sequencing technologies of 454 Life Sciences, Applied Biosystems (SOLiD sequencing), and Illumina companies have been expertly used to explore the transcriptome information in organisms. Compared with traditional sequencing technologies, next-generation sequencing technologies can provide a large amount of sequence data with a wider range and depth by spending less time and cost (Huse et al. 2007). Recently, digital gene expression (DGE) analysis has become an effective and convenient method for monitoring differences in the transcriptomic response of tissues or organs under environmental stress in aquatic crustaceans, such as P. trituberculatus (Lv et al. 2013), Eriocheir sinensis (Li et al. 2013a; Sun et al. 2014), Sinopotamon henanense (Sun et al. 2016), Macrobrachium rosenbergii (Rao et al. 2015), Procambarus clarkii (Shen et al. 2014), Litopenaeus vannamei (Guo et al. 2016) and Fenneropenaeus chinensis (Li et al. 2013b). Lu et al. (2016) performed a comparative transcriptome analysis of the hepatopancreas between controls and an ammonia-treated group of L. vannamei. 136 significantly differentially expressed genes were detected, of which 94 genes were related to the immune response and other genes were related to growth, apoptosis, moulting and osmoregulation. A study by our group has demonstrated that the response to elevated ambient ammonia-N in the gills involved a variety of physiological and metabolic pathways, mainly involving nucleobase metabolism and amino acid metabolism of P. trituberculatus (Ren and Pan 2014). These studies have considerably enriched our knowledge of the genetics and genomics of crustaceans. However, the differences in transcriptomic response in the hepatopancreas of P. trituberculatus exposed to ammonia-N have not been studied.
The typical benthic swimming crab P. trituberculatus (Crustacea: Decapoda: Brachyura) is one of the most popular aquatic products and is widely cultivated in China due to its rapid growth and high protein content (Ren et al. 2015). In the course of aquacultural practices, ammonia-N concentration may accumulate over time due to the excrement from the cultured animals and decomposition of nitrogenous organic compounds. The present study utilized a high-throughput sequencing technology to analyze transcriptome data obtained from the hepatopancreas of P. trituberculatus experimentally exposed to elevated ambient ammonia-N. The purpose of this study was to discover and investigate the complex molecular responses of P. trituberculatus under ammonia-N stress. The sequencing results are helpful for understanding the physiological functions of the hepatopancreas and provide a foundation for further study of P. trituberculatus (Table 1).
Annotation Gene ID Primer sequences (5′-3′) Myosin-VIIa-like (MYO7a) CL3307.Contig2_sanyousuozixie F:CAGGCGTGTATGTTGTGGAT
C type lectin-containing domain protein (CTLD) CL2684.Contig1_sanyousuozixie F:ACGACTGTGACCGTAACCT
Serine protease CL1409.Contig2_sanyousuozixie F:GATTCCCATCAGCCAACTC
Glutathione peroxidase (GPx) Unigene26426_sanyousuozixie F:TTGATTTGCTCGGGACAC
Vitellogenin Unigene42462_sanyousuozixie F:GCAGGCAAGAGATTGACAG
Trypsin Unigene38004_sanyousuozixie F:ACTGTGCCTGCTCATCGT
Carboxypeptidase B (CBP B) Unigene37912_sanyousuozixie F:TCGCTCGGACACCAACTCT
Ecdysteroid-regulated-like protein (ERLP) Unigene19530_sanyousuozixie F:GGCGACAGTTTACCAGGATT
Ribosomal protein L8 (RPL8) Unigene25025_sanyousuozixie F:GCGTACCACAAGTATCGCGT
Table 1. Primer sequences for amplification of target and reference genes selected from DGE
DGE analysis was performed on the hepatopancreas of P. trituberculatus in controls (C1, C2, C3) and the 5 mg/L NH4Cl-exposed group (A1, A2, A3) at 48 h. Over 14 Mio. and 15 Mio. raw reads were generated for control and experiment group libraries, respectively. After trimming the reads including ploy-N, adapter and low-quality reads, a total of 29.5 Mio. clean reads (98.61%) were obtained. Among these clean reads, the percentages of sequences that could be mapped to unigenes in the two libraries were 89.10% and 88.96%, respectively, and the error rates of base sequencing were both 0.03% (Table 2). These sequences were used for subsequent analysis.
Summary Control group Experiment group Raw data 14, 703, 023 15, 221, 253 Clean data 14, 490, 813 15, 016, 334 Error rate (%) 0.03 0.03 Q20 (%) 97.27 97.29 Q30 (%) 91.86 91.83 GC content (%) 50.57 50.67 Total mapped 12, 911, 222 (89.10%) 13, 359, 214 (88.96%)
Table 2. Summary of the DGE data collected from the hepatopancreas of Portunus trituberculatus in response to ambient ammonia-N
All tag sequences from six libraries (C1, C2, C3 and A1, A2, A3) were mapped to the P. trituberculatus transcriptome library (SRP018007), which contains 70, 569 unigenes. Reads per kilobase of exon model per million mapped reads (RPKM) was used to assess the relative gene expression levels. The percentage of genes in different RPKM intervals was shown in Fig. 1. Differential expression genes (DEGs) were identified by the DGE method. In this study, a total of 52, 280 high-quality unigenes with an average length of 678 bp were obtained in the control group and treated group and 60 genes were differentially expressed between the two experimental groups. Under ammonia-N stress, 30 of these transcripts were up-regulated and 30 were down-regulated. The top six up- and down-regulated annotated transcripts in ammonia-treated crabs were listed in Table 3. The most up-regulated transcript in ammonia-treated crabs encode a glycoprotein. The most down-regulated transcript in exposed crabs encode a zinc finger protein. Three down-regulated transcripts encode vitellogenin, which is closely related to reproduction.
Gene ID log2 ratio (48 h/0 h) Annotation The top six most up-regulated transcripts Unigene30473_sanyousuozixie 6.2404 Glycoprotein CL3307.Contig2_sanyousuozixie 5.6723 Myosin-VIIa-like CL5816.Contig1_sanyousuozixie 5.3208 Haloalkane dehalogenase CL2540.Contig1_sanyousuozixie 5.2405 Ero1-like protein CL1409.Contig2_sanyousuozixie 5.1946 Serine protease CL696.Contig9_sanyousuozixie 5.1877 β-chain spectrin-like protein The top six most down-regulated transcripts CL5171.Contig1_sanyousuozixie − 5.3263 Zinc finger protein 227-like CL6071.Contig2_sanyousuozixie − 5.0688 Protein sel-1 homolog 1-like Unigene42462_sanyousuozixie − 3.2802 Vitellogenin Unigene42751_sanyousuozixie − 3.1026 Chitinase 3 Unigene42464_sanyousuozixie − 3.1014 Vitellogenin Unigene42463_sanyousuozixie − 3.0369 Vitellogenin
Table 3. Top six most up-regulated annotated transcripts and down-regulated annotated transcripts between the exposed and unexposed libraries
Genes with altered expression covered a variety of physiological metabolic and regulatory processes. In the basis of sequence homology, 14, 368 sequences of 52, 280 unigenes (27.48%) were classified into 709 subcategories, including 541 biological process terms (76.30%), 100 molecular functions terms (14.10%) and 68 cellular component terms (9.60%). Figure 2 showed that ammonia-responsive genes participated mainly in the nucleobase metabolic process (GO:0019859/GO:0006206/GO:0072527/GO:0009112) and the amino acid metabolic process (GO:0006573/GO:0009081/GO:1901605) according to biological process, oxidoreductase activity (GO:0016620/GO:0016903), methylmalonate-semialdehyde dehydrogenase activity (GO:0004491), and malonate-semialdehyde dehydrogenase activity (GO:0018478) according to molecular functions. For the cellular component terms, no GO term was enriched significantly.
Figure 2. Functional categorization of DEGs in response to environmental ammonia-N in the hepatopancreas of P. trituberculatus-based gene ontology distribution. X-axis represents the number of differential genes of each GO subcategory. GO subcategories are on the Y-axis. "Asterisk" means significantly enriched GO subcategories
Besides GO analysis, the differentially gene expression caused by elevated ammonia-N affected a range of KEGG pathways. The number of sequences annotated in KEGG pathways ranged from 10 to 2446. The differentially expressed genes were mapped to 45 pathways in the KEGG database and the 20 most enriched KEGG pathways are presented in Fig. 3. The results showed significant enrichment of four KEGG pathways (corrected P value < 0.05). They were beta-alanine metabolism (ko00410, P value=0.00598), propanoate metabolism (ko00640, P value=0.00598), valine, leucine and isoleucine degradation (ko00280, P value=0.00790) and lysosome (ko04142, P value=0.04050).
Eight genes related to the microtubules and vesicle transport, immune system, antioxidant system, reproduction, digestion and moulting were selected to further detect the relative mRNA expression levels by qPCR. These date would verify the gene expression levels identified by DGE. Analysis of the melting curve of qPCR demonstrated that all test genes had a single product. The results showed that the expressions of the MYO7a (involved in microtubules and vesicle transport), CTLD, serine protease (involved in immune response), GPx (involved in antioxidant response), vitellogenin (involved in reproduction), trypsin, CBP B (involved in digestion), ERLP (involved in moulting) using qPCR were consistent with the DGE pattern under ammonia-N exposure (Fig. 4). In theory, the close correlation between qPCR and DGE provides a powerful reference for the quantitative accuracy of DGE method.
Figure 4. Comparison of relative fold change of DGE and qPCR results between the 0 and 5 mg/L NH4Cl groups in the hepatopancreas of P. trituberculatus. The transcript expression levels of the selected genes were each normalized to that of the ribosomal protein L8 (RPL8) gene. MYO7a Myosin-VIIa-like, CTLD C type lectin-containing domain protein, GPx glutathione peroxidase, CBP B carboxypeptidase B, ERLP ecdysteroid-regulated-like protein