Transcriptome analysis of the liver of Eospalax fontanierii under hypoxia

Hypoxia can induce cell damage, inflammation, carcinogenesis, and inhibit liver regeneration in non-adapted species. Because of their excellent hypoxia adaptation features, subterranean rodents have been widely studied to clarify the mechanism of hypoxia adaptation. Eospalax fontanierii, which is a subterranean rodent found in China, can survive for more than 10 h under 4% O2 without observable injury, while Sprague-Dawley rats can survive for less than 6 h under the same conditions. To explore the potential mechanism of hypoxia responses in E. fontanierii, we performed RNA-seq analysis of the liver in E. fontanierii exposed to different oxygen levels (6.5% 6h, 10.5% 44h, and 21%). Based on the bioinformatics analysis, 39,439 unigenes were assembled, and 56.78% unigenes were annotated using public databases (Nr, GO, Swiss-Prot, KEGG, and Pfam). In total, 725 differentially expressed genes (DEGs) were identified in the response to hypoxia; six with important functions were validated by qPCR. Those DEGs were mainly involved in processes related to lipid metabolism, steroid catabolism, glycolysis/gluconeogenesis, and the AMPK and PPAR signaling pathway. By analyzing the expression patterns of important genes related to energy associated metabolism under hypoxia, we found that fatty acid oxidation and gluconeogenesis were increased, while protein synthesis and fatty acid synthesis were decreased. Furthermore, the upregulated expression of specific genes with anti-apoptosis or anti-oxidation functions under hypoxia may contribute to the mechanism by which E. fontanierii tolerates hypoxia. Our results provide an understanding of the response to hypoxia in E. fontanierii, and have potential value for biomedical studies.

approximately 20% of resting total body oxygen consumption (details at: https://aneskey.com/liveranatomy-and-physiology/). Hypoxia induces cell damage, in ammation, carcinogenesis, and inhibits liver regeneration in non-adapted species [20]. Therefore, E. fontanierii liver is a useful model for investigations of the hypoxia tolerance.
In this study, we carried out RNA-seq of E. fontanierii liver to explore shared or unique molecular mechanisms underlying hypoxia adaptation in subterranean rodents. We analyzed the changes in gene expression following exposure to 21%, 10.5%, and 6.5% oxygen, presenting normoxic, chronic hypoxic and acute hypoxic conditions, respectively, and evaluated the molecular adaptations to hypoxia. Our results form the basis of further studies of hypoxia adaptation in E. fontanierii and have potential biomedical applications.

RNA sequencing and de novo transcriptome assembly
In E. fontanierii, we performed RNA sequencing of liver organ, and 258.4 million pair-end raw reads were generated. After removal of adapters, primer sequences, and low-quality reads, 239.2 million pair-end clean reads were retained for further analysis. The guanine and cytosine (GC) contents ranged from 49.73% to 52.48%, and Q30 (99.9% base accuracy) scores were ≥85.00% (Table 1). An average of 26.6 million pair-end reads was generated for each sample. All reads were pooled for the de novo assembly. There were 39,439 unigenes, in which the N50 length was 2,556 nt, and the median length was 1,412 nt.
The lengths of the unigenes ranged from 200~21,949, with the majority distributed in the range of 600~800 nt (Fig. S1A).

Gene function annotation and CDS prediction of unigenes
Several databases, such as GO (Fig S2), Nr, Swiss-Prot, Pfam, and KEGG, were searched for functional annotation of unigenes. In total, 56.78% (22,395) unigenes were annotated at least once in the databases searched (Table 2). Furthermore, 16,754 unigenes ≥1,000 nt in length were annotated by the different databases.
A total of 33,003 CDSs were predicted from 30,893 unigenes, of which 1,888 unigenes possessed two or more CDSs. The mean and N50 lengths of CDSs were 814.9 nt and 1,554 nt, respectively. Many short CDSs were in the range of 200-300 nt in length (Fig. S1B). Among the CDSs identi ed, 58.7% (19,378) were complete. In addition, 46.4% (15,304) and 41.1% (13,561) of the CDSs were annotated by the UniRef90 and Pfam databases (E-value < 1e-5), respectively.
Read-mapping and differentially expressed gene identi cation Based on RNA-Seq data, gene expression levels can be quanti ed by counting reads mapped to transcripts. This process is often in uenced by changes in gene length and sequencing depth, alternative splicing, and gene duplication. RSEM enables accurate transcript quanti cation for species without sequenced genomes. To compare gene expression levels in multiple samples, FPKM values were used to measure the expression levels of unigenes. Overall, expression levels of 87.7% (34,591 of 39,439) of the unigenes were available with FPKM values ≥1 in at least one sample. A majority of the most highly expressed genes in the live are very important for liver functions (Table S2). For example, apolipoproteins (APOE, APOAI, APOA2, and APOC1) are involved in the metabolism of lipoproteins and their uptake in tissues [21]. These genes showed dominant or tissue-speci c expression in the liver, with no signi cant changes in expression under hypoxia, indicating that these proteins facilitate the maintenance of liver function integrity. Hemopexin (HPX) prevents the pro-oxidant and pro-in ammatory effects of heme and also promotes its detoxi cation [22]. Fructose-bisphosphate aldolase B (ALDOB) is essential in fructose metabolism [22]. Glutathione S-transferase A1 (GSTA1) protects the cells from reactive oxygen species and the products of peroxidation [23]. HPX, ALDOB, and GSTA1 under normoxia and hypoxia could protect liver cells from oxidative damage and energy de ciency.
To identify hypoxia-induced DEGs, DESeq2 was used for comparisons between the treatment groups and control groups: 10.5% O 2 vs. 21% O 2 ; 6.5% O 2 vs. 21% O 2 ; 6.5% O 2 vs. 10.5% O 2 , Fig. S3) [24]. A total of 725 unigenes were considered to be DEGs in response to different hypoxia conditions ( Table 3, Table S3). In total, 71.2% (516) of the DEGs were identi ed in the 6.5% O 2 vs. 10.5% O 2 comparison, while only 4.83% of the DEGs (35 genes) were identi ed in the 10.5% O 2 and 21% O 2 comparison, suggesting a weak response to chronic hypoxia. Sixty-three upregulated DEGs and 62 downregulated DEGs were found in both the 6.5% vs. 10.5% and 6.5% vs. 21% comparisons. In all the DEG sets, the numbers of downregulated genes exceeded the number of upregulated genes under hypoxia when compared with the numbe. It can be speculated that reduced transcriptional activities may contribute to the tolerance to hypoxia in E. fontanierii.

GO enrichment of DEGs
To identify the functions of DEGs induced by hypoxic stress, GO terms were assigned and enriched. The GO term annotations of DEGs were mainly related to metabolic process, biological regulation, response to stimulus, catalytic activity, transporter activity, and molecular function regulator ( Fig 1A, Table S4). We found the most enriched GO terms were commonly associated with lipid metabolism (58 genes), heme binding (18 genes), oxygen binding (7 genes), and response to oxygen-containing compounds (48 genes) ( Fig. 1B&C, Fig. S4, Table S5). Many GO terms were associated with energy metabolism, such as fatty acid transporter activity, glucose metabolism, threonine metabolism, and acylglycerol metabolism. Along with the metabolism processes under hypoxia, we also identi ed some GO terms with response functions, such as response to carbohydrate, response to ethanol, response to zinc ion, response to hormone, response to hexose, and response to monosaccarides. We also identi ed GO terms associated with regulation of these metabolic and response processes, including regulation of hormone levels, negative regulation of gluconeogenesis, regulation of insulin secretion, regulation of hormone secretion, regulation of lipid metabolism, and regulation of peptide transport, of which the former one is statistically signi cant and the latter ve are not signi cant. These results showed that hypoxic environments alter metabolism in the liver in E. fontanierii at numerous molecular levels, and the corresponding responses and regulations accommodate the changes in metabolism to maintain liver cell function.

KEGG enrichment of DEGs
To further explore the interactions among DEGs, the pathways containing DEGs were annotated and enriched using the KEGG database. The DEGs were mainly involved in lipid, amino acid, and carbohydrate metabolism, signal transduction, the digestive and immune system, as well as infectious diseases, endocrine and metabolic diseases ( Fig. 2A). As crucial signaling pathways, PPAR and AMPK signaling pathways were found to be enriched among the DEG sets (Fig. 2B, Fig. S5), and both were associated with energy metabolism. In the AMPK signaling pathway, fatty acid oxidation and gluconeogenesis are enhanced under hypoxia stress, while protein synthesis and fatty acid synthesis are repressed (Fig 3A). In the PPAR signaling pathway, fatty acid oxidation, gluconeogenesis, and cholesterol metabolism were enhanced by upregulating the expression levels of related genes under hypoxic stress (Fig. 3B). Other enriched pathways playing key roles in liver functions were also detected, including steroid hormone biosynthesis, cholesterol metabolism, and bile secretion (Fig. 2B). Energy metabolism-associated pathways, such as glycolysis/gluconeogenesis, tyrosine metabolism, and linoleic acid metabolism, and pathways related to the immune system or disease, such as chemical carcinogenesis, antigen-processing and presentations, and metabolism of xenobiotics by cytochrome P450, were also observed ( Fig. 2B). Those pathways enriched for DEGs were associated with basic liver functions, which may contribute to the adaptation of the liver under hypoxic stress. Other important pathways and key genes involved in liver cell survival and functional integrity were explored.
Furthermore, Pten deletion allows nerve regeneration in mice [27]. It can be speculated that Pten downregulation inhibits liver cell apoptosis and promotes cell regeneration, thus contributing to the tolerance to acute hypoxia in E. fontanierii liver.

Glycolysis /gluconeogenesis pathway
The glycolysis-related genes Glycolysis pathway, hexokinase (HK) (4.01 fold-change and padj < 0.05, 2.85 fold-change and padj = 0.15) and glucokinase (GCK) (5.05 fold-change and padj < 0.05, 3.19 fold-change and padj < 0.1) were downregulated under acute hypoxia compared with normoxia and chronic hypoxia, respectively, which impedes the conversion of glucose to glucose-6-phosphate (G6P) under acute hypoxia (Fig. 4B). G6PC encodes glucose-6-phosphatase, which catalyzes the conversion of G6P to a phosphate group and free glucose, which is the last step in gluconeogenesis [31]. In the liver of E. fontanierii, the upregulated expression of G6PC (4.17 fold-change, padj < 0.05) under acute hypoxia compared with chronic hypoxia will facilitate gluconeogenesis (Fig. 4B). These results showed that under hypoxia, glucose consumption in the liver is reduced, while production supply for other tissues, such as the brain, is increased.

Validation of six DEGs
We selected six genes that were found to be upregulated in the liver under hypoxia by RNA-seq for validation by quantitative real-time PCR (qRT-PCR) (Fig. 5A-F). Three of the genes [very long-chain acyl-CoA synthetase (Slc27a2), carnitine O-palmitoyltransferase 1 (Cpt1a), and cholesterol 7-alphamonooxygenase (Cyp7a1)] were included in PPAR pathway, while the other three genes [Metallothionein (MT), C4b-binding protein beta chain (C4bpb), and Hyaluronidase-2 (Hyal2)] were key genes in liver functions (Fig. 3B). The qRT-PCR results were generally consistent with RNA-seq data, which showed that all of genes were signi cantly upregulated under acute hypoxia compared with normoxia or chronic hypoxia. In the qRT-PCR results, MT, Cpt1a, and Cyp7a1 were signi cantly upregulated under acute hypoxia compared with normoxia and chronic hypoxia, simultaneously. Slc27a2, C4bpb, and Hyal2 were signi cantly up-regulated in acute hypoxia and chronic hypoxia compared with normoxia. As these genes are highly important in maintaining liver function, we investigated their functions under hypoxic stress.
Very long-chain acyl-CoA synthetase (Slc27a2), which converts free long-chain fatty acids into fatty acyl-CoA esters, and plays key role in lipid biosynthesis and fatty acid degradation [32]. Carnitine Opalmitoyltransferase 1 (Cpt1a) catalyzes the transfer of the acyl group from CoA to carnitine to form palmitoylcarnitine as the rst and rate-limiting step in the carnitine palmitoyltransferase system. The acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase [33]. Slc27a2 and Cpt1a are crucial for the activation and oxidation of fatty acids. In the E. fontanierii liver transcriptome, Slc27a2 and Cpt1a were upregulated by 2.14-fold (padj < 0.05) and 3.19-fold (padj < 0.05) under acute hypoxia compared with normoxia, respectively. The results suggested that the upregulated expression of Slc27a2 and Cpt1a in the liver of E. fontanierii under acute hypoxia could increase the local energy supply through oxidation of fatty acids, especially when the levels of glucose as the energy supply in the liver were decreased.
Cholesterol 7-alpha-monooxygenase (Cyp7a1), which plays an important role in cholesterol metabolism, converts cholesterol to 7-alpha-hydroxycholesterol, in the rst and rate-limiting step in bile acid synthesis [34]. Our results showed that Cyp7a1 in the liver was upregulated by 3.29-fold (padj < 0.05) under acute hypoxia compared with normoxia, which could contribute to bile acid biosynthesis and regulation of cholesterol levels.
Metallothionein (MT) has been reported to function as a negative regulator of apoptosis [35]. In E. fontanierii liver, MT contains 61 residues, including 20 cysteine residues. As a cysteine-rich protein, MT performs important functions in the control of oxidative stress, by capturing damaging oxidant radicals, such as superoxide, and hydroxyl radicals via the cysteine residues [36]. In our study, MT was upregulated by 3.26-fold (padj < 0.05) and 3.22-fold (padj < 0.1) under acute hypoxia compared with normoxia and chronic hypoxia, respectively. These results indicate that MT protects E. fontanierii liver tissue against the harmful in uences of acute hypoxia.
C4b-binding protein beta chain (C4bpb), which is the main inhibitor of the classical complement activation pathway, accelerates the decay of C3-convertase and hydrolyzes the C4b complement fragment [37]. It also interacts with anticoagulant protein S, and binds apoptotic and necrotic cells as well as DNA to clean up after injury and limit the in ammatory potential of necrotic cells [38,39]. In this study, C4bpb was upregulated by 2.78-fold (padj < 0.05) under acute hypoxia compared with normoxia. The nding indicated that upregulated C4bpb in acute hypoxia plays a role in preventing the in ammation and blood coagulation induced by acute hypoxia. C4bpb negatively regulates complement activation, and its upregulated expression may helps to reduce coagulation under hypoxia, which is consistent with previous study in E. fontanierii heart tissue [18].
Hyaluronidase-2 (Hyal2) is thought to be involved in cell proliferation, migration, and differentiation [40,41]. Various functions have been described for the gene, such as response to reactive oxygen species, positive regulation of in ammatory response, negative regulation of protein kinase, cellular response to tumor necrosis factor, and homeostatic processes [42]. In our study, Hyal2 was upregulated by 2.85-fold (padj < 0.05) and 3.04-fold (padj < 0.05) under acute hypoxia compared with normoxia and chronic hypoxia, respectively, suggesting that upregulated Hyal2 is an important gene in regulating multiple responses to hypoxia.
DEGs with the GO term "response to hypoxia" Apart from the DEGs validated by qRT-PCR, we identi ed some DEGs assigned the GO term "response to hypoxia", which may play key roles in hypoxia adaptation in subterranean animals and have important additional functions. Compared with normoxic conditions, cystathionine beta-synthase (Cbs) and heme oxygenase 1 (Hmox1) were upregulated by 2.11-fold (padj < 0.05) and 3.29-fold (padj < 0.05) under acute hypoxia, respectively. Cbs catalyzes the rst step in the trans-sulfuration pathway, from homocysteine to cystathionine, which is the precursor of cysteine. In mammals, Cbs is a highly regulated enzyme, which contains a heme cofactor that functions as a redox sensor [43]. In our study, Cbs was assigned to multiple GO terms that may contribute to liver hypoxia tolerance, such as oxygen binding, carbon monoxide binding, cysteine synthase activity, nitrite reductase (NO-forming) activity, selenocystathionine beta-synthase activity, superoxide metabolic process and negative regulation of apoptotic. Hmox1 catabolizes free heme, produces carbon monoxide (CO), and induces the upregulation of interleukin 10 (IL-10) and interleukin 1 receptor antagonist (IL-1RA), which form the basis of its anti-in ammatory properties [44]. Certain important GO terms associated with hypoxia adaptation, such as liver regeneration, negative regulation of extrinsic apoptotic signaling pathway via death domain receptors, positive regulation of angiogenesis, erythrocyte homeostasis, regulation of blood pressure and cellular iron ion homeostasis, were assigned to Hmox1. Under hypoxia, hypoxia-inducible factor 1-alpha (HIF-1alpha) is often signi cantly upregulated [45], and is considered to be the master transcriptional regulator of cellular and developmental responses to hypoxia [45,46]. As a component of the HIF signaling pathway, HIF-1 could induce upregulation of Hmox1 expression under acute hypoxia to promote protection of the liver.
Compared with chronic hypoxia, heme oxygenase 2 (Hmox2) and eukaryotic translation initiation factor 4E-binding protein (EIF4EBP1 or 4E-BP1) were upregulated by 2.02-fold (padj < 0.05) and 3.20-fold (padj < 0.05) under acute hypoxia, respectively. As a modi er in the regulation of hemoglobin metabolism, Hmox2 has been reported to contribute to high-altitude adaptation in Tibetans [47]. EIF4EBP1 is a repressor of translation initiation that regulates eIF4E activity by preventing its assembly into the eIF4E complex [48]. In the AMPK signaling pathway, upregulated EIF4EBP1 inhibits protein synthesis and reduces cell activity to save energy (Fig. 3A).

Discussion
E. fontanierii has the ability to tolerate very low oxygen concentration, showing adaptation to the hypoxic environment underground. The extent to which genes remodel their transcriptomes under different oxygen concentrations is not clear in E. fontanierii. By pro ling the transcriptomes in the liver of E. fontanierii, we characterized the DEGs in response to different oxygen concentrations. Most DEGs were identi ed in comparison of the transcriptomes under chronic hypoxia and acute hypoxia, displaying different mechanisms underlying the response to different oxygen concentrations. In comparisons with normoxia, fewer DEGs were identi ed in response to chronic hypoxia than to acute hypoxia, suggesting only minor changes in the E. fontanierii liver transcriptome as the conditions changed to chronic hypoxia from normoxia. This indicates excellent adaptation to chronic hypoxia during the long-term evolution of E. fontanierii in subterranean conditions.
Several genes that were upregulated under acute hypoxia compared with normoxia were enriched in the GO term "negative regulation of apoptosis". This term was not present in the enriched pathways for upregulated DEGs in chronic hypoxia compared with normoxia, which may be due to the lower number of anti-apoptosis genes identi ed as DEGs under chronic hypoxia. These ndings suggest that the anti-apoptotic ability of liver cells is enhanced to survive in the adversity associated with acute hypoxia. The negative regulation of apoptosis as a response to hypoxia in the liver in E. fontanierii identi ed in this study is shared by another subterranean mole-rat Spalax, indicating different subterranean rodents may share a similar strategy for coping with hypoxia [5,49]. Hypoxia increases the generation of mitochondrial reactive oxygen species [50], which lead to a harmful oxidation effects [51]. The upregulated expression of antioxidant genes (MT, Cbs) in E. fontanierii liver under hypoxia may prevent oxidative damage. Cbs serves as a CO-sensitive modulator of H 2 S to support bile excretion and has a putative role in bile-dependent detoxi cation processes [52]. In the enriched pathways "glycine, serine and threonine metabolism" and "cysteine and methionine metabolism", Cbs catalyzes the conversion of homocysteine to cystathionine, which is converted to cysteine by gamma lyase [53]. Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death [54]. Cysteine is the rate-limiting factor in the biosynthesis of glutathione, an amino acid that is relatively rare in foods. Glutathione is one of the major endogenous antioxidants produced by cells and participates directly in the neutralization of free radicals and reactive oxygen compounds [55]. This suggests that Cbs upregulation plays roles in anti-apoptotic and anti-oxidant processes under conditions of acute hypoxia.
In this study, we characterized the E. fontanierii liver transcriptomes and pro led the changes in gene expression in the liver under different oxygen levels. Functional enrichment analysis showed that the main functions (steroid catabolic process, lipid metabolic process, primary bile acid biosynthesis, energy production and amino acid metabolic) of the liver were regulated in response to hypoxia. We identi ed multiple important DEGs underlying the potential molecular adaptation mechanisms to hypoxia, including genes associated with anti-apoptosis, energy supply, anti-in ammation, and anti-oxidation. Our study helps to understand the complexity of hypoxic adaptation in E. fontanierii liver. which was monitored using a JRC-1020 thermo-magnetic analyzer. The animals were anesthetized with an intraperitoneal injection of pentobarbital and sacri ced to collect fresh tissues and frozen into liquid nitrogen immediately. Total RNA was extracted using an RNA Simple Total RNA kit (TaKaRa) according to the instructions. The total RNA integrity was tested through 1% agarose gel electrophoresis. The RNA concentration was veri ed with a NanoDrop-2000 spectrophotometer (Thermo Fisher, USA). The cDNA library preparation was performed according to the standard protocol for Illumina sample preparation. The mRNA was enriched using magnetic beads with oligo(dT), and then random fragments of mRNA were generated by adding fragmentation buffer. The mRNA was reverse transcribed into the rst-strand cDNA using a six-base random primer, and the second-strand cDNA was synthesized by addition of dNTPs, DNA polymerase I, RNase H, and buffer. The cDNA was puri ed with Ampure XP beads to obtain double-stranded cDNA. After end repair, the products were puri ed by AMPure XP beads and were ampli ed by PCR to construct cDNA library. The concentration and insert size of cDNA libraries were evaluated by Qubit2.0 and Agilent 2100, and effective concentration was estimated precisely by qRT-PCR. After complying with the quality control criteria, cDNA libraries were sequenced using the Illumina HiSeq™ 2500 sequencing platform.

Assembly and Annotation
To obtain clean data, raw data containing adapters and primer sequences were removed, and low-quality bases were ltered by cutadapt (version 1.16) [56]. Trinity (version: 2.4) was applied for de novo assembly of Bruijn graphs and full-length transcripts (command: Trinity --seqType fq --left reads.fq --right reads_2.fq --CPU 24 --max_memory 256G) [57]. Unigenes were used to represent one or more transcripts in the same cluster. To display its sequence and avoid redundancy, the sequence of the primary mRNA with the highest expression level or the longest length was regarded as the unigene sequence when the whole genomic sequences were unknown. An isoform was rst identi ed as the unigene sequence with the highest expression (>50% total expression value). If this criterion was not satis ed, the isoform with the longest length was considered to be the unigene sequence. The unigenes with at least 10 read in at least two samples were retained by in-house perl script. Software CD-HIT (version 4.8.1) was used to cluster unigene sequences with a sequence identity threshold of 0.9. RepeatMasker (version open-4.0.7) was used to mask the repeat elements in unigene sequences with nhmmscan as engine (version 3.1b1) and Mus musculus as the query species [58]. RNA sequences from 10 species used for homology searches were downloaded from NCBI RefSeq (Table S6). BLAST (version: 2.6.0+) searches for unigene sequences were performed against Nr (blastx, E-value<1e-3, command: blastall -p blastx -i input -d nr -e 1e-3 -m 7 -v 20 -b 20 -o output) and Swiss-Prot (blastx, E-value < 1e-10) databases. To annotate the transcriptome, blastx searches against the Nr database were performed for all unigenes (E-value < 1e-3). Functional annotation with gene ontology (GO) terms was conducted using Blast2GO software, which is designed for the high-throughput and automatic functional annotation of DNA or protein sequences based on the gene ontology vocabulary. Blast2GO Command Line (version:1.3.2) (downloaded from http://www.blast2go.com/blast2go-pro/blast2go-command-line) used the BLAST output to map and annotate unigenes (E-value < 1e-6, command: Blast2GO_HOME/blast2go_cli.run -properties cli.proploadfasta input.fasta -loadblast blastResult.xml -mapping -annotation -saveb2g -savedat -annex -useobo go-basic.obo) [59]. Unigene sequences were uploaded into KOBAS3.0 to search for functional annotation in the Kyoto Encylopedia of Genes and Genomes (KEGG) [60]. The predicted peptide sequences of unigenes were searched against Pfam (E-value ≤ 1e-5, command: phmmer -E 1e-5 -cpu 8 -pfamtblout output input.pep Pfam-A.hmm) using HMMER (version: 3.1b2) [61]. TransDecoder (version: 3.0.1) [57,62] was used to predict potential con dent coding sequences (CDS, minimum length: 150 nucleotides) in unigenes and corresponding amino acid sequences based on open reading frame, log-likelihood score, and Pfam alignment information (command 1: TransDecoder.LongOrfs -t input.fasta; command 2: TransDecoder.predict -t input.fasta --retain_pfam_hits) [57]. ExTaq. Relative gene expression levels were normalized against that of an internal reference gene (β actin) and calculated using the △△Ct method. Primers were designed using Primer-BLAST on the NCBI website ( Table 4). The expression of each gene was analyzed using three biological replications for each condition. Data were presented as the mean ± standard deviation (SD). SPSS 17.0 statistical software was used for statistical analysis of the data. The the statistical signi cance of the differences between the groups were evaluated using student's t-test. P-values of <0.05 were considered to indicate statistical signi cance.

Accession number
All pair-end read data have been deposited in NCBI Sequence Read Archive (SRA) with BioProject accession: PRJNA497961 (SRA accession: SRR8090385-SRR8090393). This Transcriptome Shotgun Assembly project has been deposited at DDBJ/ENA/GenBank under the accession GHFG00000000. The version described in this paper is the rst version, GHFG01000000.