Integrated analysis of metabolomes and transcriptome between the testis of non-repropductive season and reproductive season of oriental river prawn, Macrobrachium nipponense

Background: The better understanding of male sexual differentiation and development mechanism of M. nipponense is urgently needed, in order to maintain the sustainable development of M. nipponense industry. In the present study, we aimed to identify the key genes and metabolites involved in the male sexual differentiation and development of M. nipponense through performing the integrated analysis of metabolomes and transcriptomes of testis between the reproductive season and non-reproductive season. Results: A total of 385 differentially expressed metabolites (DEMs) and 12,230 differentially expressed genes (DEGs) were identied. Glycerophospholipid metabolism and Sphingolipid metabolism were predicted to have dramatic effects on male sexual differentiation and development in M. nipponense, based on the integrated analysis of metabolomes and transcriptomes. According to the KEGG enrichment analysis of DEGs, Oxidative phosphorylation, Glycolysis/Gluconeogenesis, HIF-1 signaling pathway, Citrate cycle, Steroid hormone synthesis and Spliceosome were predicted to promote the male differentiation and development through providing ATP, promoting the synthesis of steroid hormone and providing correct gene products. SDHB, PDE1, HSDL1, Cp450, SRSF and SNRNP40 from these metabolic pathways were predicted to have dramatic effects in M. nipponene through performing the qPCR analysis in different reproductive season of testis and various tissues. Conclusion: This study provides new evidences for analyzing the process of male sexual differentiation and development in M. nipoponense. The expression level of 10 DEGs were determined in different reproductive seasons of testis and different tissues by using quantitative Real-time PCR (qPCR), in order to further analyze the gene functions in depth. These 10 selected DEGs were predicted to have potential functions in male sex-differentiation and development in M. nipponense, based on the expression fold change by RNA-Seq. The different tissues include testis, ovary, hepatopancreas, muscle, eyestalk, gill, heart and brain. The preparation of RNA samples for qRT-PCR analysis has been described in previous studies [64-65]. Bio-Rad iCycler iQ5 Real-Time PCR System was used to carry out the SYBR Green RT-qPCR assay. The procedure and statistical analysis can be also seen in previous studies [64-65].

analyzed, and their functions in male sexual differentiation and development were also identi ed, including sex-lethal, transformer-2 and extra sex comb [10][11][12]. Other male reproductive studies in M. nipponense have been conducted at the organic and cellular level [13][14][15]. However, the male sexual differentiation and development mechanism was still unclear. A reasonable explanation was that only limited unigenes were carried out, and the complete set of metabolites of M. nipponense testis was unknown.
Metabolomics is an idea technique to analyze the chemical processes involving metabolites, which is the products and small molecule intermediates of metabolism. The metabolome represents the complete set of metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes [16]. Transcriptomic and proteomic analyses reveal the gene products in the cell, representing one aspect of cellular function. Conversely, metabolic pro ling represents the instantaneous snapshot of the physiology in the cell, providing the direct physiological state of an organism. The metabolomic information, integrated with the genomics, transcriptomic, proteomic data, can provide a better understanding of cellular biology [17][18].
In this study, our objectives were to identify the key genes and metabolites capable of regulating the male sexual differentiation and development of M. nipponense through integrating the metabolomic and transcriptomic pro ling analysis of testis between thereproductive season and non-reproductive season.
This study will provide valuable evidence for better understanding the molecular mechanisms, determining the male sex-differentiation and development in M. nipponense.

Histological observation
The morphological difference between the testis of non-reproductive and reproductive season was analyzed by histological observation (Fig. 1). According to the histological observation, both of nonreproductive and reproductive season include the spermatogonium, spermatocyte and sperm. Over 70% of the cells in the testis of reproductive season are sperms, whereas spermatogonium and spermatocyte are the dominant cells in the testis of non-reproductive season.

Metabolic transcriptome analysis
The metabolic pro ling analysis of the testis between non-reproductive season and reproductive season were performed, and the overall structure of the GS/MS dataset was assessed by principal component analysis (PCA) (Fig. 2-A). The metabolic pro le was also checked by using the orthogonal projections to latent structures-discriminate analysis (OPLS-DA) (Fig. 2-B). The criterion of the robustness and predictive ability of the model was set as seven-fold cross-validation, and further validation was performed using permutation tests. The R2 and Q2 intercept values were 0.934 and -0.896 after 200 permutations of the treatments. The low Q2 intercept values show strong robustness and reliability of the model and the risk of over tting was low, indicating the metabolites related to male sexual differentiation and development is represented by different metabolic patterns in the testis of non-reproductive season and reproductive season. A total of 268 differentially expressed metabolites (DEMs) were identi ed between the testis of non-reproductive and reproductive season, including 171 up-regulated metabolites and 98 downregulated metabolites in the testis of reproductive season, using the criterion of >1.2 as up-regulated metabolites and <0.8 as down-regulated metabolites (Additional les 1 Table S1). The most 10 upregulated and 10 down-regulated metabolites were listed in Table 2. These DEMs were assigned to 22 metabolic pathways. According to the integrated analysis of metabolomes and transcriptomes, Glycerophospholipid metabolism (Additional les 2 Figure S1) and Sphingolipid metabolism (Additional les 2 Figure S2) were the most enriched metabolic pathways in both of the metabolomes and transcriptome analysis.
To identify their putative functions, all of the assembled unigenes were compared with the non-redundant protein database and nucleotide sequences in NCBI using Blastp and Blastx at an E-value of <10 -5 in the priority order of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Gene Ontology (GO) and Cluster of Orthologous Groups (COG) database. A total of 16,472 (33.95%) unigenes were annotated in Nr database, while the other unannotated unigenes represent novel genes whose functions were still not clear.
GO and COG analysis aimed to provide a structured vocabulary to describe gene products. A total of 11,306 and 10,594 unigenes were highly matched the protein with known functions in GO and COG database, respectively. GO analysis comprised of 55 functional groups, in which Cell, Cell parts, Cellular process, Binding and Organelle with the number of unigenes > 7,000 represent the largest functional groups (Fig. 4). The matched unigenes in COG database were classi ed into 25 functional categories, in which General function prediction only, Signal transduction mechanisms and Posttranslational modi cation, protein turnover, chaperones with number of unigenes >1000 represent the largest groups (Fig. 5). KEGG analysis aimed to match the assembled unigenes in biological pathways in the ladybird. A total of 7,621 unigenes were highly matched the known genes in KEGG databse, mapped onto 273 metabolic pathways.
Identi cation of candidate male differentiation and development related genes and pathways The transcriptome pro ling analysis between the testis of non-reproductive season and reproductive season was performed, in order to select candidate male sexual differentiation and development related genes in M. nipponense. A total of 12,230 unigenes were differentially expressed between the testis of non-reproductive and reproductive season, including 6,151 up-regulated unigenes and 6,079 downregulated unigenes in the testis of non-reproductive season, compared with those of reproductive season, using the criterion of >1.5 as up-regulated unigenes and <0.66 as down-regulated unigenes (Additional les 1 Table S3).
A total of 1,136 DEGs highly match the known proteins in KEGG pathway database, mapped onto 181 predicted metabolic pathways. Purine metabolism, Spliceosome, Endocytosis and Lysosome represent the most enriched metabolic pathways, in which the number of DEGs were more than 40.
Several important candidate enriched metabolic pathways were selected, which may affect the male sexual differentiation and development. These metabolic pathways include Oxidative phosphorylation, Glycolysis/Gluconeogenesis, HIF-1 signaling pathway, Citrate cycle, Steroid hormone synthesis and Spliceosome (Table 3). Oxidative phosphorylation, Glycolysis/Gluconeogenesis, HIF-1 signaling pathway, Citrate cycle may promote the male sexual differentiation and development through providing ATP. Steroid hormone synthesis synthesizes steroid hormone and spliceosome ensures the correct alternative splicing product, in order to promote the male sexual differentiation and development in M. nipponense. Several DEGs were also selected which may play essential roles in these metabolic pathways (Table 3). SDHB, PDE1 and Gadph-PA are involved in the at least two ATP-releasing related metabolic pathways. qPCR analysis qPCR analysis was rstly used to verify the expression pattern of these 10 DEGs in the testis of nonreproductive season and reproductive season. According to the qPCR analysis, these 10 DEGs showed the same expression pattern with those of RNA-Seq. All of these 10 DEGs were up-regulated in the testis of reproductive season, compared with those in the testis of non-reproductive season (Fig. 6).
qPCR analysis was then used to verify the expression of these DEGs in different tissues, in order to analyze the functions of these 10 DEGs in depth (Fig. 7). SDHB, PDE1 and Gadph-PA were the ATPreleasing related genes. SDHB and PDE1 had the highest expression levels in testis, and showed the signi cant expression difference with those in other tested tissues (P < 0.05). Gadph-PA showed the highest expression level in heart, whereas the expression in testis was relatively low. HSDL1 and CYP450 were steroid hormone synthesis-related genes. HSDL1 and CYP450 showed the similar expression pattern that these two genes had the expression levels in hepatopancreas, followed by testis, and the expressions in testis showed signi cant difference with those in other tested tissues (P < 0.05). SRSF showed the highest expression level in testis, and showed signi cant difference with other tested tissues (P < 0.05).
The highest expression level of SNRNP40 was observed in ovary, followed by testis, and showed signi cant difference with other tested tissues (P < 0.05). However, the expression levels of the other tested genes in testis were dramatically low. The expression levels of XDO and MHC were dramatically low in testis. XDO was dramatically expressed in hepatopancreas and brain (P < 0.05). The peak expression of MHC was observed in muscle (P < 0.05).

Discussion
The present study aimed to select the candidate metabolites and genes involved in the male sexual differentiation and development in M. nipponense, through the integrated analysis of metabolomics and transcriptomic analysis of the testis between reproductive season and non-reproductive season. The morphological differences between the testis of reproductive season and non-reproductive season were revealed by histological observation. The dominant cells in the testis of reproductive season were sperms, whereas spermatogonium and spermatocyte were the majority cells in the testis of non-reproductive season. Thus, identi cations of DEMs and DEGs are urgently needed, in order to fully understand the male sexual differentiation and development mechanism in M. nipponense. A testis cDNA library of M. nipponense was constructed in previous study using Roche 454 sequencing platform. A total of 5,539 EST were sequenced with an average length of 989 bp, and 52 sex-or reproduction-related genes were identi ed [9]. To the best of our knowledge, there is no previous study related to the metabolic transcriptome analysis of testis in M. nipponense. In the present study, a total of 16,472 unigenes were identi ed in the testis transcriptome of M. nipponense, providing more basic information for the male sexual differentiation and development analysis in M. nipponense than previous study. In addition, 385 metabolites and 12,230 genes were differentially expressed between the testis of reproductive season and non-reproductive season. These DEMs and DEGs were predicted to dramatically affect the male sexual differentiation and development in M. nipponense, based on the dramatic morphological difference between the testis of reproductive season and non-reproductive season.
Glycerophospholipid metabolism and Sphingolipid metabolism were the most enriched metabolic pathways in both of the metabolomes and transcriptome analysis. The glycerophospholipids of the plasma membrane bilayer play an important role in the generation of both extracellular and intracellular signals. Intact glycerophos-pholipids and primary and secondary glycerophospholipid metabolites have been implicated in the regulation of many aspects of cell function [19][20][21]. Sphingolipids have been proven to be involved in many aspects, including the regulation of cell growth, differentiation, and programmed cell death. This action is performed through interacting of complex sphingolipids (such as gangliosides) with growth factor receptors, neighboring cells, and the extracellular matrix [22]. The signi cant enrichment in both of metabolomes and transcriptome analysis indicated that lipid metabolism play essential roles in male sexual development in M. nipponense.
Some other important metabolic pathways, involved in the male sexual differentiation and development, were also identi ed through transcriptome analysis. Spliceosome is the most enriched metabolic pathway in this study with 49 DEGs. The spliceosome removes introns from a transcribed pre-mRNA, in order to produce necessary transcripts. This process cuts out irrelevant or incorrect introns from the initial pre-mRNA and sends the processed version to the director for the nal cut [23][24]. The dramatic enrichment of DEGs in the metabolic pathway of spliceosome indicated that this metabolic pathway plays essential roles in producing necessary gene products involved in the male sexual differentiation and development. The signi cant DEGs in this metabolic pathway are the expected candidate genes involved in the male sexual differentiation and development. Serine/arginine-rich splicing factor RS2Z32 (SRSF) is the most signi cant DEG in spliceosome with 2.63-folder higher in the testis of reproductive season. SRSF is an important splicing factor, playing essential roles in pre-mRNA splicing [25][26][27]. SRSF is necessary for all splicing reactions to in uence splice site selection, resulting in alternative splicing [26]. Heat shock 70kDa protein 8 (HSPA8), coding for heat shock protein 70 (HSC70), plays vital roles in regulating cellular functions through interacting with many molecules. HSC70 has been proven to be synthesized in haploid cells during spermatogenesis and are mainly activated at the spermatid stage [28]. The other main functions of HSC70 include the protecting of cells from stressful damage [29], regulating of protein folding [30][31] and promoting of ATP synthesis [32][33].
Adenosine triphosphate (ATP) is an unstable high-energy compound, which is the most direct energy source in organisms. Every activity in an organism needs ATP to provide energy, including the male differentiation and development. In the present study, 4 important ATP-related metabolic pathways were found, including Oxidative phosphorylation, Glycolysis/Gluconeogenesis, HIF-1 signaling pathway and Citrate cycle (TCA cycle). Oxidative phosphorylation is the metabolic pathway, almost found in all aerobic organisms. Cells use enzyme to oxidize nutrients, releasing energy to produce ATP [34]. Glycolysis/Gluconeogenesis converts glucose (C6H12O6) into pyruvate (CH3COCOO− + H+), releasing free energy to form the high-energy molecules ATP and NADH (reduced nicotinamide adenine dinucleotide) [35]. TCA cycle is found in all aerobic organisms using a series of chemical reactions to release stored energy into ATP. The energy is released through oxidation of acetyl-CoA derived from carbohydrates, proteins, and fats [36]. Several genes are up-regulated in HIF-1 signaling pathway, when hypoxic conditions are stabilized, in order to promote survival in low-oxygen conditions [37]. These upregulated genes include glycolysis enzymes, promoting ATP synthesis in an oxygen-independent manner. The DEGs in these metabolic pathways may integrate to promote the male sex-differentiation and development in M. nipponense (Fig. 8). Several ATP synthesis-related DEGs were identi ed, which are involved in at least two metabolic pathways. Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial (SDHB) is a DEG with 3.72-folder higher expression in the testis of reproductive season, playing vital roles in Oxidative phosphorylation and Citrate cycle (TCA cycle). SDHB is one of four protein subunits forming succinate dehydrogenase, which catalyzes the oxidation of succinate [38][39]. Pyruvate dehydrogenase E1 is involved in the Glycolysis/Gluconeogenesis, HIF-1 signaling pathway and Citrate cycle (TCA cycle) with 1.7-folder higher expression in the testis of reproductive season. The pyruvate dehydrogenase complex (PDC) plays essential roles in transforming pyruvate into acetyl-CoA through pyruvate decarboxylation [40][41]. Acetyl-CoA may then be used in the citric acid cycle to produce ATP. Pyruvate dehydrogenase is the rst component enzyme of PDC. Pyruvate dehydrogenase E1 performs the rst two reactions within PDC, including a decarboxylation of pyruvate and a reductive acetylation of lipoic acid [42][43]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a DEG with 1.65-folder higher expression in the testis of reproductive season, enriched in Glycolysis/Gluconeogenesis and HIF-1 signaling pathway. GADPH generates products, catalyzing a vital energy-yielding step. It occurs through the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate when inorganic phosphate and NAD are present [44]. Energy and molecular carbon are released when GADPH is catalyzed to break down glucose [45].
Steroid hormone has been proven to play essential roles in sexual development. Steroid hormone generally divided into ve main classes, including glucocorticoids, mineralocorticoids, androgens, oestrogens and progestogens. The natural steroid hormones, which are lipids, are generally synthesized from cholesterol in the gonads and adrenal glands [46][47]. The steroid hormone biosynthesis metabolic pathway was one of the main enriched pathways in the present study, including 17 DEGs. Hydroxysteroid dehydrogenase like 1 gene (HSDL1) is the most signi cant DEG in the metabolic pathway of steroid hormone synthesis, which the expression in the testis of reproductive season is 12.58-folder higher than that of non-reproductive season. HSDL1 is a novel human gene, which is highly expression in reproductive tissues. HSDL1 is highly expressed in testis and ovary, revealed by Northern blot. In situ hybridization analysis indicated that HSDL1 is predominantly expressed in the prostate cancer compared with the normal prostate cancer [48]. In addition, this gene has been proven to be involved in the development of sheep fetus in late gestation [49]. Cytochrome P450 (CYP450) is another main DEG, which is 10.24-folder higher in the testis of reproductive season than that of non-reproductive season.
The previous studies have been proven that a series of CYP enzymes play important roles in the synthesis of steroid hormones ( steroidogenesis) by the adrenals, gonads, and peripheral tissue [50][51][52][53]. For instance, CYP19A is involved in the aromatization of androgens to estrogens [54][55]. The dramatically higher expression of HSDL1 and CYP450 in the testis of reproductive season than that of non-reproductive season indicated that these two genes are strong candidate genes in male sexual differentiation and development in M. nipponense.
The expression levels of the 10 selected DEGs in the testis of non-reproductive season and reproductive season were veri ed in qPCR. The expression patterns of these 10 DEGs were as the same as that of RNA-Seq, based on the qPCR analysis. All of these DEGs were up-regulated in the testis of reproductive season, indicating the results of RNA-Seq in the present study were correct. The expression levels of these 10 DEGs were further investigated in different tissues of M. nipponense. Pyruvate dehydrogenase E1α (PDE1 α) is a testis-speci c form in human, and it has been proven to be expressed in postmeiotic spermatogenic cells [42]. SDHB and PDE1 were ATP-releasing related genes, showed the highest expression levels in the testis of M. nipponense, indicating SDHB and PDE1 may have dramatic effects on male sexual differentiation and development in M. nipponense. HSDL1 and Cp450, which were selected from the metabolic pathway of steroid hormone synthesis, were highly expressed in hepatopancreas, followed by testis. HADL1 has been proven to be highly expressed in hepatopancreas and testis, and played vital roles in hepatic growth [49,56]. Liver Cytochrome P450 Metabolism has been proven to be involved in the endogenous steroid hormones, bile acids, and fatty acids synthesis [57]. In addition, Vitellogenin (Vg) is the precursor of yolk protein, functioning as a nutritive resource and playing essential roles in embryonic growth and gonad development. Vg has been proven to be only expressed in the female hepatopancreas, hemolymph, and ovary in M. nipponense [58]. The highly expressed of HSDL1 and Cp450 in male hepatopancreas and testis in present study predicted that HSDL1 and Cp450 may play similar roles in male sexual differentiation and development in M. nipponene as that of Vg in female sexual development. RSRF and SNRNP40 were signi cant DEGs in the metabolic pathway of spliceosome in present study. The highest expression level of RSRF was observed in testis, indicating RSRF has positive effects on producing correct gene products to promote male sexual differentiation and development in M. nipponense. The highest expression level of SNRNP40 was observed in ovary, followed by testis. This result implies SNRNP40 may dramatically promote gonad development in M. nipponense. However, the expression levels of the other tested DEGs in testis were dramatically lower than the other tested tissues, implying these DEGs may also have other important physiology functions in M. nipponenes, which needs further analysis.

Conclusion
In the present study, we selected the male sexual differentiation and development related metabolites and genes through integrating the metabolomic and transcriptomic pro ling analysis of testis between thereproductive season and non-reproductive season. A total of 385 metabolites and 12,230 genes were differentially expressed between the testis of reproductive season and non-reproductive season, which may promote the male sexual differentiation and development in M. nipponense. In addition, Oxidative phosphorylation, Glycolysis/Gluconeogenesis, HIF-1 signaling pathway and Citrate cycle were predicted to promote the male differentiation and development through providing ATP, whereas Steroid hormone synthesis and Spliceosome may promote through promoting the synthesis of steroid hormone and providing correct gene products, respectively. SDHB, PDE1, HSDL1, CYP450, SRSF and SNRNP40 were predicted to have dramatic effects on male sexual differentiation and development in M. nipponene, according the qPCR analysis of 10 DEGs in different reproductive season of testis and various tissues.

Sample collection
Male specimens of oriental river prawn at nonreproductive season and reproductive season (body weights, 2.67-5.37 g) were respectively collected from a wild population in Tai Lake, Wuxi, China (120°13′44″E, 31°28′ 22″N) in winter and summer. According to the previous study, the specimens at the water temperature of 15°C or below were collected as non-reproductive season, while the specimens at the water temperature of 28°C or above were collected as reproductive season. All the samples were transferred to a 500 L tank and maintained in aerated freshwater. The testes were respectively collected from the specimens from non-reproductive season and reproductive season, and immediately preserved in liquid nitrogen until used for metabolomics and transcriptomic analysis.

Morphological observation
Hematoxylin and eosin (HE) staining was used to observe the morphological difference between the testis of non-reproductive season and reproductive season. The testis of non-reproductive season and reproductive season were respectively collected at the water temperature of 15°C or below and 28°C or above. The procedure of HE staining has been well-described in previous studies [59][60]. The histological slides were observed under an Olympus SZX16 microscope.

Metabolomic pro ling analysis
The comparative metabolic analysis of testis between the non-reproductive and reproductive season were performed, in order to select candidate metabolites involved in the male sexual differentiation and development in M. nipponense. In order to ensure the su cient amount of RNA samples, at least 0.5g testes were pooled to form one biological replicate, and eight biological replicates were sequenced for the testes from both of the non-reproductive and reproductive season. Differentially expressed metabolites between the testis of non-reproductive season and reproductive season involved in the male sexual differentiation and development were determined using liquid chromatography-mass spectrometry (LC/MS) analysis [61].

Transcriptomic pro ling analysis
The comparative transcriptome analysis of testis between the non-reproductive and reproductive season were performed, in order to select candidate genes and pathways involved in the male sexual differentiation and development in M. nipponense. In order to ensure the su cient amount of RNA samples, at least 30 testes were pooled to form one biological replicate, and three biological replicates were sequenced for the testes from both of the non-reproductive and reproductive season.
Total RNA, RNA samples for comparative transcriptome analysis were prepared followed by the previous studies [62][63]. Illumina HiSeq 2500 platform was employed to performed the transcriptome analysis.
The detailed procedure has been well described in previous studies [62][63]. The bioinformatics analysis, including the transcriptome assemble and annotation, was also described in the previous studies [62][63].

Quantitative real-time PCR (qRT-PCR) analysis
The expression level of 10 DEGs were determined in different reproductive seasons of testis and different tissues by using quantitative Real-time PCR (qPCR), in order to further analyze the gene functions in depth. These 10 selected DEGs were predicted to have potential functions in male sex-differentiation and development in M. nipponense, based on the expression fold change by RNA-Seq. The different tissues include testis, ovary, hepatopancreas, muscle, eyestalk, gill, heart and brain. The preparation of RNA samples for qRT-PCR analysis has been described in previous studies [64][65]. Bio-Rad iCycler iQ5 Real-Time PCR System was used to carry out the SYBR Green RT-qPCR assay. The procedure and statistical analysis can be also seen in previous studies [64][65]. Additional File Legend Table S1: Differentially expressed metabolites between the testis of non-reproductive season and reproductive season.      Cluster of orthologous groups (COG) classi cation of putative proteins.