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BackgroundThe oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China, even in whole of Asia. The androgenic gland produces hormones that play crucial roles in sexual differentiation to maleness. This study is the first de novo M. nipponense transcriptome analysis using cDNA prepared from mRNA isolated from the androgenic gland. Illumina/Solexa was used for sequencing.Methodology and Principal FindingThe total volume of RNA sample was more than 5 ug. We generated 70,853,361 high quality reads after eliminating adapter sequences and filtering out low-quality reads. A total of 78,408 isosequences were obtained by clustering and assembly of the clean reads, producing 57,619 non-redundant transcripts with an average length of 1244.19 bp. In total 70,702 isosequences were matched to the Nr database, additional analyses were performed by GO (33,203), KEGG (17,868), and COG analyses (13,817), identifying the potential genes and their functions. A total of 47 sex-determination related gene families were identified from the M. nipponense androgenic gland transcriptome based on the functional annotation of non-redundant transcripts and comparisons with the published literature. Furthermore, a total of 40 candidate novel genes were found, that may contribute to sex-determination based on their extremely high expression levels in the androgenic compared to other sex glands,. Further, 437 SSRs and 65,535 high-confidence SNPs were identified in this EST dataset from which 14 EST-SSR markers have been isolated.ConclusionOur study provides new sequence information for M. nipponense, which will be the basis for further genetic studies on decapods crustaceans. More importantly, this study dramatically improves understanding of sex-determination mechanisms, and advances sex-determination research in all crustacean species. The huge number of potential SSR and SNP markers isolated from the transcriptome may shed the lights on research in many fields, including the evolution and molecular ecology of Macrobrachium species.

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Forkhead box protein L2 (Foxl2) has dramatic effects on early differentiation and development of the female gonad in mammals and fish through the repression of SOX9 expression. In this study, we aimed to isolate a full‐length cDNA sequence encoding Foxl2 from the Macrobrachium nipponense (Mn‐Foxl2) and investigated its gene function. The full‐length cDNA sequence of Mn‐Foxl2 was 2097 bp with an open reading frame of 1740 bp, encoding 579 amino acids. Phylogenetic analysis revealed considerable evolutionary divergence between Mn‐Foxl2 and Foxl2 from fish. Mn‐Foxl2 may be involved in gonadal sex differentiation, based on its dramatically high expression during the sex‐differentiation sensitive period. Tissue distributions indicated that Mn‐Foxl2 mRNA expression was higher in the testis than that in the ovary and higher in males than in females in the same tissues, implying that Foxl2 may promote both male and female sexual development in M. nipponense. In situ hybridization analysis and quantitative polymerase chain reaction analysis in the gonad during the reproductive cycle further confirmed the important roles of Foxl2 in early gonad development in M. nipponense. This study advances our understanding of the role of Foxl2 in M. nipponense and provides the basis for further studies of Foxl2 in crustaceans.

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

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