Marine Biotechnology

, Volume 20, Issue 1, pp 20–34 | Cite as

Comparative Transcriptome Analysis Provides Insights into Differentially Expressed Genes and Long Non-Coding RNAs between Ovary and Testis of the Mud Crab (Scylla paramamosain)

  • Xiaolong Yang
  • Mhd Ikhwanuddin
  • Xincang Li
  • Fan Lin
  • Qingyang Wu
  • Yueling Zhang
  • Cuihong You
  • Wenhua Liu
  • Yinwei Cheng
  • Xi Shi
  • Shuqi Wang
  • Hongyu MaEmail author
Original Article


The molecular mechanism underlying sex determination and gonadal differentiation of the mud crab (Scylla paramamosain) has received considerable attention, due to the remarkably biological and economic differences between sexes. However, sex-biased genes, especially non-coding genes, which account for these differences, remain elusive in this crustacean species. In this study, the first de novo gonad transcriptome sequencing was performed to identify both differentially expressed genes and long non-coding RNAs (lncRNAs) between male and female S. paramamosain by using Illumina Hiseq2500. A total of 79,282,758 and 79,854,234 reads were generated from ovarian and testicular cDNA libraries, respectively. After filtrating and de novo assembly, 262,688 unigenes were produced from both libraries. Of these unigenes, 41,125 were annotated with known protein sequences in public databases. Homologous genes involved in sex determination and gonadal development pathways (Sxl-Tra/Tra-2-Dsx/Fru, Wnt4, thyroid hormone synthesis pathway, etc.) were identified. Three hundred and sixteen differentially expressed unigenes were further identified between both transcriptomes. Meanwhile, a total of 233,078 putative lncRNAs were predicted. Of these lncRNAs, 147 were differentially expressed between sexes. qRT-PCR results showed that nine lncRNAs negatively regulated the expression of eight genes, suggesting a potential role in sex differentiation. These findings will provide fundamental resources for further investigation on sex differentiation and regulatory mechanism in crustaceans.


Scylla paramamosain RNA sequencing Unigenes lncRNAs Sex differentiation 



long non-coding RNAs


differentially expressed genes


quantitative reverse transcription polymerase chain reaction




Coding Potential Calculator


Coding-Non-Coding Index


Gene Ontology


Kyoto Encyclopedia of Genes and Genomes


open reading frames


epididymal sperm-binding protein 1


insulin-like growth factor-binding protein complex acid labile chain


epidermal growth factor receptor substrate 15-like 1


hormone receptor-like 97b


estrogen receptor


follistatin-related protein 1


steroid receptor RNA activator 1


muscle myosin heavy chain


female sterile homeotic


paternally expressed gene 3


Funding Information

This study was supported by the STU Scientific Research Foundation for Talents (No. NTF17006), the National Program for Support of Top-Notch Young Professionals, the National Natural Science Foundation of China (No. 31772837), and the “Sail Plan” Program for the Introduction of Outstanding Talents of Guangdong Province, China.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

10126_2017_9784_MOESM1_ESM.docx (81 kb)
Online Resource 1 Fig. S1. Expression level of partial lncRNAs and its putative target genes (DOCX 81 kb)
10126_2017_9784_MOESM2_ESM.docx (23 kb)
Online Resource 2 Table S1. Primers used in validation of Illumina sequencing by qRT-PCR (DOCX 22 kb)
10126_2017_9784_MOESM3_ESM.docx (30 kb)
Online Resource 3 Table S2. Candidate genes involved in the regulation of gonadal development in S. paramamosain (DOCX 30 kb)
10126_2017_9784_MOESM4_ESM.xls (18 kb)
Online Resource 4 Table S3. Analysis results of differential expressed unigenes (XLS 17 kb)
10126_2017_9784_MOESM5_ESM.xls (8 kb)
Online Resource 5 Table S4. Analysis results of differential expressed lncRNAs (XLS 8 kb)


  1. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106CrossRefPubMedPubMedCentralGoogle Scholar
  2. Brown JP, Bullwinkel J, Baron-Luhr B, Billur M, Schneider P, Winking H, Singh PB (2010) HP1γ function is required for male germ cell survival and spermatogenesis. Epigenet Chromatin 3(1):9Google Scholar
  3. Chen S, Zhang G, Shao C, Huang Q, Liu G, Zhang P, Song W, An N, Chalopin D, Volff JN, Hong Y, Li Q, Sha Z, Zhou H, Xie M, Yu Q, Liu Y, Xiang H, Wang N, Wu K, Yang C, Zhou Q, Liao X, Yang L, Hu Q, Zhang J, Meng L, Jin L, Tian Y, Lian J, Yang J, Miao G, Liu S, Liang Z, Yan F, Li Y, Sun B, Zhang H, Zhang J, Zhu Y, Du M, Zhao Y, Schartl M, Tang Q, Wang J (2014) Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nat Genet 46(3):253–260CrossRefPubMedGoogle Scholar
  4. China Fishery Statistical Yearbook (2016) Fisheries Bureau. Department of Agriculture of China, Beijing, pp 30–31. (In Chinese)Google Scholar
  5. da Fonseca RR, Albrechtsen A, Themudo GE, Ramos-Madrigal J, Sibbesen JA, Maretty L, Zepeda-Mendoza ML, Campos PF, Heller R, Pereira RJ (2016) Next-generation biology: sequencing and data analysis approaches for non-model organisms. Mar Genom 30:3–13Google Scholar
  6. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44(D1):D279–D285CrossRefPubMedGoogle Scholar
  7. Gao J, Wang X, Zou Z, Jia X, Wang Y, Zhang Z (2014) Transcriptome analysis of the differences in gene expression between testis and ovary in green mud crab (Scylla paramamosain). BMC Genomics 15(1):585CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gong J, Huang C, Shu L, Bao C, Huang H, Ye H, Zeng C, Li S (2016) The retinoid X receptor from mud crab: new insights into its roles in ovarian development and related signaling pathway. Sci Rep 6(1):23654CrossRefPubMedPubMedCentralGoogle Scholar
  9. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adicois X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29(7):644–652CrossRefPubMedPubMedCentralGoogle Scholar
  10. He L, Jiang H, Cao D, Liu L, Hu S, Wang Q (2013) Comparative transcriptome analysis of the accessory sex gland and testis from the Chinese mitten crab (Eriocheir sinensis). PLoS One 8(1):e53915CrossRefPubMedPubMedCentralGoogle Scholar
  11. Huang HY, Ye HH, Han SZ, Wang GZ (2009) Profiles of gonadotropins and steroid hormone-like substances in the hemolymph of mud crab Scylla paramamosain during the reproduction cycle. Mar Freshw Behav Physiol 42(4):297–305CrossRefGoogle Scholar
  12. Ikhwanuddin M, Bachok Z, Hilmi MG, Ghazali A, Zakaria MZ (2010) Species diversity, carapace width-body weight relationship, size distribution and sex ratio of mud crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. J Sustain Sci Manag 5:97–109Google Scholar
  13. Jia XW, Chen YD, Zou ZH, Lin P, Wang YL, Zhang ZP (2013) Characterization and expression profile of vitellogenin gene from Scylla paramamosain. Gene 520(2):119–130CrossRefPubMedGoogle Scholar
  14. Jiang W, Ma HY, Ma CY, Li SJ, Liu YX, Qiao ZG, Ma LB (2014) Characterization of growth traits and their effects on body weight of G1 individuals in the mud crab (Scylla paramamosain). Genet Mol Res 13(3):6050–6059CrossRefPubMedGoogle Scholar
  15. Jung H, Yoon BH, Kim WJ, Kim DW, Hurwood DA, Lyons RE, Salin KR, Kim HS, Baek I, Chand V, Mather PB (2016) Optimizing hybrid de novo transcriptome assembly and extending genomic resources for giant freshwater prawns (Macrobrachium rosenbergii): the identification of genes and markers associated with reproduction. Int J Mol Sci 17(5):690CrossRefPubMedCentralGoogle Scholar
  16. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40(D1):D109–D114CrossRefPubMedGoogle Scholar
  17. Kong L, Zhang Y, Ye ZQ, Liu XQ, Zhao SQ, Wei L, Gao G (2007) CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res 35(suppl_2):W345–W349CrossRefPubMedPubMedCentralGoogle Scholar
  18. Leelatanawit R, Sittikankeaw K, Yocawibun P, Klinbunga S, Roytrakul S, Aoki T, Hirono I, Menasveta P (2009) Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon. Comp Biochem Physiol A Mol Integr Physiol 152(1):66–76CrossRefPubMedGoogle Scholar
  19. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12(1):323CrossRefPubMedPubMedCentralGoogle Scholar
  20. Li Y, Zhang L, Sun Y, Ma X, Wang J, Li R, Zhang M, Wang S, Hu X, Bao Z (2016) Transcriptome sequencing and comparative analysis of ovary and testis identifies potential key sex-related genes and pathways in scallop Patinopecten yessoensis. Mar Biotechnol 18(4):453–465CrossRefPubMedGoogle Scholar
  21. Lin MF, Jungreis I, Kellis M (2011) PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27(13):I275–I282CrossRefPubMedPubMedCentralGoogle Scholar
  22. Liu Y, Hui M, Cui ZX, Luo DL, Song CW, Li YD, Liu L (2015) Comparative transcriptome analysis reveals sex-biased gene expression in juvenile Chinese mitten crab Eriocheir sinensis. PLoS One 10(7):e0133068CrossRefPubMedPubMedCentralGoogle Scholar
  23. Luo CW, Tsementzi D, Kyrpides N, Read T, Konstantinidis KT (2012) Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample. PLoS One 7(2):e30087CrossRefPubMedPubMedCentralGoogle Scholar
  24. Ma HY, Chen SL, Yang JF, Ji XS, Chen SQ, Tian YS, Bi JZ (2010) Isolation of sex-specific AFLP markers in spotted halibut (Verasper variegatus). Environ Biol Fish 88(1):9–14CrossRefGoogle Scholar
  25. Ma HY, Ma CY, Ma LB, Xu Z, Feng NN, Qiao ZG (2013) Correlation of growth-related traits and their effects on body weight of the mud crab (Scylla paramamosain). Genet Mol Res 12(4):4127–4136CrossRefPubMedGoogle Scholar
  26. Ma HY, Li SJ, Feng NN, Ma CY, Wang W, Chen W, Ma LB (2016) First genetic linkage map for the mud crab (Scylla paramamosain) constructed using microsatellite and AFLP markers. Genet Mol Res 15:gmr.15026929Google Scholar
  27. Mantione KJ, Kream RM, Kuzelova H, Ptacek R, Raboch J, Samuel JM, Stefano GB (2014) Comparing bioinformatic gene expression profiling methods: microarray and RNA-Seq. Med Sci Monit Basic Res 20:138–142CrossRefPubMedPubMedCentralGoogle Scholar
  28. Meng XL, Liu P, Jia FL, Li J, Gao BQ (2015) De novo transcriptome analysis of Portunus trituberculatus ovary and testis by RNA-Seq: identification of genes involved in gonadal development. PLoS One 10(6):e0128659CrossRefPubMedPubMedCentralGoogle Scholar
  29. Mistry J, Finn RD, Eddy SR, Bateman A, Punta M (2013) Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res 41(12):e121CrossRefPubMedPubMedCentralGoogle Scholar
  30. Nagaraju GP (2011) Reproductive regulators in decapod crustaceans: an overview. J Exp Biol 214(1):3–16CrossRefPubMedGoogle Scholar
  31. Ponting CP, Belgard TG (2010) Transcribed dark matter: meaning or myth? Hum Mol Genet 19(R2):R162–R168CrossRefPubMedPubMedCentralGoogle Scholar
  32. Qiao H, Xiong YW, Jiang SF, Fu HT, Sun S, Jin SB (2015) Gene expression profile analysis of testis and ovary of oriental river prawn, Macrobrachium nipponense, reveals candidate reproduction-related genes. Genet Mol Res 14(1):2041–2054CrossRefPubMedGoogle Scholar
  33. Quackenbush LS (1992) Yolk synthesis in the marine shrimp, Penaeus vannamei. Comp Biochem Physiol A Physiol 103(4):711–714CrossRefGoogle Scholar
  34. Reddy PS, Reddy PR, Nagaraju GPC (2004) The synthesis and effects of prostaglandins on the ovary of the crab Oziotelphusa senex senex. Gen Comp Endocrinol 135(1):35–41CrossRefPubMedGoogle Scholar
  35. Ryner LC, Goodwin SF, Castrillon DH, Anand A, Villella A, Baker BS, Hall JC, Taylor BJ, Wasserman SA (1996) Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell 87(6):1079–1089CrossRefPubMedGoogle Scholar
  36. Sagi A, Silkovsky J, Fleisher-Berkovich S, Danon A, Chayoth R (1995) Prostaglandin E2 in previtellogenic ovaries of the prawn Macrobrachium rosenbergii: synthesis and effect on the level of cAMP. Gen Comp Endocrinol 100(3):308–313CrossRefPubMedGoogle Scholar
  37. Shen Y, Lai Q (1994) Present status of mangrove crab (Scylla serrata (Forskal)) culture in China. Naga. ICLARM Quarterly 17:28–29Google Scholar
  38. Sheng Y, Zhao W, Song Y, Li Z, Luo M, Lei Q, Cheng H, Zhou R (2015) Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal. Sci Rep 5(1):10176CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sun L, Luo H, Bu D, Zhao G, Yu K, Zhang C, Liu Y, Chen R, Zhao Y (2013) Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Res 41(17):e166CrossRefPubMedPubMedCentralGoogle Scholar
  40. Thongbuakaew T, Siangcham T, Suwansa-ard S, Elizur A, Cummins SF, Sobhon P, Sretarugsa P (2016) Steroids and genes related to steroid biosynthesis in the female giant freshwater prawn, Macrobrachium rosenbergii. Steroids 107:149–160CrossRefPubMedGoogle Scholar
  41. Tilghman SM (1999) The sins of the fathers and mothers: genomic imprinting in mammalian development. Cell 96(2):185–193CrossRefPubMedGoogle Scholar
  42. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7(3):562–578CrossRefPubMedPubMedCentralGoogle Scholar
  43. Waiho K, Fazhan H, Shahreza MS, Moh JHZ, Noorbaiduri S, Wong LL, Sinnasamy S, Ikhwanuddin M (2017) Transcriptome analysis and differential gene expression on the testis of orange mud crab, Scylla olivacea, during sexual maturation. PLoS One 12(1):e0171095CrossRefPubMedPubMedCentralGoogle Scholar
  44. Wimuttisuk W, Tobwor P, Deenarn P, Danwisetkanjana K, Pinkaew D, Kirtikara K, Vichai V (2013) Insights into the prostanoid pathway in the ovary development of the penaeid shrimp Penaeus monodon. PLoS One 8(10):e76934CrossRefPubMedPubMedCentralGoogle Scholar
  45. Xie X, Zhou YQ, Liu MX, Tao T, Jiang QH, Zhu DF (2016) The nuclear receptor E75 from the swimming crab, Portunus trituberculatus: cDNA cloning, transcriptional analysis, and putative roles on expression of ecdysteroid-related genes. Comp Biochem Physiol B 200:69–77CrossRefPubMedGoogle Scholar
  46. Xu Z, Ma H, Ma C, Feng N, Li X, Li S, Jiang W, Ma L (2013) Cloning and tissue expression of β-actin in the mud crab (Scylla paramamosain) and its utility as an endogenous control. Biotechnol Bull 8:105–112. (In Chinese)Google Scholar
  47. Xu ZQ, Zhao MZ, Li XG, Lu QP, Li YH, Ge JC, Pan JL (2015) Transcriptome profiling of the eyestalk of precocious juvenile Chinese mitten crab reveals putative neuropeptides and differentially expressed genes. Gene 569(2):280–286CrossRefPubMedGoogle Scholar
  48. Zhang WY, Jiang SF, Xiong YW, Fu HT, Qiao H, Sun S, Gong YS, Jin SB (2015) Molecular cloning and expression analysis of female sterile homeotic gene (Fsh) in the oriental river prawn Macrobrachium nipponense. Genet Mol Res 14(2):4318–4330CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Guangdong Provincial Key Laboratory of Marine BiotechnologyShantou UniversityShantouChina
  2. 2.Institute of Tropical AquacultureUniversiti Malaysia TerengganuKuala TerengganuMalaysia
  3. 3.East China Sea Fisheries Research InstituteChinese Academy of Fishery SciencesShanghaiChina

Personalised recommendations