, Volume 825, Issue 1, pp 121–136 | Cite as

Analysis of candidate gene expression patterns of adult male Macrobrachium rosenbergii morphotypes in response to a social dominance hierarchy

  • Dania AzizEmail author
  • Md. Lifat Rahi
  • David A. Hurwood
  • Peter B. Mather


In this study, we investigated the relative gene expression pattern of 12 candidate genes from giant freshwater prawn (GFP) using hepatopancreas, eyestalk and testis tissues to compare expression profiles among adult male morphotypes [blue claw (BC), orange claw (OC) and small males (SM)] that reflect a social hierarchy. In particular, we focused our analysis on genes documented in other invertebrate taxa that are known to influence (i) inter-male aggressive behaviour, (ii) visual systems and (iii) olfactory genes. Genes examined here were normalised to 18S rRNA as a reference. Differences in gene expression patterns among male morphotypes and tissues were highly significant (P < 0.0001) with higher expression levels in eyestalk tissue compared with testis and hepatopancreas in all morphotypes. This might imply that differences in expression pattern of key candidate genes in the eyestalk can potentially provide cues to directly influence the formation of the male social dominance hierarchy. Expression stabilities of genes were evaluated using the RefFinder analytical tool, which revealed that STRPC-short transient receptor and BDP-beadex dlmo protein showed relatively similar expression levels. LW OPSIN, a visual system gene, appeared to be directly involved in suppression of subordinate male gene expression related to the social dominance hierarchy in male GFP. This finding could potentially be important for developing technologies that allow male morph frequencies to be manipulated at harvest in farmed stocks.


Gene expression QRT-PCR Social dominance hierarchy GFP 



The authors would like to gratefully acknowledge the support provided by Marie Curie International Research Staff exchange Scheme Fellowship within the 7th European Community Framework Programme (612296-DeNuGReC) and the help from Central Analytical Research Facility (CARF) at the Queensland University of Technology with the qRT-PCR labwork and analysis. We would also like to thank the staff of the Marine Science Center, Port Dickson in Malaysia for the help in the sample collection, and Vincent Chand for his assistance and technical support in QUT’s CARF-genomics lab. The manuscript has been greatly improved through helpful comments from two anonymous reviewers. This project was supported by an International Postgraduate Research Scholarship (Australia) and an Australia Postgraduate Award Grant awarded to Dania Aziz (N8724768).

Supplementary material

10750_2018_3721_MOESM1_ESM.pdf (46 kb)
Supplementary material 1 (PDF 46 kb)


  1. Andersen, C. L., J. L. Jensen & T. F. Ørntoft, 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64: 5245–5250.CrossRefGoogle Scholar
  2. Atema, J. & R. Voigt, 1995. Behaviour and sensory biology. In Factor, J. R. (ed.), The Biology of the Lobster, Homarus americanus. Academic Press, New York: 313–348.CrossRefGoogle Scholar
  3. Aziz, D., T. V. Nguyen, M. L. Rahi, D. A. Hurwood & P. B. Mather, 2017. Identification of genes that potentially affect social dominance hierarchy in adult male giant freshwater prawns (Macrobrachium rosenbergii). Aquaculture 476: 168–184.CrossRefGoogle Scholar
  4. Barlow, G. W., 1977. Modal action patterns. In Sebeok, T. A. (ed.), How Animals Communicate. Indiana University Press, Indianapolis: 98–134.Google Scholar
  5. Barlow, H. B., 1982. What causes trichromacy? A theoretical analysis using comb-filtered spectra. Vision Research 22: 635–643.CrossRefGoogle Scholar
  6. Berens, A. J., J. H. Hunt & A. L. Toth, 2014. Comparative transcriptomics of convergent evolution: different genes but conserved pathways underlie case phenotype across lineages of eusocial insects. Molecular Biology and Evolution 32: 690–703.CrossRefGoogle Scholar
  7. Berry, F. & T. Breithaupt, 2008. Development of behavioural and physiological assays to assess discrimination of male and female odours in crayfish, Pacifastacus leniusculus. Behaviour 145: 1427–1446.CrossRefGoogle Scholar
  8. Berry, F. C. & T. Breithaupt, 2010. To signal or not to signal? Chemical communication by urine-borne signals mirrors sexual conflict in crayfish. BMC Biology 8: 25.CrossRefGoogle Scholar
  9. Bruski, C. A. & D. W. Dunham, 1987. The importance of vision in agonistic communication of the crayfish Orconectes rusticus. Behaviour 103: 83–107.CrossRefGoogle Scholar
  10. Bushmann, P. J., 1999. Concurrent signals and behavioural plasticity in blue crab (Callinectes sapidus Rathbun) courtship. The Biological Bulletin 197: 63–71.CrossRefGoogle Scholar
  11. Christy, J. H. & D. Rittschof, 2010. Deception in visual and chemical communication in crustaceans. Chemical Communication in Crustaceans. Springer, New York: 313–333.CrossRefGoogle Scholar
  12. Christy, J. H., P. R. Y. Backwell & U. Schober, 2003. Interspecific attractiveness of structures built by courting male fiddler crabs: experimental evidence of a sensory trap. Behavioral Ecology and Sociobiology 53: 84–91.Google Scholar
  13. Cong, L., F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. A. Marraffini & F. Zhang, 2013. Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823.CrossRefGoogle Scholar
  14. Coohill, T. P., C. K. Bartell & M. Fingerman, 1970. Relative effectiveness of ultraviolet and visible light in eliciting pigment dispersion directly in melanophores of the fiddler crab, Uca pugilator. Physiological Zoology 43: 232–239.CrossRefGoogle Scholar
  15. Cowing, J. A., S. Poopalasundaram, S. E. Wilkie, P. R. Robinson, J. K. Bowmaker & D. M. Hunt, 2002. The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments. Biochemical Journal 367: 129–135.CrossRefGoogle Scholar
  16. De Man, J. G., 1879. On some species of the genus Palaemon Fabr. with descriptions of two new forms. Notes from the Leyden Museum 41: 165–184.Google Scholar
  17. Diaz, E. R. & M. Thiel, 2004. Chemical and visual communication during mate searching in rock shrimp. The Biological Bulletin 206: 134–143.CrossRefGoogle Scholar
  18. Donner, K., P. Zak, M. Viljanen, M. Lindström, T. Feldman & M. Ostrovsky, 2016. Eye spectral sensitivity in fresh-and brackish-water populations of three glacial-relict Mysis species (Crustacea): physiology and genetics of differential tuning. Journal of Comparative Physiology A 202: 297–312.CrossRefGoogle Scholar
  19. Dunham, P. J., 1978. Sex pheromones in Crustacea. Biological Reviews 53: 555–583.CrossRefGoogle Scholar
  20. Gaj, T., C. A. Gersbach & C. F. Barbas, 2013. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31: 397–405.CrossRefGoogle Scholar
  21. George, D. & P. Mallery, 2016. IBM SPSS Statistics 23 Step by Step: A Simple Guide and Reference. Routledge, New York.CrossRefGoogle Scholar
  22. Grafals, M., M. A. Sosa, C. M. Hernandez & J. A. Inserni, 2000. Role of serotonin and octopamine in aggressive behavior in the freshwater prawn Macrobrachium rosenbergii. FASEB Journal 14: A546.Google Scholar
  23. Hellemans, J., G. Mortier, A. De Paepe, F. Speleman & J. Vandesompele, 2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology 8: R19.CrossRefGoogle Scholar
  24. Herberholz, J., M. M. Sen & D. H. Edwards, 2003. Parallel changes in agonistic and non-agonistic behaviours during dominance hierarchy formation in crayfish. Journal of Comparative Physiology A 189: 321–325.Google Scholar
  25. Henze, M. J. & T. H. Oakley, 2015. The dynamic evolutionary history of Pancrustacean eyes and opsins. Integrative and Comparative Biology 55: 830–842.CrossRefGoogle Scholar
  26. Hunt, D. M., J. A. Cowing, S. E. Wilkie, J. W. Parry, S. Poopalasundaram & J. K. Bowmaker, 2004. Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates. Photochemical and Photobiological Sciences 3: 713–720.CrossRefGoogle Scholar
  27. Itagaki, H. & J. H. Thorp, 1981. Laboratory experiments to determine if crayfish can communicate chemically in a flow-through system. Journal of Chemical Ecology 7: 115–126.CrossRefGoogle Scholar
  28. Jiang, H., Z. Qian, W. Lu, H. Ding, H. Yu, H. Wang & J. Li, 2015. Identification and characterization of reference genes for normalizing expression data from red swamp crawfish Procambarus clarkii. International Journal of Molecular Sciences 16: 21591–21605.CrossRefGoogle Scholar
  29. Jiang, H., X. Li, Y. Sun, F. Hou, Y. Zhang, F. Li, Z. Gu & X. Liu, 2016. Insights into sexual precocity of female oriental river prawn Macrobrachium nipponense through transcriptome analysis. PloS ONE 11: e0157173.CrossRefGoogle Scholar
  30. Kim, M., M. Gee, A. Loh & N. Rachatasumrit, 2010. Ref-finder: A refactoring reconstruction tool based on logic query templates. In: Proceedings of the Eighteenth ACM SIGSOFT International Symposium on Foundations of Software Engineering, FSE 2010. ACM, New York: 371–372.Google Scholar
  31. Kravitz, E. A. & R. Huber, 2003. Aggression in invertebrates. Current opinion in neurobiology 13: 736–743.CrossRefGoogle Scholar
  32. Leelatanawit, R., K. Sittikankeaw, P. Yocawibun, S. Klinbunga, S. Roytrakul, T. Aoki, I. Hirono & P. Menasveta, 2009. Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology 152: 66–76.CrossRefGoogle Scholar
  33. Leelatanawit, R., A. Klanchui, U. Uawisetwathana & N. Karoonuthaisiri, 2012. Validation of reference genes for real-time PCR of reproductive system in the black tiger shrimp. PLoS ONE 7: e52677.CrossRefGoogle Scholar
  34. Li, J. Y., Z. L. Guo, X. H. Gan, D. L. Wang, M. F. Zhang & Y. L. Zhao, 2011. Effect of different dietary lipid sources on growth and gonad maturation of pre-adult female Cherax quadricarinatus (von Martens). Aquaculture Nutrition 17: e853–e860.CrossRefGoogle Scholar
  35. Livak, K. J. & T. D. Schmittgen, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)) Method. In: Methods. San Diego, CA: 25: 402–408.Google Scholar
  36. Maroufi, A., E. Van Bockstaele & M. De Loose, 2010. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Molecular Biology 11: 15.CrossRefGoogle Scholar
  37. Maruska, K. P. & R. D. Fernald, 2011. Social regulation of gene expression in the hypothalamic-pituitary-gonadal axis. Physiology 26: 412–423.CrossRefGoogle Scholar
  38. Mocellin, S. & M. Provenzano, 2004. RNA interference: learning gene knock-down from cell physiology. Journal of Translational Medicine 2: 1–6.CrossRefGoogle Scholar
  39. New, M. B., 2009. History and global status of freshwater prawn farming. In: Freshwater Prawns: Biology and Farming. Blackwell Science, Oxford: 194: 16–40.Google Scholar
  40. New, M. B. & C. M. Nair, 2012. Global scale of freshwater prawn farming. Aquaculture Research 43: 960–969.CrossRefGoogle Scholar
  41. Palaoro, A. V., L. A. Peres & S. Santos, 2013. Modulation of male aggressiveness through different communication pathways. Behavioral Ecology and Sociobiology 67 (2): 283–292.CrossRefGoogle Scholar
  42. Pan, D., N. He, Z. Yang, H. Liu & X. Xu, 2005. Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp (Penaeus japonicus) by suppression subtractive hybridization. Developmental and Comparative Immunology 29: 103–112.CrossRefGoogle Scholar
  43. Peltier, H. J. & G. J. Latham, 2008. Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA 14: 844–852.CrossRefGoogle Scholar
  44. Pfaffl, M. W., A. Tichopad, C. Prgomet & T. P. Neuvians, 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper–Excel-based tool using pair-wise correlations. Biotechnology Letters 26: 509–515.CrossRefGoogle Scholar
  45. Porter, M. L., D. I. Speiser, A. K. Zaharoff, R. L. Caldwell, T. W. Cronin & T. H. Oakley, 2013. The evolution of complexity in the visual systems of stomatopods: insights from transcriptomics. Integrative and Comparative Biology 53: 39–49.CrossRefGoogle Scholar
  46. Ra’anan, Z. & A. Sagi, 1985. Alternative mating strategies in male morphotypes of the freshwater prawn Macrobrachium rosenbergii (De Man). The Biological Bulletin 169: 592–601.CrossRefGoogle Scholar
  47. Rahi, M. L., 2017. Understanding the molecular basis of adaptation to freshwater environments by prawns in the genus Macrobrachium. PhD Thesis, Science and Engineering Faculty, Queensland University of Technology.
  48. Rahi, M. L., S. Amin, P. B. Mather & D. A. Hurwood, 2017. Candidate genes that have facilitated freshwater adaptation by palaemonid prawns in the genus Macrobrachium: identification and expression validation in a model species M. koombooloomba. PeerJ 5: e2977.CrossRefGoogle Scholar
  49. Salmon, M., 1983. Courtship mating system and sexual selection in decapods. In Rebach, S. & D. Dunham (eds), Studies in Adaptations. John Wiley and Sons, New York: 143–169.Google Scholar
  50. Schmittgen, T. D. & K. J. Livak, 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols 3: 1101–1108.CrossRefGoogle Scholar
  51. Schneider, R. A., R. Huber & P. A. Moore, 2001. Individual and status recognition in the crayfish, Orconectes rusticus: the effects of urine release on fight dynamics. Behaviour 138: 137–153.CrossRefGoogle Scholar
  52. Stebbing, P. D., M. G. Bentley & G. J. Watson, 2003. Mating behaviour and evidence for a female released courtship pheromone in the signal crayfish Pacifastacus leniusculus. Journal of Chemical Ecology 29: 465–475.CrossRefGoogle Scholar
  53. Takahashi, Y. & T. G. Ebrey, 2003. Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment. Biochemistry 42: 6025–6034.CrossRefGoogle Scholar
  54. Vandesompele, J., K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe & F. Speleman, 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3: 1–12.CrossRefGoogle Scholar
  55. Von Martens, E., 1868. Ueber einige neue Crustaceen und ueber die neuhollaendischen Suessvasserkrebse. Monatsbericte der Deutschen Akademie der Wissenschaften zu Berlin 1868: 608–619.Google Scholar
  56. Webster, S. G., R. Keller & H. Dircksen, 2012. The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction. General and Comparative Endocrinology 175: 217–233.CrossRefGoogle Scholar
  57. Wyatt, T. D., 2014. Pheromones and Animal Behaviour: Chemical Signals and Signatures, 2nd ed. Cambridge University Press, Cambridge: 419.Google Scholar
  58. Yang, Y. N., H. Ye, H. Huang, S. Li, X. Zeng, J. Gong & X. Huang, 2013. Characterization and expression of SpHsp60 in hemocytes after challenge to bacterial, osmotic and thermal stress from the mud crab Scylla paramamosain. Fish and Shellfish Immunology 35: 1185–1191.CrossRefGoogle Scholar
  59. Zito, I., D. L. Thiselton, M. B. Gorin, J. T. Stout, C. Plant, A. C. Bird, S. S. Bhattacharya & A. J. Hardcastle, 1999. Identification of novel RPGR (retinitis pigmentosa GTPase regulator) mutations in a subset of X-linked retinitis pigmentosa families segregating with the RP3 locus. Human Genetics 105: 57–62.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Science and Engineering Faculty, School of Earth, Environmental and Biological SciencesQueensland University of TechnologyBrisbaneAustralia

Personalised recommendations