Cytogenetic of Brachyura (Decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species




aquaculture, chromosomes, confocal microscope, mangrove crabs, DAPI, metaphase


Brachyura is one of the most specious infra-order belonging to Decapoda and it plays a central role from an ecological and economic point of view. Despite its importance, cytogenetic studies on Brachyura (Decapoda) are extremely limited due to the difficulties in obtaining chromosome preparations of good quality. Molecular cytogenetic have proven to provide basic knowledge on the genome organization of species and in methods for manipulating genomes. It is also very useful to support aquaculture. In this study we focused on six semi-terrestrial mangrove crab species to test several variations of technical steps to produce chromosome preparations in Brachyura. The best results were obtained using cells from early stage embryos incubated with 0.0005% nocodazole or 0.05% colchicine and hypotonized with 0.075 M KCl solution. The best method to analyze the chromosomes was the use of confocal microscope after DAPI staining. We recorded a high chromosome number for the six study species. Similar chromosome morphology was recorded for allied species likely due to phylogenetic relationship. Variable results with cytogenetic treatments in different species suggest that there may be a species-specific response to the techniques we tested. Chromosome number ranges reported in this study will be useful in future genome sequencing studies, i.e. to assess the quality of sequencing assemblies.


Download data is not yet available.


Abdelrahman H, ElHady M, Alcivar-Warren A, Allen S, Al-Tobasei R, Bao L, Beck B, Blackburn H, Bosworth B, Buchanan J, Chappell J. 2017. Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research. BMC Genomics. 18:191.
Adiyodi RG. 2008. Reproduction and Development. In: Burggren WW, McMahon BR, editors. Biology of Land Crabs. Cambridge, New York: Cambridge University Press; pp. 139-185.
Aiyun D, Siliang Y. 1991. Crabs of the China seas. Beijing: China Ocean Press and Berlin: Springer-Verlag.
Anger K. 2001. The Biology of Decapod Crustacean Larvae. In: Anger K, Balkema AA, editors. Crustacean Issues 14. Lisse: AA Balkema Publishers; pp. 1-420.
Awodiran MO, Fajana HO, Yusuf AO. 2016. Chromosomal and ecological studies of freshwater crabs, Sudanonautes aubryi (H. Milne-Edward, 1853) (Brachyura: Potamonautidae) from Ile-Ife, Nigeria. Zool Ecol. 26:295-300.
Bartley DM, Rana K, Immink AJ. 2001. The use of inter-specific hybrids in aquaculture and fisheries. Rev Fish Biol Fish. 10:325-337.
Burton JN, Adey A, Patwardhan RP, Qiu R, Kitzman JO, Shendure J. 2013. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol 31:1119.
Campos-Ramos R. 1997. Chromosome studies on the marine shrimps Penaeus Uannamei and P. Californiensis (Decapoda). J Crust Biol. 17:666-673.
Cannicci S, Simoni R, Giomi F. 2011. Role of the embryo in crab terrestrialisation: an ontogenetic approach. Mar Ecol Prog Ser. 430:121-131.
Charmantier G. 1998. Ontogeny of osmoregulation in crustaceans: a review. Invert Reprod Dev. 33:177-190.
Coghlan A, Eichler EE, Oliver SG, Paterson AH, Stein L. 2005. Chromosome evolution in eukaryotes: a multi-kingdom perspective. Trends Genet. 21:673-82.
Colombo L, Barbaro A, Francescon A, Libertini A, Bortolussi M, Argenton F, Dalla Valle L, Vianello S, Belvedere P. 1998. Towards an integration between chromosome set manipulation, intergeneric hybridization and gene transfer in marine fish culture. In: Bartley D, Basurco B, editors. Genetics and Breeding of Mediterranean Aquaculture Species. Zaragoza: Ciheam; pp. 77-122 (Cahiers Options Méditerranéennes; n. 34).
Coluccia E, Cannas R, Cau A, Deiana AM, Salvadori S. 2004. B chromosomes in Crustacea Decapoda. Cytogenet Genome Res. 106:215-221.
Cui Z, Hui M, Liu Y, Song C, Li X, Li Y, Liu L, Shi G, Wang S, Li F, et al. 2015. High-density linkage mapping aided by transcriptomics documents ZW sex determination system in the Chinese mitten crab Eriocheir sinensis. Heredity. 115:206-215.
De Grave S, Pentcheff ND, Ahyong ST, Chan T, Crandall KA, Dworschak PC, Felder DL, Feldmann RM, Fransen CHJM, Goulding LYD, et al. 2009. A classification of living and fossil genera of decapods crustaceans. Raff Bull Zool. 21:1-109.
FAO. 2016. The State of World Fisheries and Aquaculture 2016. Contributing to food security and nutrition for all. Rome.
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3:294-299.
Galetti PM, Aguilar CT, Molina WF. 2000. An overview of marine fish cytogenetics. In: Solè-Cava AM, Russo CAM, Thorpe JP, editors. Marine Genetics. Dordrecht: Springer; pp. 55-62.
González-Tizón AM, Rojo V, Menini E, Torrecilla Z, Martínez-Lage A. 2013. Karyological analysis of the shrimp Palaemon serratus (Decapoda: Palaemonidae). J Crust Biol. 33:843-848.
Gui J, Zhu Z. 2012. Molecular basis and genetic improvement of economically important traits in aquaculture animals. Chin Sci Bull. 57:1751-1760.
Guo L, Accorsi A, He S, Guerrero-Hernández C, Sivagnanam S, McKinney S, Gibson M, Sánchez Alvarado A. 2018. An adaptable chromosome preparation methodology for use in invertebrate research organisms. BMC Biology. 16:25.
Hong Y, Yang X, Cheng Y, Liang P, Zhang J, Li M, Shen C, Yang Z, Wang C. 2013. Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the mitten crab, Eriocheir sinensis. In Vitro Cell Dev Biol Anim. 49:716-727.
Indy JR, Arias-Rodriguez L, Páramo-Delgadillo S, Hernández-Vidal U, Álvarez-González CA, Contreras-Sánchez WM. 2010. Mitotic karyotype of the tropical freshwater crayfish Procambarus (Austrocambarus) llamasi (Decapoda: Cambaridae). Rev Biol Trop. 58:655-662.
Jazayeri A, Papahn F, Moslehabadi Z. 2010. The study of karyological of Blue crab (Portunus plagicus). J Mar Biol. 2:37-44.
Lécher P, Defaye D, Noel P. 1995. Chromosomes and nuclear DNA of Crustacea. Invertebr Reprod Dev. 27:85-114.
Lee TH, Naitoh N, Yamazaki F. 2004. Chromosome studies on the mitten crabs Eriocheir japonica and E. sinensis. Fish Sci. 70:211-214.
Lee TH, Shirayama S. 2008. A preliminary study on the problems in the preparation of Artemia parthenogenetica chromosomes for scanning electron microscopy. J Crust Biol, 28:167-170.
Ma C, Chen H, Xin M, Yang R, Wang X. 2012. KGBassembler: a karyotype-based genome assembler for Brassicaceae species. Bioinformatics. 28:3141-3143.
Martin P, Thonagel S, Scholtz G. 2016. The parthenogenetic Marmorkrebs (Malacostraca: Decapoda: Cambaridae) is a triploid organism. J Zool Syst Evol Res. 54:13-21.
Mittal OP, Dhall U. 1971. Chromosome studies in three species of freshwater decapods (Crustacea). Cytologia. 36:633-638.
Mlinarec J, Porupski I, Maguire I, Klobučar G. 2016. Comparative karyotype investigations in the white-clawed crayfish Austropotamobius pallipes species complex and stone crayfish A. torrentium (Decapoda: Astacidae). J Crust Biol. 36:87-93.
Moralli D, Yusuf M, Mandegar MA, Khoja S, Monaco ZL, Volpi EV. 2011. An improved technique for chromosomal analysis of human ES and iPS cells. Stem Cell Rev Rep. 7:471-477.
Ng PKL, Guinot D, Davie PJF. 2008. Systema Brachyurorum: part I. An annotated checklist of extant Brachyuran crabs of the world. Raff Bull Zool. 17:1-286.
Niiyama H. 1942. On the chromosomes of four species of crabs (Brachyura, Decapoda) (in Japanese). Zool Mag. 54:302-304.
Niiyama H. 1959. A comparative study of the chromosomes in decapods, isopods and amphipods, with some remarks on cytotaxonomy and sex-determination in the Crustacea. Mem Fac Fish Hokkaido Univ. 7:1-60.
Niiyama H. 1966. The chromosomes of two species of edible crabs (Brachyura, Decapoda, Crustacea). Mem Fac Fish Hokkaido Univ. 16:201-205.
Salvadori S, Coluccia E, Deidda F, Cau A, Cannas R, Deiana AM. 2012. Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences. Genetica. 140:429-437.
Salvadori S, Coluccia E, Deidda F, Cau A, Cannas R, Lobina C, Sabatini A, Deiana AM. 2014. Karyotype, ribosomal genes, and telomeric sequences in the crayfish Procambarus clarkii (Decapoda: Cambaridae). J Crust Biol. 34:525-531.
Sashikumar A, Desai PV. 2008. Development of primary cell culture from Scylla serrata. Cytotech. 56:161-169.
Scalici M, Solano E, Gibertini G. 2010. Karyological analyses on the Australian crayfish Cherax destructor (Decapoda: Parastacidae). J Crust Biol. 30:528-530.
Schubart CD, Huber MGJ. 2006. Genetic comparisons of German populations of the stone crayfish, Austropotamobius torrentium (Crustacea: Astacidae). Bull Fr Peche Prot Milieux Aquat. 380-381:1019-1028.
Sharakhov IV. 2014. Protocols for Cytogenetic Mapping of Arthropod Genomes. Boca Raton: CRC Press.
Shih H, Ng PKL, Davie PJF, Schubart CD, Türkay M, Naderloo R, Jones D, Liu MY. 2016. Systematics of the family Ocypodidae Rafinesque, 1815 (Crustacea: Brachyura), based on phylogenetic relationships, with a reorganization of subfamily rankings and a review of the taxonomic status of Uca Leach, 1814, sensu lato and its subgenera. Raff Bull Zool. 64:139-175.
Shpak N, Manor R, Aflalo ED, Sagi A. 2017. Three generations of cultured prawn without W chromosome. Aquaculture. 467:41-48.
Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 87:651-701.
Simoni R, Cannicci S, Anger K, Pörtner HO, Giomi F. 2011. Do amphibious crabs have amphibious eggs? A case study of Armases miersii. J Exp Mar Biol Ecol. 409:107-113.
Sola L, Cataudella S, Capanna E. 1981. New developments in vertebrate cytotaxonomy III. Karyology of bony fishes: a review. Genetica. 54:285-328.
Sumner AT. 2008. Chromosomes: organization and function. 1st ed. New Jersey: John Wiley & Sons.
Swagatika S, Kumar MP. 2014. Karyological analysis of mud crab and flower crab of Odisha. Int Res J Biol Sci. 3:51-56.
Tan X, Qin JG, Chen B, Chen L, Li X. 2004. Karyological analyses on redclaw crayfish Cherax quadricarinatus (Decapoda: Parastacidae). Aquaculture. 234:65-76.
Thiriot-Quievreux C. 2002. Review of the literature on bivalve cytogenetics in the last ten years. Cah Biol Mar. 43:17-26.
Thiriot-Quiévreux C. 2003. Advances in chromosomal studies of gastropod molluscs. J Molluscan Stud. 69:187-202.
Torrecilla Z, Martínez-Lage A, Perina A, González-Ortegón E, González-Tizón AM. 2017. Comparative cytogenetic analysis of marine Palaemon species reveals a X1X1X2X2/X1X2Y sex chromosome system in Palaemon elegans. Front Zool. 14:47.
Toullec JY. 1999. Crustacean primary cell culture: a technical approach. Methods Cell Sci. 21:193-198.
Trentini M, Corni MG, Froglia C. 1989. The chromosomes of Liocarcinus vernalis (Risso, 18 16) and Liocarcinus depurator (L., 1758) (Decapoda, Brachyura, Portunidae). Biol Zent Bl. 108:163-166.
Trentini M, Corni MG, Froglia C. 1992. The chromosomes of Carcinus mediterraneus Czerniavsky, 1884 Liocarcinus maculatus (Risso, 1827) and Necora puber (L., 1767) (Decapoda, Brachyura, Portunidae). Zool Anz. 228:39-44.
Vishnoi DN. 1972. Studies on the chromosomes of some Indian Crustacea. Cytologia. 37:43-51.
Zeng H, Ye H, Li S, Wang G, Huang J. 2010. Hepatopancreas cell cultures from mud crab, Scylla paramamosain. In Vitro Cell Dev Biol Anim. 46:431-437.
Zhu DF, Wang CL, Li ZQ. 2005. Karyotype analysis on Portunus trituberculatus. J Fish China. 25:649-653.




How to Cite

Iannucci, A., Cannicci, S., Lin, Z. ., Yuen, K. W. ., Ciofi, C., Stanyon, R., & Fratini, S. (2020). Cytogenetic of Brachyura (Decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species. Caryologia, 73(2), 39-49.