Open Access Research article

Molecular cloning of doublesex genes of four cladocera (water flea) species

Kenji Toyota1, Yasuhiko Kato12, Masaru Sato1, Naomi Sugiura13, Shinichi Miyagawa1, Hitoshi Miyakawa1, Hajime Watanabe12, Shigeto Oda1, Yukiko Ogino1, Chizue Hiruta1, Takeshi Mizutani1, Norihisa Tatarazako4, Susanne Paland5, Craig Jackson5, John K Colbourne56 and Taisen Iguchi1*

Author Affiliations

1 Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, and Department of Basic Biology, Faculty of Life Science, Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan

2 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

3 Toyota Nishi High School, 14-65 Kosaka, Toyota, Aichi, 471-0035, Japan

4 National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki, 305-8506, Japan

5 The Center for Genomics and Bioinformatics, Indiana University, 915 East Third Street, Bloomington, IN, 47405, USA

6 Present address: School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK

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BMC Genomics 2013, 14:239  doi:10.1186/1471-2164-14-239

Published: 10 April 2013



The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species.


We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5’ upstream regulatory elements are preserved in D. magna and D. pulex.


The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.

Cladocera; Doublesex genes; Environmental sex determination; Gene duplication; Annotated regulatory regions