Sex determination is a fundamental developmental process, affecting the sexual differentiation of gonads, and leads to sex-specific differences in behavior, physiology and morphology. Sex-determining systems can be divided into two categories: genotypic sex determination (GSD) and environmental sex determination (ESD) [1–3]. GSD is attributed to the genetic segregation of genes, often residing on sex chromosomes that initiate alternate sex-determining developmental pathways. In contrast, ESD has repeatedly arisen during animal evolution  and is initiated by diverse environmental cues, such as temperature, photoperiod, nutrition and population density, that trigger alternative genetic signals, resulting in the regulation of male or female sex-determining genes [5, 6].
Natural selection of rare mutational variants has been suggested to mediate the transitions between GSD and ESD [7, 8]. A previous phylogenetic analysis revealed that there have been at least three independent switches from GSD to ESD in lizards, and six transitions from ESD to GSD in turtles . Moreover, previous experiments using temperature-sensitive mutations created artificially in Caenorhabditis elegans demonstrated how GSD could rapidly evolve into ESD as a consequence of a mutation in key sex determining genes . Orthologs of GSD genes such as dmrt1, sox9 and cyp19a (aromatase) are expressed in the gonads during the temperature-sensitive period in ESD of reptiles . Thus, according to the current interpretation of these data, ESD mechanisms are likely to share many sex-determining components with GSD .
Sex determination systems in insects vary considerably in key factors and regulatory mechanisms to develop sex-specific traits. The sex determination mechanism in Drosophila melanogaster is best understood. The ratio of X chromosomes to autosomes (X:A ratio) is thought to provide the initial signal for the activation of sex-lethal (sxl), a master gene of the sex determination cascade. Then, sxl is produced as the sex-specific splicing isoforms. Sxl in female acts on the pre-mRNA of transformer (tra) resulting in reproduction of functional Tra. The functional Tra in the female, in concert with Tra-2, regulates the production of female-specific doublesex (dsx) mRNA. The male-specific splice form of dsx mRNA is the default splice-variant in D. melanogaster. Dsx regulates the various sex-specific traits such as gonads. Recently, sex determination mechanisms have also been demonstrated in various insect lineages such as Diptera (Musca domestica and Ceratitis capitata), Hymenoptera (Apis mellifera and Nasonia vitripennnis) and Coleoptera (Tribolium castaneum). These studies revealed that tra and dsx are highly conserved among insects [11–14]. However, in case of Lepidoptera, Bombyx mori, tra and tra-2 are assumed not to be required for the sex-specific splicing of Bmdsx pre-mRNA, because Bmdsx has no Tra/Tra-2 binding motif. Recently, it has been revealed that binding of the BmPSI, a Bombyx homolog of P-element somatic inhibitor, to the exonic splicing suppressor sequence on expected region is involved in sex-specific splicing of Bmdsx. These data suggest that upstream genetic cascades of dsx might be diverse among insects.
The Cladocera (commonly called water fleas) is an ancient clade of branchiopod crustaceans comprising 16 or 18 family lineages [15, 16] that all reproduce by cyclical parthenogenesis involving ESD . The most well studied species are from the family Daphniidae, particularly of the genus Daphnia. Daphnia inhabit freshwater ponds and lakes on all continents and are known to switch between parthenogenetic and sexual reproduction when environmental conditions for growth and reproduction deteriorate. During normal growing conditions, populations are most often entirely composed of females. However, shortened photoperiod, a lack of food and/or increased population density all lead to the clonal production of males that are genetically identical to their sisters and mothers . First instar male juveniles are easily distinguished from the females . During maturation, daphniids undergo morphological sexual differentiation of various somatic tissues, including the armament of a first thoracic leg with the copulatory hook in males, which becomes larger during the fifth instar . Gonads develop and finally settle at both sides of the gut during embryogenesis in both sexes . The appearance of males allows sexual reproduction to occur [22, 23], when females begin producing haploid eggs requiring fertilization.
Recently, we and others found that male production occurred independently of environmental cues by treatment with exogenous juvenile hormone (JH) or its analogs [24, 25]. Exposure of D. magna to JH analogs at the stage corresponding to the environmentally-sensitive period for sex determination of a cladoceran species of the family Moinidae , produced exclusively male broods, suggesting that JH could be a key molecule for understanding mechanisms of ESD [24, 27, 28].
A doublesex (dsx) gene was originally identified in D. melanogaster as a critical and terminal transcription factor in the fly sex determining cascade. Dsx is spatially and temporally transcribed into two sex-specific splice forms conferring sexually dimorphic traits during development [29, 30]. The dsx gene contains two conserved domains: the Dsx/Mab-3 (DM) domain at the N-terminus and the oligomerization domain at the C-terminus . Genes encoding DM-domain (DM-domain genes) were discovered to play a related role in C. elegans[32, 33] and also in vertebrates [34–36]. In contrast, results from numerous studies have shown that other genes in the genetic sex-determination cascade widely diversified among species [1, 2, 37].
To understand the molecular and evolutionary relationships between GSD and ESD, we previously identified and analyzed three DM-domain genes (DMRT11E, DMRT93B and DMRT99B) from D. magna, displaying sexual dimorphic gene expression patterns in adult gonads . However, none of these DM-domain genes exhibited sexually dimorphic expression patterns during embryonic development, suggesting that they are not involved in sex determination . Two additional DM-domain genes were later found in the D. magna expressed sequence tags (ESTs) database . Therefore, we analyzed the function of these two genes from D. magna using gene manipulations that we developed . These experiments revealed that two dsx genes in D. magna were obtained by lineage-specific duplication, and then one of the paralogs, Daphnia magna dsx1 (DapmaDsx1), plays an important role in directing the major sexually dimorphic development of D. magna. In contrast, specific function of Daphnia magna dsx2 (DapmaDsx2) remains unknown. These newly identified dsx genes showed greater sequence similarity at the amino acid sequence level to known insect dsx genes than to the previously identified DM-domain containing genes in D. magna. A genome-wide study of gene functions in D. pulex suggested that lineage-specific duplicated genes are most responsive to varying environmental conditions . In the present study, we investigated the sequence and functional conservation of the two dsx genes in a broader taxonomic sampling of cladocerans by cloning dsx homologs, and determining their sex specific expression in four species representing two families and three genera. We also analyzed the structures of cloned dsx genes of D. magna and D. pulex including their putative regulatory motifs and putative transcription factor binding sites in the 5’ upstream regions of these duplicated dsx genes.