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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)
Korean J. Environ. Biol. Vol.39 No.2 pp.160-168

Identification and molecular characterization of doublesex and mab-3-related transcription factor (dmrt ) in brackish water flea, Diaphanosoma celebensis, exposed to bisphenol analogs

Hayoung Cho, Min Jeong Jeon, Young-Mi Lee*
Department of Biotechnology, College of Convergence Engineering, Sangmyung University, Seoul 03016, Republic of Korea
* Corresponding author Young-Mi Lee Tel. 02-2298-5448 E-mail.
12/04/2021 10/05/2021 13/05/2021


Doublesex and mab-3 related transcription factor (dmrt) play crucial roles in sex determination and sex differentiation in vertebrates and invertebrates. Although dmrt genes have been identified in vertebrates, little is known about aquatic invertebrates. In this study, two dmrt genes, namely, Dc_dmrt93B and Dc_dmrt99B, were identified from brackish water flea, Diaphanosoma celebensis. Transcriptional changes were observed in the dmrt genes when the flea was exposed to bisphenol (BP), an endocrine disruptor. Sequence and phylogenetic analyses showed that both dmrt genes contained two conserved domains, namely, DM and DMA, closely clustered with those of Daphnia spp. Additionally, a significant increase in the Dc_dmrt99B mRNA expression level was observed upon exposure to intermediate concentrations of BP (bisphenol A>bisphenol S=bisphenol F, p<0.05), while the expression of Dc_dmrt93B mRNA was slightly modulated. These findings imply that the two dmrt genes may be involved in sex differentiation of D. celebensis. Furthermore, it was found that the ability of BP to modulate dmrt genes could affect development and reproduction. This study provides a basis for understanding the function of the dmrt genes and the molecular mode of action of BP in small crustaceans.



    Bisphenols (BPs) are chemicals with two phenolic hydro- xyl functional groups. They are widely used as industrial additives to produce polycarbonate plastics and epoxy resins (Ruan et al. 2015;Hu et al. 2019). Bisphenol A (BPA) is most often used in the production of food containers, ther- mal receipts, toys, medical equipment, and electronics (Caballero- Casero et al. 2016). However, the use of BPA has been restricted or regulated in many industrial fields because it can cause ROS production, DNA damage, gene mutagenesis; inhibit reproductive development; impair glucose and lipid metabolism; and disrupt the endocrine system (Meli et al. 2020). Therefore, BPA analogs such as bisphenol S (BPS) and bisphenol F (BPF) have been developed as alternative substances to replace the use of BPA in a variety of consumer products (Morales et al. 2020). BPS is used to make epoxy glues, baby bottles, and artistic organs etc.; BPF to make lacquers and dental sealants (Hu et al. 2019;Liu et al. 2021). However, it is essential to assess the risks associated with the use of BPA analogs, as several studies have demonstrated that BPA analogs also can cause similar to or higher toxic effects than BPA in genetic, cellular, and reproductive level (Chen et al. 2016;Wu et al. 2018;Liu et al. 2021). Owing to their wide usage, BP analogs are easily detected in the environment. In the Han River in South Korea, BPA (141 ng L-1), BPS (41 ng L-1), and BPF (633 ng L-1) have been detected (Yamazaki et al. 2015). In addition, BPA (1520 ng g-1 dw), BPS (44.9 ng g-1 dw), and BPF (384 ng g-1 dw) were detected in sludge from sewage treatment plants in Korea (Lee et al. 2015). The decomposition products of BP are discharged into the aquatic ecosystems via industrial wastewater, landfill leachate, urban sewage, and sludge (Caballero-Casero et al. 2016;Liu et al. 2021). Therefore, studies on the toxicity of BP to aquatic organisms are important.

    Reports highlight the detrimental effects of BP analogs on reproduction in aquatic organisms. For example, zebrafish (Danio rerio) showed abnormal sex ratios with an excess of females after ingesting food containing BPA (200 mg kg-1) (Drastichová et al. 2005). Only a few studies exist on the effects of BP on crustaceans. Superfeminization syndrome was found in the freshwater invertebrate Marisa cornuarietis after chronic exposure to low BPA concentrations (1 μg L-1) (Oehlmann et al. 2006). When D. rerio was exposed to 10 and 100 μg L-1 of BPS, the proportion of females increased by approximately 12% and 20%, respectively, and the spawning rate, hatching rate, and sperm count decreased (Naderia et al. 2014). Liu et al. (2019) found a gradual decrease in the number of neonates and suppressed reproduction in Daphnia magna during single and mixed exposures of BPA, BPF, and BPS for 21 days. Thus, BP may induce sexual disturbances and affect the reproduction of invertebrates (Canesi and Fabbri 2015). However, little information is available on the effects of BP at the molecular level in small crustaceans.

    The doublesex and mab-3 related transcription factor (dmrt) family is generally known to be involved in development and sex determination in vertebrates (Kopp 2012). In mammals, eight dmrt genes (dmrt1-dmrt8) have been identified; dmrt1 gene encoding the doublesex (dsx) and mab-3 proteins plays a key role in sex determination and differentiation (Bellefroid et al. 2013). In vertebrates, four dmrt genes (dmrt2a/2b, dmrt3, dmrt4/5 (dmrt99B), and dmrt93B) are commonly found (Mawaribuchi et al. 2019). Three dmrt genes (dmrt11E (2a/2b), dmrt99B (4/5), and dmrt93B) found in Arthropoda have been suggested to be originated from a common ancestor of bilaterian animals (dmrt2a/2b, dmrt4/5, and dmrt93B, respectively). In clado- cerans such as D. magna, environmental sex determination may be related to the expression of dmrt93B only in the testis (Kato et al. 2008). On the contrary, dmrt99B was suggested to be involved in vitellogenesis due to its higher expression in the ovary. However, the role of dmrt genes in aquatic invertebrates, particularly cladocerans, remains largely unknown.

    Cladocerans play an important role in the food chain as primary consumers; they are supplied as live food to higher order animals (Marcial and Hagiwara 2007). Diaphanosoma celebensis, a euryhaline cladoceran, can inhabit conditions with a wide range of temperature (15-35°C) and salinity (7-32 ppt). It is easy to maintain in a laboratory owing to its short life cycle (4-5 days) and small size in adulthood (Marcial and Hagiwara 2007;Kim et al. 2018;Yoo et al. 2019). Recently, D. celebensis was proposed as a model organism for studies on the effects of BP analogs on molting and reproduction of aquatic organisms (In et al. 2019, 2020).

    In the present study, two dmrt genes were identified in D. celebensis. Changes in the dmrt gene expression were further investigated after exposure to BP analogs to understand the role of dmrt and the effects of BPs on the organism. This study holds implications in understanding the effects of BP on reproduction in aquatic invertebrates.


    1. Experimental organism and culture maintenance

    The experimental organism, D. celebensis were obtained from the Korea Institute of Ocean Science & Technology (KIOST; South Korea) and maintained in Molecular Toxicology Laboratory at Sangmyung University since 2016. The cultured medium used 15 psu (practical salinity unit) of artificial seawater using filtered Instant Ocean (Aquarium systems, France) with 0.2-μm filters (Whatman, UK) and renewed once a week. The culture conditions were as follows: 25±1°C and a photoperiod of 12 h : 12 h light/dark. D. celebensis fed with 1.0-3.0×108 cells L-1 of Chlorella vulgaris daily as a food source.

    2. Reagents

    All chemical reagents were purchased from Sigma-Aldrich Co. (Saint Louis, USA). The stock solutions were prepared as follows: BPA (3 mg L-1), BPS (23 mg L-1), and BPF (5 mg L-1) in dimethyl sulfoxide (DMSO). All oligonucleotide synthesis and DNA sequencing were performed at Macrogen (Seoul, South Korea) and Bionics (Seoul, South Korea), respectively.

    3. Exposure test

    The D. celebensis (4-day-old; 200 individuals per concentration) was exposed to BPA (0.12, 0.6, and 3 mg L-1), BPS (0.92, 4.6, and 23 mg L-1), BPF (0.2, 1, and 5 mg L-1) for 48 h, based on the previous study (In et al. 2019). D. celebensis were not fed during the exposure period. The DMSO solvent concentration did not exceed 0.05%, where no mortality was observed.

    4. Total RNA extraction and cDNA synthesis

    After exposure to BP analogs, D. celebensis were harvested, total RNA was extracted using 500 μL of Trizol reagents, according to manufacturer’s instruction. The purity and quantity of the extracted total RNA were checked by gel ele- ctrophoresis and nanodrop spectrophotometry (Maestrogen Inc., Taiwan) and stored at -80°C until use. The cDNA was synthesized from 500 ng of the total RNA using Revert Aid First strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., USA) and kept at -20°C.

    5. Polymerase chain reaction (PCR)

    The cDNA sequences of dmrt99B and dmrt93B were obtai- ned from the local database of D. celebensis transcriptome (Molecular Toxicology Laboratory, Sangmyung University). The sequence was confirmed using Touchdown PCR. All PCR reactions contained 5 μL of 10X reaction buffer, 5 μL of dNTP (2 mM), 4 μL of 10 pmol primer set, 2 μL of cDNA, 0.5 μL of Geneall Taq (5 U μL-1), and 33.5 μL of PCR-grade water. The PCR primer was designed using the NetPrimer ( and Primer3 ( Primer sequence inf- ormation is shown in Table 1. PCR condition was as follows: 15 cycles of 95°C/3 min, 95°C/30 sec, 65.5°C/45 sec, 72°C/1 min 30 sec; 20 cycles of 95°C/30 sec, 54°C/45 sec, 72°C/1 min 30 sec; and finally, a final extension step at 72°C/5 min. The PCR products were confirmed to 1% agarose gel electrophoresis and directly sequenced. Conserved domains/motifs were analyzed by the conserved domain searching of National Center for Biotechnology Information (NCBI).

    6. Phylogenetic analysis

    Deduced amino acid sequences of D. celebensisdmrt99B and dmrt93B were aligned with those of other species retrie- ved from GenBank using Clustal X (version 1.83) and visual- ized using GeneDoc (version 2.6.002). GenBank accession numbers of other species dmrt99B and dmrt93B are indica- ted in Table 2. A phylogenetic analysis was performed to confirm the systematic location of D. celebensis dmrts, and tree was constructed by 1000 replication bootstraps with neighbor- joining methods using MEGA (version 6.0).

    7. Quantitative real time - polymerase chain reaction (qRT-PCR)

    We performed qRT-PCR to investigate the mRNA expression changes of dmrt99B and dmrt93B by exposure to BPs using the CFX96TM real-time PCR system. The reactant was 3 μL of cDNA (500 ng), 2 μL of 10 pmol primer set. The PCR product was analyzed on a 1% agarose gel under UV transilluminator to check the single band for confirming the specific amplification of target genes. The PCR efficiency is included in the range of 90-110% (92.9% for 18S; 102.8% for dmrt93B; and 107.7% for dmrt99B). PCR cycle condition was as follow: 95°C/10 min, followed by 33 cycles of 95°C/15 sec, 60°C/1 min. Finally, in order to check the amplification of a specific product, the melting curve reaction was increased by 0.5°C every 5 sec from 65°C to 95°C. All the experiments used SYBR master mix (KAPA Bioassay System, USA), and were performed in triplicate. The threshold cycle (Ct) was normalized by 18SrRNA. The fold changes were calculated using the 2-ΔΔCt method.

    8. Statistical analysis

    Data from all the experiments were represented as the mean±standard deviation (S.D.) of three replicates. Comparison of relative mRNA expression level was analyzed using one-way analysis of variance (one-way ANOVA) followed a Tukey’s test and t-test. The PASW Statistics ver. 18.0 program (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. A p-value below 0.05 was considered as statistically significant.


    1. Sequence analysis and phylogenetic relationship of D. celebensisdmrt93B and dmrt99B

    The partial cDNA sequence of dmrt93B was 1236 bp in length and consisted of a 33 bp long 5′-untranslated region (UTR) and 1203 bp long open reading frame (ORF) with complete domains that encode a putative polypeptide of 401 amino acids (aa) (Suppl. Fig. 1). The complete cDNA sequence of dmrt99B was 2422 bp in length with 5′-UTR of 140 bp, an ORF of 1755 bp encoding a putative polypeptide of 584 aa, and a 3′-UTR of 527 bp (Suppl. Fig. 2). The length of the dmrt93B polypeptide was 321 aa and 400 aa in the rotifer Brachionus koreanus (Kim et al. 2014) and D. magna (Kato et al. 2008), respectively. Notably, dmrt99B has been previously cloned and characterized in B. koreanus (381 aa; Kim et al. 2014), D. magna (603 aa; Kato et al. 2008), and the giant prawn Macrobrachium rosenbergii (618 aa; Yu et al. 2014).

    D. celebensis dmrt93B and dmrt99B proteins had two conserved domains; the DM domain and the DMA domain (Suppl. Figs. 1 and 2). Multiple alignments of the deduced amino acid sequences of these domains from D. celebensis and other species revealed their highly conserved nature across species (Fig. 1). Additionally, the DM domain was found to contain a nuclear localization signal (NLS; KGHKR, 18-22 aa) and two zinc (Zn2+) binding sites (CCHC and HCCC). The DM domain is also called the DM DNA-binding domain and is named from doublesex (dsx) and mab-3. where dsx has one amino-terminal domain and mab-3 has two amino-terminal domains. The dsx DM domain binds to and dimerizes palindromic DNA (Narendra et al. 2002). The DMA domain can be identified in the carboxyl-terminal of the DM domain; a DM domain protein with this motif is referred to as a DMRTA protein, and is considered as the DMRTA motif. The function of the DMA domain remains unknown.

    In the present study, sequence alignments of the domain amino acids demonstrated that the DM domain was highly conserved whereas the DMA domain exhibited low identity across species. The DM domains of Dc_dmrt93B and Dc_ dmrt99B shared 80% identity. Dc_dmrt93B and dmrt99B had the highest identity with those of D. magna (100% and 97%, respectively) and Daphnia pulex (100% and 100%, respectively) (Suppl. Fig. 3). Considering the DMA domain, Dc_dmrt93B shared a low identity of 34% with Dc_dmrt99B. Dc_dmrt93B showed the highest identity with that of D. magna (61% for 93B; 71% for 99B) and D. pulex (60% for 93B; 72% for 99B) (Suppl. Fig. 4). Regions other than the DM domain showed low similarity, which may contribute to distinct phylogenetic clusters (Suppl. Fig. 5).

    Phylogenetic analysis revealed that the dmrts of D. celebensis closely clustered with those of the other invertebrates, particularly Daphnia spp. (Fig. 2). As expected, the dmrt93B group was separated from the dmrt99B group in invertebrates, in which dmrts (dmrt1 to dmrt5) of mammals were dis- tinctly clustered from those of invertebrates. Kato et al. (2008) also found similar results wherein Daphnia dmrt99B and dmrt93B were grouped separately. Each gene was clustered with that of the insects and was distinct from those of the vertebrates. Notably, the Bayesian tree of bilaterian dmrt genes depicts the same clustering pattern (Mawaribuchi et al. 2019).

    2. Transcriptional modulation of D. celebensisdmrt93B and dmrt99B after exposure to BP analogs

    The changes in the dmrt genes were investigated at the transcriptional level in D. celebensis after 48 h of exposure to three BP (BPA, BPS, and BPF). Both genes showed similar expression patterns after exposure. When compared with the control group, the lowest (BPA 0.12 mg L-1, BPS 0.92 mg L-1, and BPF 0.2 mg L-1) and highest (BPA 3 mg L-1, BPS 23 mg L-1, and BPF 5 mg L-1) concentrations of each chemical did not significantly modulate the expression level of these genes (Fig. 3). However, at their intermediate concentrations (BPA 0.6 mg L-1, BPS 4.6 mg L-1, and BPF 1 mg L-1), the expression of both dmrt genes was upregulated (p<0.05); the Dc-dmrt99B mRNA level was higher than that of Dc-dmrt93B. In particular, the expression of dmrt99B was approximately 3.5-fold higher than that of the control group at 0.6 mg L-1 of BPA (p<0.001). On the contrary, mRNA levels of both dmrt genes were lower (>2-fold change, p<0.05) upon exposure to BPS and BPF when compared with those after exposure to BPA. The U- or inver- ted U-shaped curve obtained at different concentrations of BP analogs indicates non-monotonic dose response effects and is a characteristic of endocrine-disrupting compounds (EDCs), in particular BPA (Vandenberg 2013;Zhang et al. 2016). In this case, no response or decreased response are detected at low and high concentration of EDCs, which can be challenging for risk assessment of their effects (Beronius and Vandenberg 2015;Lagarde et al. 2015).

    The dmrt plays an essential role in the development and differentiation of male testicular in crustaceans such as the Chinese mitten crab Eriocheir sinensis (Zhang et al. 2010). This family is involved in germline development, embryonic development, and sexual maturation in the river prawn, Macrobrachium nipponense (Wang et al. 2019). Moreover, the dmrt of D. magna contributes to environmental sex determination and reproduction (Kato et al. 2008, 2011). However, the role of dmrt in D. celebensis remains unknown.

    Several studies strongly support the fact that sex different-iation-related genes are potential molecular targets for EDCs in vertebrates and invertebrates (Iguchi et al. 2006;Zhang et al. 2008;Rhee et al. 2011;Toyota et al. 2021). Kim et al. (2014) studied the effects of benzo[a]pyrene (a known reproductive toxin) in monogonont rotifer B. koreanus. They observed that a decrease in the mRNA levels of dmrt11E, dmrt93B, and dmrt99B may be related to growth retardation and reproduction inhibition. In D. magna exposed to fenoxycarb, a juvenile hormone analog, the testisspecific dmrt93B mRNA expression was upregulated (Kim et al. 2011). Together with our results, these findings suggest that the dmrt family of proteins may be a molecular target for environmental chemicals that interrupt the steroid hormone pathway and result in negative effects on the growth, development, and reproduction of cladocerans.

    Kato et al. (2008) have suggested a sex-dependent, dimor- phic expression of dmrt93B and dmrt99B in D. magna. Here, dmrt93B was testis-specific and dmrt99B was ovary-specific. In particular, they assumed that dmrt99B might be invol- ved in the production of vitellogenin. In the present study, the higher expression of Dc_dmrt99B compared to that of Dc_dmrt93B upon BP exposure seems to be related to the reproductive strategy of parthenogenesis adopted by D. celebensis. In our previous study, vitellogenine mRNA levels were upregulated upon exposure to BPA and BPS but reduced in the BPF-exposed group (In et al. 2020). These findings suggest that BP may affect reproduction in D. celebensis by regulating the Dc_dmrt99B expression by different modes. However, the modulation of additional genes and endogenous hormones during development and reproduction needs to be further studied for a better understanding of the molecular mechanisms underlying sex differentiation, and the effect of EDCs such as BP on D. celebensis.

    In conclusion, we identified the dmrt93B and dmrt99B genes from the brackish water flea D. celebensis. The proteins encoded by these genes contained two conserved domains and were closely clustered with those of Daphnia spp. This result indicates their evolutionarily conserved function in sex differentiation of D. celebensis. Our findings suggest that BP analogs, in particular BPA, may affect the development and reproduction by modulating dmrt93B and dmrt99B in different modes in this organism.


    This work was supported by a grant from Sangmyung University (2020) funded to Young-Mi Lee.



    Multiple alignments of the deduced amino acid sequences of (A) DM domain and (B) DMA domain of Diaphanosoma celebensisdmrt93B and dmrt99B genes using Clustal X and GenDoc. The red box represents the conserved nuclear localization signal (NLS). The asterisk and the cross indicate two conserved zinc (Zn2+) binding sites for site I (CCHC) and site II (HCCC), respectively. The shaded region indicates conserved residues. Abbreviations: Dc (Diaphanosoma celebensis), Dmag (Daphina magna), Dpul (Daphnia pulex), Bk (Brachionus koreanus), Dmel (Drosophila melanogaster), Hs (Homo sapiens).


    Phylogenetic analysis of the deduced amino acid sequences of Diaphanosoma celebensisdmrt93B and dmrt99B with those of other species retrieved from GenBank (Table 2). The phylogenetic tree was constructed by the neighbor-joining method using MEGA (version 6.0). Bootstrapping replications of 1,000.


    mRNA expression of dmrt93B and dmrt99B in Diaphanosoma celebensis exposed to BPA (0.12, 0.6, and 3 mg L-1), BPS (0.92, 4.6, and 23 mg L-1), and BPF (0.2, 1, and 5 mg L-1) for 48 h. Different letters indicate significant differences among concentrations determined using one-way ANOVA followed by Tukey’s test. Asterisks represent significant differences between genes using t-test. p-value below 0.05 was considered as statistically significant.


    Primer sets used in this study

    GenBank accession numbers of the sequences used for phylogenetic analysis


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