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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Physiol.</journal-id>
<journal-title>Frontiers in Physiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Physiol.</abbrev-journal-title>
<issn pub-type="epub">1664-042X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">1201846</article-id>
<article-id pub-id-type="doi">10.3389/fphys.2023.1201846</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Physiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Analyses of the <italic>Dmrt</italic> family in a decapod crab, <italic>Eriocheir sinensis</italic> uncover new facets on the evolution of DM domain genes</article-title>
<alt-title alt-title-type="left-running-head">Zhang et al.</alt-title>
<alt-title alt-title-type="right-running-head">
<ext-link ext-link-type="uri" xlink:href="https://doi.org/10.3389/fphys.2023.1201846">10.3389/fphys.2023.1201846</ext-link>
</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Zhang</surname>
<given-names>Peng</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2256335/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yang</surname>
<given-names>Yanan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1389182/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Xu</surname>
<given-names>Yuanfeng</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1882016/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Cui</surname>
<given-names>Zhaoxia</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1744024/overview"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>School of Marine Sciences</institution>, <institution>Ningbo University</institution>, <addr-line>Ningbo</addr-line>, <country>China</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Laboratory for Marine Biology and Biotechnology</institution>, <institution>Pilot Qingdao National Laboratory for Marine Science and Technology (Qingdao)</institution>, <addr-line>Qingdao</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>
<bold>Edited by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1737926/overview">Yafei Duan</ext-link>, South China Sea Fisheries Research Institute, China</p>
</fn>
<fn fn-type="edited-by">
<p>
<bold>Reviewed by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/487118/overview">Yilei Wang</ext-link>, Jimei University, China</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/2123695/overview">Jianjian Lv</ext-link>, Chinese Academy of Fishery Sciences (CAFS), China</p>
</fn>
<corresp id="c001">&#x2a;Correspondence: Zhaoxia Cui, <email>cuizhaoxia@nbu.edu.cn</email>
</corresp>
</author-notes>
<pub-date pub-type="epub">
<day>26</day>
<month>05</month>
<year>2023</year>
</pub-date>
<pub-date pub-type="collection">
<year>2023</year>
</pub-date>
<volume>14</volume>
<elocation-id>1201846</elocation-id>
<history>
<date date-type="received">
<day>07</day>
<month>04</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>16</day>
<month>05</month>
<year>2023</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2023 Zhang, Yang, Xu and Cui.</copyright-statement>
<copyright-year>2023</copyright-year>
<copyright-holder>Zhang, Yang, Xu and Cui</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>DM domain genes are a group of transcription factors that are integral to sexual development and its evolution in metazoans. Their functions and regulatory mechanisms are not well understood in Malacostraca (crabs and crayfish) while these sex regulators have been widely identified in the past decade. In this study, the <italic>Dmrt</italic> family was investigated in the decapod crab, <italic>Eriocheir sinensis</italic>. We find that most members of the <italic>EsDmrt</italic> family begin to enrich around the juvenile 1 stage. In reproductive organs, <italic>EsDsx1</italic>, <italic>EsDsx2</italic>, <italic>EsiDMY</italic> and <italic>EsiDmrt1a</italic> highly express in the male-specific androgenic gland (AG), while <italic>EsDmrt-like</italic>, <italic>EsDsx-like</italic>, <italic>EsDmrt11E</italic>, and <italic>EsiDmrt1b</italic> show relatively high expression in testis. Also, we find the highly aberrant expression of <italic>EsiDMY</italic> and <italic>EsiDmrt1a</italic> in the chimeric AG, strongly indicating their function in AG development. Moreover, RNA interference of <italic>EsDsx1</italic>, <italic>EsiDMY</italic>, and <italic>EsiDmrt1a</italic> results in a significant decrease in transcription of the <italic>Insulin-like androgenic hormone</italic> (<italic>IAG</italic>), respectively. Our findings suggest that <italic>Dmrt</italic> genes in <italic>E. sinensis</italic> primarily function in male sexual differentiation, especially in AG development. Besides, this study identifies two unique groups of <italic>Dmrt</italic> genes in Malacostraca: <italic>Dsx</italic> and <italic>iDmrt1</italic>. In Malacostraca <italic>Dsx</italic>, we uncover a cryptic mutation in the eight zinc motif-specific residues, which were firmly believed to be invariant across the <italic>Dmrt</italic> family. This mutation sets the Malacostraca <italic>Dsx</italic> apart from all the other <italic>Dmrt</italic> genes and implies a different way of transcriptional regulation. Genes from the <italic>iDmrt1</italic> group show phylogenetical limitation to the malacostracan species and underwent positive selection, suggesting their highly specialized gene function to this class. Based on these findings, we propose that <italic>Dsx</italic> and <italic>iDmrt1</italic> in Malacostraca have developed unique transcriptional regulation mechanisms to facilitate AG development. We hope that this study would contribute to our understandings of sexual development in Malacostraca and provide new insights into the evolutionary history of the <italic>Dmrt</italic> family.</p>
</abstract>
<kwd-group>
<kwd>sexual development</kwd>
<kwd>
<italic>Dmrt</italic>
</kwd>
<kwd>evolution</kwd>
<kwd>malacostraca</kwd>
<kwd>
<italic>Eriocheir sinensis</italic>
</kwd>
</kwd-group>
<contract-sponsor id="cn001">National Natural Science Foundation of China<named-content content-type="fundref-id">10.13039/501100001809</named-content>
</contract-sponsor>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Aquatic Physiology</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1">
<title>1 Introduction</title>
<p>Animals have evolved to become sexual dimorphism in the power of sexual selection (<xref ref-type="bibr" rid="B8">Darwin, 1888</xref>). Despite the various sexually dimorphic traits, the upstream regulators that contribute to these discrepancies are highly conserved (<xref ref-type="bibr" rid="B53">Zarkower, 2001</xref>). Recent years, the center of these regulators converge on the family of DM domain genes, also known as the <italic>Doublesex and male abnormal-3 related transcription factor</italic> (<italic>Dmrt</italic>) (<xref ref-type="bibr" rid="B38">Volff et al., 2003</xref>; <xref ref-type="bibr" rid="B12">Hong et al., 2007</xref>; <xref ref-type="bibr" rid="B16">Kopp, 2012</xref>). The essence of the <italic>Dmrt</italic> family is a group of transcription factors that contain the DM DNA binding domain, which was firstly identified in the <italic>Drosophila melanogaster Dsx</italic> (<xref ref-type="bibr" rid="B11">Hildreth, 1965</xref>) and <italic>Caenorhabditis elegans mab-3</italic> (<xref ref-type="bibr" rid="B34">Shen and Hodgkin, 1988</xref>) genes. The discovery of the evolutionary conservation of these two genes in structure and function soon leads to numerous identifications in other species (<xref ref-type="bibr" rid="B30">Raymond et al., 1998</xref>).</p>
<p>The <italic>Dmrt</italic> family is widely distributed across the animal kingdom and includes various groups (<xref ref-type="bibr" rid="B46">Wexler et al., 2014</xref>; <xref ref-type="bibr" rid="B28">Picard et al., 2015</xref>). In vertebrates, the <italic>Dmrt1</italic> gene is essential to sexual development: promoting postnatal testis differentiation in mammalian (<xref ref-type="bibr" rid="B29">Raymond et al., 2000</xref>; <xref ref-type="bibr" rid="B18">Matson et al., 2011</xref>), maintaining sexual bias in avian (<xref ref-type="bibr" rid="B35">Smith et al., 2009</xref>), and preventing sexual transition in fish (<xref ref-type="bibr" rid="B47">Wu et al., 2012</xref>). Sometimes, the mutated <italic>Dmrt1</italic> homologue <italic>DM domain on Y</italic> (<italic>DMY</italic>) (<xref ref-type="bibr" rid="B15">Kobayashi et al., 2004</xref>) and <italic>DM domain on W</italic> (<italic>DMW</italic>) (<xref ref-type="bibr" rid="B51">Yoshimoto et al., 2008</xref>) could take over sex determination due to the amino acid substitution (<xref ref-type="bibr" rid="B26">Ogita et al., 2020</xref>).</p>
<p>In invertebrates, the genes of <italic>Dmrt</italic> family are broadly divided into four groups: <italic>Dsx</italic>, <italic>Dmrt11E</italic>, <italic>Dmrt93B</italic> and <italic>Dmrt99B</italic> (<xref ref-type="bibr" rid="B27">Panara et al., 2019</xref>). While the functions of some of these genes are not yet well understood, the <italic>Dsx</italic> gene has been found to have some similarities in expression pattern and functionality to the vertebrate <italic>Dmrt1</italic> gene (<xref ref-type="bibr" rid="B19">Matson and Zarkower, 2012</xref>). Besides, a unique group of <italic>Dmrt</italic> genes with two DM domains was specifically found in the Malacostraca. Initial research of the group was conducted on <italic>Sagmariasus verreauxi</italic> (<xref ref-type="bibr" rid="B5">Chandler et al., 2017</xref>) and soon extended to other species, such as <italic>E. sinensis</italic> (<xref ref-type="bibr" rid="B7">Cui et al., 2021</xref>), <italic>Macrobrachium rosenbergii</italic> (<xref ref-type="bibr" rid="B1">Abayed et al., 2019</xref>) and <italic>Scylla paramamosain</italic> (<xref ref-type="bibr" rid="B40">Wan et al., 2021</xref>).</p>
<p>The <italic>Dmrt</italic> family is a group of transcription factors with a conserved DNA-binding motif known as the DM domain (<xref ref-type="bibr" rid="B30">Raymond et al., 1998</xref>). The DM domain consists of two intertwined structures that chelate two atoms of zinc and binds to the minor groove of target gene in transcriptional regulation (<xref ref-type="bibr" rid="B56">Zhu et al., 2000</xref>). In Pancrustacea, the <italic>Dmrt11E</italic>, <italic>Dmrt93B</italic>, and <italic>Dmrt99B</italic> genes have been shown to play less important roles in sexual development compared to the <italic>Dsx</italic> gene. (<xref ref-type="bibr" rid="B13">Kato et al., 2011</xref>; <xref ref-type="bibr" rid="B27">Panara et al., 2019</xref>). In Hexapoda, <italic>Dsx</italic> is alternatively spliced into sex-specific isoforms (<xref ref-type="bibr" rid="B6">Chikami et al., 2022</xref>), which control the transcription of the target gene <italic>yolk protein</italic>/<italic>vitellogenin</italic> (<xref ref-type="bibr" rid="B3">Burtis et al., 1991</xref>), contributing to the sexual differentiation of both genders. In Branchiopoda, the targeted gene of <italic>Dsx</italic> remains unclear, but a recent transcriptomic analysis suggests a vitellogenin receptor could be the downstream element of <italic>Dsx</italic> (<xref ref-type="bibr" rid="B25">Nong et al., 2020</xref>). In Branchiopoda and Malacostraca, <italic>Dsx</italic> transcribes male-specifically, controls the development of male-specific structures, and is dispensable for female sexual differentiation (<xref ref-type="bibr" rid="B45">Wexler et al., 2019</xref>). In Malacostraca, the common regulator of male sexual differentiation is <italic>IAG</italic> (<xref ref-type="bibr" rid="B4">Chandler et al., 2018</xref>), which is secreted by the class-specific endocrine organ androgenic gland (AG, <xref ref-type="bibr" rid="B37">Ventura et al., 2011</xref>). And putative binding sites of <italic>Dmrt</italic> have been found on the promoter of <italic>IAG</italic> in many malacostracan species (<xref ref-type="bibr" rid="B17">Li et al., 2018</xref>; <xref ref-type="bibr" rid="B7">Cui et al., 2021</xref>; <xref ref-type="bibr" rid="B41">Wan et al., 2022</xref>; <xref ref-type="bibr" rid="B44">Wei et al., 2022</xref>; <xref ref-type="bibr" rid="B42">Wang et al., 2023</xref>). Moreover, homologue of the <italic>vitellogenin</italic> was also found in the <italic>S. paramamosain</italic>, but no <italic>Dmrt</italic> binding site was found on the promoter (<xref ref-type="bibr" rid="B41">Wan et al., 2022</xref>).</p>
<p>In this study, we investigated the <italic>Dmrt</italic> family in <italic>E. sinensis</italic> and analyzed its function in AG development. Additionally, we conducted evolutionary analyses of the <italic>Dmrt</italic> genes, reconstructed their ancestral sequences, and proposed a hypothesis for the evolution of the family. The discovery of the Malacostraca-specific <italic>Dsx</italic> and <italic>iDmrt1</italic> groups supports the idea that the <italic>Dmrt</italic> gene family in this class may have undergone a unique evolutionary event that differs from that of Hexapoda and Branchiopoda.</p>
</sec>
<sec sec-type="materials|methods" id="s2">
<title>2 Materials and methods</title>
<sec id="s2-1">
<title>2.1 Crab sample and RNA preparation</title>
<p>We reasoned that male reproductive tissues were likely to express <italic>Dmrt</italic>, including splicing isoforms of these genes that might exist. To obtain different stages of testis and androgenic gland, crabs were purchased separately in August and October from a local market in Ningbo city (Zhejiang, China). Crabs were anesthetized on ice for 15&#xa0;min before dissection. Testis at the spermatid stage and AG at the synthesis stage were gently dissected from crabs bought in August, and testis at the sperm stage and AG at the secretion stage were gently dissected from crabs bought in October. The fresh reproductive organs were plunged into TRIzol reagent (Invitrogen, United States) and stored at &#x2212;80&#xb0;C for RNA extraction. Total RNA was extracted by grinding tissue in TRIzol reagent (Invitrogen, United States) and processed following the manufacturer&#x2019;s instructions. RNA quality was guaranteed by NanoOne and 1.2% agarose gel. The integrity of the RNA was determined with the Agilent 2100 Bioanalyzer (Agilent Technologies, United States). Besides, chimeric crabs, collected in local crab farms, are genetically females whereas exhibit male characteristics such as AG and male gonopods (<xref ref-type="bibr" rid="B55">Zhu et al., 2022</xref>).</p>
</sec>
<sec id="s2-2">
<title>2.2 PacBio sequencing</title>
<p>Four SMRT libraries were generated from collected tissues, each corresponding to a specific stage of testis or AG development: T_S2 (testis at the spermatid stage), T_S3 (testis at the sperm stage), AG_S2 (AG at the synthesis stage), and AG_S3 (AG at the secretion stage). The libraries were constructed using the SMRTbell Template Prep Kit from Pacbio (United States). The mRNA was first converted to cDNA using the SMARTer PCR cDNA Synthesis Kit from Takara (United States). The cDNA was then amplified by PCR and purified using AMPure PB beads from Pacbio (United States). Finally, the resulting templates were sequenced on a Sequel II instrument from Pacbio (United States).</p>
</sec>
<sec id="s2-3">
<title>2.3 Identification and characterization of the <italic>EsDmrt</italic> family</title>
<p>To get a comprehensive understanding of the <italic>EsDmrt</italic> family, we annotated a broad transcriptomic library of <italic>E. sinensis</italic> (annotation details are listed in <xref ref-type="sec" rid="s10">Supplementary Table S1</xref>), spanning from embryonic and larva stages, normal tissues of adult crab, and reproductive organs after eyestalk ablation and <italic>IAG</italic> knockdown (accession numbers of transcriptomes used for annotation are listed in <xref ref-type="sec" rid="s10">Supplementary Table S2</xref>). After annotation, we extracted all clusters that were annotated as DM domain genes in at least one database, serving as a query to conduct BLASTn against the newly published genome (GCA_013436485.1) of <italic>E. sinensis</italic>. The genomic locations of potential <italic>EsDmrt</italic> genes were marked on the general feature format (GFF) file. Then we aligned the merged SMRT library to the genome using Minimap2 v2.17 with the command &#x201c;minimap2 -ax map-pb.&#x201d; Corresponding full-length transcripts of <italic>EsDmrt</italic> genes were fetched (name of transcripts are listed in <xref ref-type="sec" rid="s10">Supplementary Table S3</xref>). Gene structures and alternative splicing events were analyzed on IGV using the binary sequence alignment/map (BAM) file obtained from the alignment. ORFs and deduced amino acids were predicted by Expasy Translate (<ext-link ext-link-type="uri" xlink:href="https://web.expasy.org/translate/">https://web.expasy.org/translate/</ext-link>). Functional domains were predicted by SMART program (<ext-link ext-link-type="uri" xlink:href="http://smart.embl-heidelberg.de/">http://smart.embl-heidelberg.de/</ext-link>). Genomic structure visualization was performed by GSDS v2.0 (<ext-link ext-link-type="uri" xlink:href="http://gsds.gao-lab.org/">http://gsds.gao-lab.org/</ext-link>). Visualization of chromosome location was conducted by MapChart (<xref ref-type="bibr" rid="B39">Voorrips, 2002</xref>). Multiple sequence alignment was conducted by MAFFT v7.407 (<xref ref-type="bibr" rid="B14">Katoh and Standley, 2013</xref>) and visualized by GeneDoc (<xref ref-type="bibr" rid="B24">Nicholas, 1997</xref>).</p>
</sec>
<sec id="s2-4">
<title>2.4 Molecular phylogenetic analysis</title>
<p>Phylogenetic analysis of DM domain genes was performed using amino acid sequences of the N-terminal DM domain (<italic>Dmrt</italic> genes used for the analysis are listed in <xref ref-type="sec" rid="s10">Supplementary Table S4</xref>; amino acid sequences used for alignment can be found in <xref ref-type="sec" rid="s10">Supplementary Material</xref>). We aligned the sequences using MAFFT with the L-INS-I option (most accurate model) and built a maximum-likelihood (ML) tree with 1,000 bootstrap values in the IQ-TREE software (<xref ref-type="bibr" rid="B20">Minh et al., 2020</xref>). The substitution model was automatically selected using &#x201c;-MPF&#x201d; and LG&#x2b;I&#x2b;G4 was the best-fit. Finally, the tree was visualized and annotated using GGTREE (<xref ref-type="bibr" rid="B52">Yu et al., 2017</xref>).</p>
</sec>
<sec id="s2-5">
<title>2.5 Gene expression analysis using transcriptomic data</title>
<p>To investigate the function of the <italic>EsDmrt</italic> family in sexual development, we conducted a wide-ranging analysis of the expression levels in gonads, early-stage transcriptomic library (Illumina data) and chimeric tissues (NanoPore data). Illumina data (SRA numbers are listed in <xref ref-type="sec" rid="s10">Supplementary Table S2</xref>) were generated from our previous studies or downloaded from NCBI and then trimmed by Trimmomatic v0.39. The clean data were mapped to the genome with Hisat2 v2.1.0. Consequent BAM and GFF files were used to calculate the transcripts per million (TPM) values by Stringtie v2.1.3. NanoPore data (Unpublished data from our lab) were aligned to the genome using minimap2 v2.17 after quality control and consequent BAM files were used to calculate the TPM values as described above. All TPM values were presented as mean &#xb1; SD. Statistical analyses were performed by the one-way analysis of variance (ANOVA) or <italic>T</italic>-test using SPSS 28 software. <italic>p-</italic>values less than 0.05 and 0.01 were considered significant and extremely significant. Each group contained three biological repetitions. The results were visualized by ggplot2 (<ext-link ext-link-type="uri" xlink:href="https://ggplot2.tidyverse.org">https://ggplot2.tidyverse.org</ext-link>).</p>
</sec>
<sec id="s2-6">
<title>2.6 RNAi and RT-qPCR</title>
<p>The siRNA of <italic>EsDsx1</italic>
<sup>
<italic>M1</italic>
</sup>, <italic>EsDsx1</italic>
<sup>
<italic>M2</italic>
</sup>, <italic>EsiDmrt1a</italic>, <italic>EsiDMY</italic>
<sup>
<italic>L</italic>
</sup> and siControl were chemically synthesized by GenePharma (Shanghai, China) (sequences of the siRNAs and control siRNA are listed in <xref ref-type="sec" rid="s10">Supplementary Table S5</xref>). Crab saline was synthesized in the lab (2.570&#xa0;g NaCl, 0.084&#xa0;g KCl, 0.148&#xa0;g CaCl<sub>2</sub>, 0.248&#xa0;g MgCl<sub>2</sub>, 0.327&#xa0;g Na<sub>2</sub>SO<sub>4</sub>, 0.238&#xa0;g HEPES, pH 7.4, total 100&#xa0;mL). The siRNA was incubated with GP-transfect for 30&#xa0;min before injection. The experiment included three treatments, namely, siRNAi, siControl and saline, with each treatment performed in six replicates (<italic>n</italic> &#x3d; 6). A total of 40&#xa0;&#x3bc;g/individual siRNA was injected into the fifth walking leg&#x2019;s sinus. After 24&#xa0;h of injection, only AG was collected in three treatments. Animal dissection and RNA extraction were conducted in the same way as described above. RT-qPCR was performed to investigate the expression level. <italic>Dmrt</italic> and <italic>IAG</italic> expression relative to the siControl and saline were determined by the 2<sup>&#x2212;&#x394;&#x394;Ct</sup> method. The Shapiro-Wilk test and Levene&#x2019;s test were used to check the normality and homogeneity of variance assumptions, respectively. Independent sample <italic>T</italic>-tests were used to analyze <italic>Dmrt</italic> and <italic>IAG</italic> expression between RNAi and siControl groups. <italic>p-</italic>values less than 0.05 and 0.01 were considered significant and extremely significant. Results of statistical tests are listed in <xref ref-type="sec" rid="s10">Supplementary Table S6</xref>.</p>
</sec>
<sec id="s2-7">
<title>2.7 Consensus analysis of DM domain</title>
<p>To gain insights into the conservation and variation of the DM domain across different species and groups including <italic>Dmrt1</italic> from various metazoans, four conserved groups from Pancrustacea and <italic>iDmrt1</italic> from Malacostraca, we conducted amino acid sequence alignments and showed their conservation using Weblogo v3.7.4 (<ext-link ext-link-type="uri" xlink:href="http://weblogo.threeplusone.com/">http://weblogo.threeplusone.com/</ext-link>). Accession numbers of sequences used for the analysis are listed in <xref ref-type="sec" rid="s10">Supplementary Table S7</xref>.</p>
</sec>
<sec id="s2-8">
<title>2.8 Positive selection analysis and ancestral sequence reconstruction</title>
<p>The ratio of nonsynonymous (<italic>d</italic>
<sub>N</sub>) and synonymous (<italic>d</italic>
<sub>S</sub>) substitution rates (<italic>&#x3c9;</italic> &#x3d; <italic>d</italic>
<sub>N</sub>/<italic>d</italic>
<sub>S</sub>) were evaluated using codeML in PAML version 4.9 (<xref ref-type="bibr" rid="B50">Yang, 2007</xref>). For the <italic>iDmrt1</italic> group, we used site models that assume different <italic>&#x3c9;</italic> varies among sites but not branches, including model M0 (one ratio), M1a (neutral), M2a (selection), M3 (discrete), M7 (beta), and M8 (beta and <italic>&#x3c9;</italic> &#x3e; 1) (<xref ref-type="bibr" rid="B49">Yang et al., 2000</xref>). The reference tree was built first (tree diagram is shown in <xref ref-type="sec" rid="s10">Supplementary Figure S1</xref>; <italic>Dmrt</italic> genes used for the analysis are listed in <xref ref-type="sec" rid="s10">Supplementary Table S8</xref>; nucleotide sequences used for the alignment can be found in <xref ref-type="sec" rid="s10">Supplementary Material</xref>), then pairwise comparisons of M0 versus M3, M1a versus M2a, and M7 versus M8 were used to perform likelihood ratio test (LRT). For the <italic>Dsx</italic> group, we used the branch-site model that assumes <italic>&#x3c9;</italic> to vary both among sites and branches, to compare the model A against the model Null (fixed <italic>&#x3c9;</italic> of 1). Also, we built a reference tree first (tree diagram is shown in <xref ref-type="sec" rid="s10">Supplementary Figure S2</xref>; <italic>Dmrt</italic> genes used for the analysis are listed in <xref ref-type="sec" rid="s10">Supplementary Table S9</xref>; nucleotide sequences used for the alignment can be found in <xref ref-type="sec" rid="s10">Supplementary Material</xref>), then the comparison of model A versus model Null was used to perform LRT. Both significance tests were performed using the <italic>&#x3c7;</italic>
<sup>2</sup> distribution with degrees of freedom of 2 and 1 for <italic>iDmrt1</italic> and <italic>Dsx</italic> groups, respectively. If the results of LRT showed significance, Bayes empirical Bayes (BEB) analysis would be conducted to identify positively selected sites with a posterior probability greater than 0.95. Meanwhile, ancestral sequences of <italic>Dsx</italic> were reconstructed using the empirical Bayes approach implemented in PAML (<xref ref-type="bibr" rid="B48">Yang et al., 1995</xref>).</p>
</sec>
<sec id="s2-9">
<title>2.9 Protein structure prediction and motif prediction</title>
<p>The reconstructed ancestral sequences of <italic>Dsx</italic> were utilized as inferred protein sequences to model structural domains using the Alphafold2-based algorithm (ColabFold: <xref ref-type="bibr" rid="B21">Mirdita et al., 2022</xref>) with the default settings. The accuracy of predicted models was assessed using the predicted Local distance difference test (plDDT) score, which was automatically calculated on the ColabFold platform (diagram of plDDT score is shown in <xref ref-type="sec" rid="s10">Supplementary Figure S3</xref>). The PyMOL Molecular Graphics System, Version 2.0 (<xref ref-type="bibr" rid="B33">Schr&#xf6;dinger, 2015</xref>) was used to visualize the 3D models of the predicted structures and we highlighted the zinc motif-specific residues cysteine and histidine with red and yellow, respectively. Then we used the online software ZincExplorer to detect the zinc binding ability of the reconstructed amino acids. Moreover, we modelled the <italic>Dsx</italic> protein from several malacostracan species using Swiss-Model.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>3 Results</title>
<sec id="s3-1">
<title>3.1 PacBio sequencing of <italic>Eriocheir sinensis</italic>
</title>
<p>We constructed four SMRT libraries (T_S2, T_S3, AG_S2 and AG_S3) using testis at spermatid and sperm stages, and AG at synthesis and secretion stages. We obtained a total of 171,436, 348,229, 187,041 and 204,937 circular consensus sequences from raw reads, of which 58,046,178,242,118,195 and 116,436 were identified as full length non-chimeric reads. After clustering, we identified 35,011, 103,219, 54,833 and 68,126 high-quality isoforms with an average length of 1,092, 1,633, 904 and 1,429 bp, respectively. Finally, four libraries were merged, resulting in a total of 197,160 nonredundant full-length transcript. The complete datasets were deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR24067590, SRR24067589, SRR24067592 and SRR24067591.</p>
</sec>
<sec id="s3-2">
<title>3.2 Identification and characterization of <italic>EsDmrt</italic> genes</title>
<p>Our study identified nine members of the <italic>Dmrt</italic> family in <italic>E. sinensis</italic>, including <italic>EsDsx1</italic>, <italic>EsDsx2</italic>, <italic>EsDsx-like</italic>, <italic>EsiDmrt1a</italic>, <italic>EsiDmrt1b</italic>, <italic>EsiDMY</italic>, <italic>EsDmrt-like</italic>, <italic>EsDmrt11E</italic> and <italic>EsDmrt93B</italic> (<xref ref-type="fig" rid="F1">Figure 1A</xref>). The nine genes exhibit highly conserved DM domains, except for <italic>EsDsx1</italic> and <italic>EsDsx2</italic>, in which a point mutation was both detected in the zinc motif-specific residues (<xref ref-type="fig" rid="F1">Figure 1B</xref>). <italic>EsDsx1</italic> and <italic>EsiDMY</italic> are alternatively spliced (<xref ref-type="fig" rid="F1">Figure 1A</xref>), and no sex-specific splicing isoforms were detected. While we found two <italic>EsDsx1</italic> isoforms (<italic>EsDsx1</italic>
<sup>
<italic>M1</italic>
</sup> and <italic>EsDsx1</italic>
<sup>
<italic>M2</italic>
</sup>) specifically expressed in AG and one isoform (<italic>EsDsx1</italic>
<sup>
<italic>C</italic>
</sup>) that was commonly expressed (<xref ref-type="sec" rid="s10">Supplementary Figure S4</xref>). Splicing isoforms of <italic>EsiDMY</italic> encoded three proteins of different lengths (<italic>EsiDMY</italic>
<sup>
<italic>S</italic>
</sup>, <italic>EsiDMY</italic>
<sup>
<italic>M</italic>
</sup> and <italic>EsiDMY</italic>
<sup>
<italic>L</italic>
</sup>), and their presence was confirmed by RT-PCR (<xref ref-type="sec" rid="s10">Supplementary Figure S5</xref>). Besides, most <italic>EsDmrt</italic> genes were located on a single chromosome, while <italic>EsDsx1</italic> and <italic>EsDsx2</italic> were adjacent to each other (<xref ref-type="fig" rid="F1">Figure 1C</xref>).</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption>
<p>Characterization of <italic>Dmrt</italic> family genes in <italic>E. sinensis</italic>. <bold>(A)</bold> Structure features of nine <italic>EsDmrt</italic> genes. All elements are shown to scale except for introns. The alternative splicing events of <italic>EsDsx1</italic> and <italic>EsiDMY</italic> are displayed above the genes with dotted lines. <bold>(B)</bold> Alignment of deduced amino acid sequences of DM domains (N-terminal domain of <italic>EsiDmrt1a</italic>, <italic>EsiDmrt1b</italic> and <italic>EsiDMY</italic> are used in this alignment). Identical amino acids are highlighted in black and similar ones in grey. Eight conserved motif-specific residues are indicated with asterisks and the point mutation in <italic>EsDsx1</italic> and <italic>EsDsx2</italic> is indicated with a top solid triangle. <bold>(C)</bold> Genome loci of nine <italic>EsDmrt</italic> genes. Except for the <italic>EsDsx-like</italic>, all genes have their clear chromosome locations.</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g001.tif"/>
</fig>
</sec>
<sec id="s3-3">
<title>3.3 Molecular phylogenetic analysis of DM domain genes</title>
<p>We built a ML tree based on 91 DM domain genes to reconstruct the phylogenetic relationship of the <italic>EsDmrt</italic> family (<xref ref-type="fig" rid="F2">Figure 2</xref>). Our analysis revealed that five <italic>EsDmrt</italic> genes were distributed in four conserved clades of Pancrustacea: <italic>Dsx</italic>, <italic>Dmrt11E</italic>, <italic>Dmrt93B</italic>, and <italic>Dmrt99B</italic>. Notably, the <italic>Dsx</italic> clade was classified into three reliable subclades, including Hexapoda <italic>Dsx</italic> clade, Branchiopoda <italic>Dsx</italic> clade and Malacostraca <italic>Dsx</italic> clade. Besides, we found that three <italic>EsDmrt</italic> genes fall into a monophyletic branch consisting of <italic>iDmrt1</italic> orthorlogues from malacostracan species. The phylogeny of <italic>EsDsx-like</italic> remained unclear in our study.</p>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption>
<p>Molecular phylogeny of <italic>Dmrt</italic> family genes in <italic>E. sinensis</italic>. Phylogenetic analysis was based on amino acid sequences of DM domain and performed by IQ-TREE following multiple sequence alignment using MAFFT. The maximum-likelihood method was applied. The 91 taxonomic units conducted for this analysis are listed in <xref ref-type="sec" rid="s10">Supplementary Table S4</xref>. Numerical value on each node indicates bootstrap value that is &#x3e;70%. <italic>EsDmrt</italic> genes are indicated with brown dots. Malacostraca <italic>Dsx</italic> and <italic>iDmrt1</italic> groups are colored in light steel blue and light coral.</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g002.tif"/>
</fig>
</sec>
<sec id="s3-4">
<title>3.4 Expression pattern of <italic>EsDmrt</italic> genes in three transcriptomic libraries</title>
<p>We analyzed expression patterns of the <italic>EsDmrt</italic> family using transcriptomic data from three libraries. The embryonic and larval transcriptomes show that most <italic>EsDmrt</italic> genes start to enrich around the juvenile 1 stage (<xref ref-type="fig" rid="F3">Figure 3A</xref>), while <italic>EsDsx1</italic>
<sup>
<italic>M1</italic>
</sup>, <italic>EsDsx1</italic>
<sup>
<italic>M2</italic>
</sup> and <italic>EsDsx2</italic> show relatively high expression in gastrula and blastula stages. In reproductive organs, <italic>EsDsx1</italic>, <italic>EsDsx2</italic>, <italic>EsiDMY</italic> and <italic>EsiDmrt1a</italic> are co-expressed predominantly in male organs, particularly in two stages of AG, where their expression levels are significantly higher than testis and ovary (<xref ref-type="fig" rid="F3">Figure 3B</xref>, one-way ANOVA, <italic>p</italic> &#x3c; 0.05). Besides, the overall expression levels of <italic>EsiDmrt1b</italic>, <italic>EsDmrt11E</italic>, <italic>EsDmrt-like</italic> and <italic>EsDsx-like</italic> are relatively low, but they still show a significantly higher expression in testis (One-way ANOVA, <italic>p</italic> &#x3c; 0.05). The expression level of <italic>EsDmrt93B</italic> is too low to show. In chimeric crabs, we find a significantly higher expression of <italic>EsiDMY</italic> in the chimeric AG compared to the normal ones (<xref ref-type="fig" rid="F3">Figure 3C</xref>, independent sample <italic>T</italic>-test, &#x2a;<italic>p</italic> &#x3c; 0.05). Additionally, <italic>EsDsx1</italic>
<sup>
<italic>C</italic>
</sup>, <italic>EsDsx2</italic> and <italic>EsiDMY</italic>
<sup>
<italic>L</italic>
</sup> show significantly higher expression levels in the chimeric eyestalk compared to the normal ones (independent sample <italic>T</italic>-test, &#x2a;<italic>p</italic> &#x3c; 0.05).</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption>
<p>Expression level of the <italic>Dmrt</italic> family genes in three different transcriptomic libraries of <italic>E. sinensis</italic>. <bold>(A)</bold> Expression pattern of <italic>EsDmrt</italic> genes in embryonic, larval and juvenile stages. The heatmap was shown as normalized TPM value. <bold>(B)</bold> Expression pattern of <italic>EsDmrt</italic> genes (including splicing isoforms) in reproductive organs (AG, testis and ovary). The bar plot was shown as TPM value. Vertical bars represented mean &#xb1; SD, <italic>n</italic> &#x3d; 3. Results were analyzed by one-way ANOVA and different letters (a-d) above the bars presented significant differences between groups (<italic>p</italic> &#x3c; 0.05). AG_SY, androgenic gland at synthesis stage; AG_SE, androgenic gland at secretion stage. <bold>(C)</bold> Expression pattern of the <italic>EsDmrt</italic> genes in normal and chimeric <italic>E. sinensis</italic>. The bar plot was shown as TPM value. Vertical bars represented mean &#xb1; SD, <italic>n</italic> &#x3d; 3. Tissues from left to right: AG-androgenic gland (chimeric, normal male); ovary (chimeric, normal female); eyestalk (chimeric, normal female and male). Tissue and organ from chimeric, normal female and male were colored with purple, red and blue. Vertical bars represented mean &#xb1; SD, <italic>n</italic> &#x3d; 3. Results were analyzed by independent <italic>T</italic>-test comparing chimeric and normal tissue and organ (&#x2a;<italic>p</italic> &#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic> &#x3c; 0.01).</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g003.tif"/>
</fig>
</sec>
<sec id="s3-5">
<title>3.5 Regulation of <italic>EsDmrt</italic> for <italic>EsIAG</italic> expression</title>
<p>We conducted siRNA interference to investigate the role of the <italic>EsDmrt</italic> family in AG development, with <italic>EsIAG</italic> serving as a molecular marker. The results prove successful silencing of <italic>EsDsx1</italic>
<sup>
<italic>M1</italic>
</sup>, <italic>EsDsx1</italic>
<sup>
<italic>M2</italic>
</sup>, <italic>EsiDmrt1a</italic> and <italic>EsiDMY</italic>
<sup>
<italic>L</italic>
</sup> genes (<xref ref-type="fig" rid="F4">Figure 4A</xref>, independent sample <italic>T-</italic>test, <italic>p</italic> &#x3d; 4.11E-03 in siEsDsx1<sup>M1</sup> group, 2.00E-03 in siEsDsx1<sup>M2</sup> group, 4.97E-02 in siEsiDmrt1a group, and 3.80E-02 in siEsiDMY<sup>L</sup> group). Furthermore, all RNAi groups show a significant decrease in <italic>EsIAG</italic> expression (<xref ref-type="fig" rid="F4">Figure 4B</xref>, independent sample <italic>T-</italic>test, <italic>p</italic> &#x3d; 4.40E-02 in siEsDsx1<sup>M1</sup> group, 3.00E-03 in siEsDsx1<sup>M2</sup> group, 3.50E-02 in siEsiDmrt1a group and 3.00E-03 in siEsiDMY<sup>L</sup> group).</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption>
<p>Function of <italic>Dmrt</italic> genes for <italic>IAG</italic> expression in <italic>E. sinensis</italic>. Transcript abundance was measured through RT-qPCR. Box plot is show as log-scale relative values of expression levels to the reference gene, <italic>Es&#x3b2;-actin</italic>. Each dot represents the expression level of each individual. Independent <italic>T</italic>-test was performed to ascertain the difference between siRNA and siControl groups, with significance levels indicated (&#x2a;<italic>p</italic> &#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic> &#x3c; 0.01) Statistical results are listed in the <xref ref-type="sec" rid="s10">Supplementary Table S6</xref>. Expression levels of <italic>EsDmrt</italic> genes <bold>(A)</bold> and <italic>EsIAG</italic> gene <bold>(B)</bold> in RNAi and control groups are shown separately.</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g004.tif"/>
</fig>
</sec>
<sec id="s3-6">
<title>3.6 Consensus analysis of DM domain in different <italic>Dmrt</italic> genes</title>
<p>In the analysis of the conservation of DM domains (<xref ref-type="fig" rid="F5">Figure 5</xref>). We find that <italic>Dsx</italic> and <italic>iDmrt1</italic> groups exhibit great variability in the amino acids, which is different from the conservation observed in <italic>Dmrt1</italic>, <italic>Dmrt11E</italic>, <italic>Dmrt93B</italic> and <italic>Dmrt99B</italic> groups. The eight zinc motif-specific residues remain invariant in all tested <italic>Dmrt</italic> genes, except for the Malacostraca <italic>Dsx</italic>, where a cryptic mutation appears due to a substitution, from cysteine to histidine. Furthermore, there are some certain amino acids, including positions 9, 15, 16, 37, 38, and 53 that also distinguish the Malacostraca <italic>Dsx</italic> from other <italic>Dmrt</italic> genes.</p>
<fig id="F5" position="float">
<label>FIGURE 5</label>
<caption>
<p>Consensus analysis of DM domains of the <italic>Dmrt</italic> gene family. Less conserved points are discarded. The cryptic mutation in Malacostraca <italic>Dsx</italic> is annotated below. Amino acids are colored according to their chemical properties: red (D, E) for acidic, green (C, G, S, T, Y) for polar, blue (H, K, R) for basic, black (A, F, I, L, M, V, W) for hydrophobic, and purple (Q, N) for neutral. n: number of unique sequences. m: number of different species.</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g005.tif"/>
</fig>
</sec>
<sec id="s3-7">
<title>3.7 Selection analysis of <italic>iDmrt1</italic> group</title>
<p>We used the site codon model in PAML to investigate ongoing selection pressure on the <italic>iDmrt1</italic> group. The selection test between M8 and M7 site models shows evidence of positive selection for the <italic>iDmrt1</italic> group (LRT, <italic>p</italic> &#x3d; 0.0494, <xref ref-type="table" rid="T1">Table 1</xref>). The BEB analysis demonstrates that position 30 on the DM domain is under positive selection with a posterior probability greater than 0.95 (<xref ref-type="table" rid="T1">Table 1</xref>). Additionally, the comparison between M3 and M0 site models suggests variable <italic>&#x3c9;</italic> values among different positions (LRT, <italic>p</italic> &#x3c; 10<sup>&#x5e;&#x2212;10</sup>, <xref ref-type="table" rid="T1">Table 1</xref>).</p>
<table-wrap id="T1" position="float">
<label>TABLE 1</label>
<caption>
<p>Analysis of positive selection using random site models on <italic>iDmrt1</italic> tree.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th align="left">Site model</th>
<th align="left">lnL</th>
<th align="left">Site class</th>
<th align="left">Proportion (p)</th>
<th align="left">
<italic>&#x3c9;</italic>
</th>
<th align="left">Positively selected sites</th>
<th align="left">LRT <italic>p</italic> value</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left">M0</td>
<td align="left">&#x2212;1192.424219</td>
<td align="left">0</td>
<td align="left">NA</td>
<td align="left">0.05351</td>
<td align="left">NA</td>
<td align="left"/>
</tr>
<tr>
<td align="left">M1a</td>
<td align="left">&#x2212;1159.966028</td>
<td align="left">0</td>
<td align="left">0.71966</td>
<td align="left">0.28034</td>
<td align="left">NA</td>
<td align="left"/>
</tr>
<tr>
<td align="left"/>
<td align="left"/>
<td align="left">1</td>
<td align="left">0.03453</td>
<td align="left">1</td>
<td align="left"/>
<td align="left"/>
</tr>
<tr>
<td align="left">M2a</td>
<td align="left">&#x2212;1159.966028</td>
<td align="left">0</td>
<td align="left">0.71966</td>
<td align="left">0.03453</td>
<td align="left">30</td>
<td align="left">&#x3e;0.99 (M2a vs. M1a)</td>
</tr>
<tr>
<td align="left"/>
<td align="left"/>
<td align="left">1</td>
<td align="left">0.12545</td>
<td align="left">1</td>
<td align="left"/>
<td align="left"/>
</tr>
<tr>
<td align="left"/>
<td align="left"/>
<td align="left">2</td>
<td align="left">0.15489</td>
<td align="left">1</td>
<td align="left"/>
<td align="left"/>
</tr>
<tr>
<td align="left">M3</td>
<td align="left">&#x2212;1136.263954</td>
<td align="left">0</td>
<td align="left">0.28696</td>
<td align="left">0</td>
<td align="left">NA</td>
<td align="left">&#x3c;10<sup>&#x2013;10</sup> (M3 vs. M0)</td>
</tr>
<tr>
<td align="left"/>
<td align="left"/>
<td align="left">1</td>
<td align="left">0.42242</td>
<td align="left">0.0314</td>
<td align="left"/>
<td align="left"/>
</tr>
<tr>
<td align="left"/>
<td align="left"/>
<td align="left">2</td>
<td align="left">0.29062</td>
<td align="left">0.23911</td>
<td align="left"/>
<td align="left"/>
</tr>
<tr>
<td align="left">M7</td>
<td align="left">&#x2212;1137.344442</td>
<td align="left">0&#x2013;9</td>
<td align="left">0&#x2013;9: 0.1</td>
<td align="left">0.00003 0.00089 0.00409 0.01124 0.02424 0.04557 0.07903 0.13152 0.21904 0.40415</td>
<td align="left">NA</td>
<td align="left"/>
</tr>
<tr>
<td align="left">M8</td>
<td align="left">&#x2212;1134.337947</td>
<td align="left">0&#x2013;10</td>
<td align="left">0&#x2013;9: 0.09810, 10: 0.01903</td>
<td align="left">0.00006 0.00108 0.00432 0.01089 0.02208 0.03960 0.06626 0.10733 0.17562 0.32419 5.26235</td>
<td align="left">30</td>
<td align="left">0.0494 (M8 vs. M7)</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>lnL, ln likelihood; LRT, likelihood ratio test; BEB, bayes empirical bayes.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s3-8">
<title>3.8 Evolutionary history of <italic>Dsx</italic>
</title>
<p>The LRT result shows that Model A fits the reference tree significantly better than the Model Null (LRT, <italic>p</italic> &#x3d; 0.046384445, <xref ref-type="table" rid="T2">Table 2</xref>), indicating selection pressure acting on the foreground branch. The BEB analysis demonstrates that five positions on the DM domain, including the cryptic mutation shown in <xref ref-type="fig" rid="F5">Figure 5</xref>, are under positive selection. Ancestral sequence reconstruction reveals that the cryptic mutation is also present in the common ancestor of Pancrustacea, but not in the common ancestor of Hexapoda and Branchiopoda (<xref ref-type="fig" rid="F6">Figure 6A</xref>). According to the prediction of ZincExplorer (<xref ref-type="sec" rid="s10">Supplementary Figure S6</xref>), the <italic>Dsx</italic> protein in the common ancestor of Pancrustacea could bind only one zinc atom (<xref ref-type="fig" rid="F6">Figure 6B</xref>), which is consistent with the three-dimension structures of Malacostraca <italic>Dsx</italic> modelled using Swiss-Model (<xref ref-type="sec" rid="s10">Supplementary Figure S7</xref>).</p>
<table-wrap id="T2" position="float">
<label>TABLE 2</label>
<caption>
<p>Analysis of positive selection using branch-site models on <italic>Dsx</italic> tree.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th align="left">Model</th>
<th align="left">lnL</th>
<th align="left">Site class</th>
<th align="left">Proportion</th>
<th align="left">Background <italic>&#x3c9;</italic>
</th>
<th align="left">Foreground <italic>&#x3c9;</italic>
</th>
<th align="left">Positively selected sites</th>
<th align="left">LRT <italic>p</italic> value</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td rowspan="4" align="left">A</td>
<td rowspan="4" align="left">&#x2212;7704.554096</td>
<td align="left">0</td>
<td align="left">0.51052</td>
<td align="left">0.04305</td>
<td align="left">0.04305</td>
<td rowspan="8" align="left">6, 14, 32, 52, 54</td>
<td align="left"/>
</tr>
<tr>
<td align="left">1</td>
<td align="left">0.10762</td>
<td align="left">1</td>
<td align="left">1</td>
<td align="left"/>
</tr>
<tr>
<td align="left">2a</td>
<td align="left">0.31537</td>
<td align="left">0.04305</td>
<td align="left">999</td>
<td align="left"/>
</tr>
<tr>
<td align="left">2b</td>
<td align="left">0.06648</td>
<td align="left">1</td>
<td align="left">999</td>
<td align="left"/>
</tr>
<tr>
<td rowspan="4" align="left">Null</td>
<td rowspan="4" align="left">&#x2212;7706.537885</td>
<td align="left">0</td>
<td align="left">0.53548</td>
<td align="left">0.04193</td>
<td align="left">0.04193</td>
<td align="left">0.046384445</td>
</tr>
<tr>
<td align="left">1</td>
<td align="left">0.11237</td>
<td align="left">1</td>
<td align="left">1</td>
<td align="left"/>
</tr>
<tr>
<td align="left">2a</td>
<td align="left">0.29107</td>
<td align="left">0.04193</td>
<td align="left">1</td>
<td align="left"/>
</tr>
<tr>
<td align="left">2b</td>
<td align="left">0.06108</td>
<td align="left">1</td>
<td align="left">1</td>
<td align="left"/>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>lnL, ln likelihood; LRT, likelihood ratio test; BEB, bayes empirical bayes.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<fig id="F6" position="float">
<label>FIGURE 6</label>
<caption>
<p>Ancestral sequence reconstruction of the DM domain from <italic>Dsx</italic> in Pancrustacea. <bold>(A)</bold> Alignment of the ancestral amino acid sequences of the DM domain from different common ancestor. Positions assumed to be positively selected are colored in red. Conserved residues are indicated with asterisks and mutation is indicated with a triangle. Es_Dsx1, <italic>Dsx1</italic> of <italic>E. sinensis</italic>, is shown as a reference. <bold>(B)</bold> Predicted protein structures of <italic>Dsx</italic> in different common ancestors. The phylogenetic relationship is based on topology from <xref ref-type="bibr" rid="B32">Rota-Stabelli et al. (2013)</xref>. 3D images in the midst indicate modelled proteins of DM domains with the motif-specific residues highlighted in red (cysteine) and yellow (histidine). Chelation of zinc atoms is visualized as a result of ZincExplore.</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g006.tif"/>
</fig>
</sec>
</sec>
<sec sec-type="discussion" id="s4">
<title>4 Discussion</title>
<p>In our previous studies, we have confirmed the names of <italic>EsDmrt11E</italic> (<xref ref-type="bibr" rid="B9">Du et al., 2019</xref>) and <italic>EsiDMY</italic> (<xref ref-type="bibr" rid="B7">Cui et al., 2021</xref>). <italic>EsDmrt-like</italic> and <italic>EsDsx-like</italic> were reported in <xref ref-type="bibr" rid="B54">Zhang and Qiu (2010)</xref> and <xref ref-type="bibr" rid="B42">Wang et al. (2023)</xref>, respectively. In this study, we rename three genes previously identified in <xref ref-type="bibr" rid="B9">Du et al. (2019)</xref> based on their phylogenetic relationships, which are <italic>EsDmrt93B</italic>, <italic>EsiDmrt1a</italic> and <italic>EsiDmrt1b</italic> (formerly known as <italic>EsDmrt3</italic>, <italic>EsIdmrt1-1</italic> and <italic>EsDsx2</italic>). Furthermore, we find the duplicated <italic>Dsx</italic> genes, <italic>EsDsx1</italic> and <italic>EsDsx2</italic>, which form a tandem gene cluster on the genome. As a result, we identify a total of nine members of the <italic>Dmrt</italic> family in the crab.</p>
<p>In invertebrates, <italic>Dmrt</italic> genes are known to play a crucial role in sexual differentiation, driving sex-specific development in varied tissues and cell types (<xref ref-type="bibr" rid="B19">Matson and Zarkower, 2012</xref>). In crustaceans, <italic>Dmrt</italic> genes have been found to transcribe in a strongly male-biased way, indicating their specific involvement in male morphogenesis (<xref ref-type="bibr" rid="B45">Wexler et al., 2019</xref>). Our transcriptomic analyses of the <italic>Dmrt</italic> family in <italic>E. sinensis</italic> support and expand on previous findings, suggesting that the <italic>EsDmrt</italic> family primarily functions in male sexual differentiation, specifically promoting the development of testis and AG. Previous studies have meticulously described the function of <italic>EsDmrt-like</italic> (<xref ref-type="bibr" rid="B54">Zhang and Qiu, 2010</xref>) and <italic>EsDsx-like</italic> (<xref ref-type="bibr" rid="B42">Wang et al., 2023</xref>) during spermatogenesis. In our research, we find the abundant expression of <italic>EsDsx1</italic>, <italic>EsDsx2</italic>, <italic>EsiDmrt1a</italic> and <italic>EsiDMY</italic> in AG, as well as the aberrant transcription of <italic>EsiDmrt1a</italic> and <italic>EsiDMY</italic> in the chimeric AG. Furthermore, RNA knockdown on the transcripts from <italic>EsDsx1</italic>, <italic>EsiDmrt1a</italic> and <italic>EsiDMY</italic> could result in a significant decrease in <italic>EsIAG</italic> transcription. Taken together, our findings propose that in <italic>E. sinensis</italic>, both genes from <italic>Dsx</italic> and <italic>iDmrt1</italic> groups may function in AG development.</p>
<p>In Malacostraca, <italic>Dsx</italic> genes have been extensively studied and are known to upregulate <italic>IAG</italic> in various species, such as <italic>F. chinensis</italic> (<xref ref-type="bibr" rid="B17">Li et al., 2018</xref>), <italic>S. paramamosain</italic> (<xref ref-type="bibr" rid="B41">Wan et al., 2022</xref>) and <italic>P. monodon</italic> (<xref ref-type="bibr" rid="B44">Wei et al., 2022</xref>). And <italic>iDmrt1</italic> genes has also showed closed relationship with AG in species such as <italic>M. rosenbergii</italic> (<xref ref-type="bibr" rid="B1">Abayed et al., 2019</xref>), where the <italic>MroiDmrt1b</italic> and <italic>MroiDmrt1c</italic> genes are highly enriched in AG, and <italic>Portunus trituberculatus</italic>, where the <italic>PtiDmrt1</italic> gene upregulates the <italic>PtIAG</italic> expression (<xref ref-type="bibr" rid="B43">Wang et al., 2020</xref>). These findings suggest that the involvement of both <italic>Dsx</italic> and <italic>iDmrt1</italic> genes in AG development might be a common feature of Malacostraca.</p>
<p>Our phylogenetic tree showed that the <italic>Dsx</italic> and <italic>iDmrt1</italic> genes in Malacostraca form two distinct monophyletic clades. On the one hand, the <italic>iDmrt1</italic> gene was first identified in the <italic>S. verreauxi</italic> (<xref ref-type="bibr" rid="B36">Ventura et al., 2020</xref>), and subsequently identified solely in malacostracan species. Our selection analysis (<xref ref-type="table" rid="T1">Table 1</xref>) reveals that the <italic>iDmrt1</italic> group is subjected to positive selection, suggesting a unique function that is specific to the Malacostraca, i.e., promoting AG development. On the other hand, the Malacostraca <italic>Dsx</italic> shows unique amino acid residues at position 9, 38 and 53 (<xref ref-type="fig" rid="F5">Figure 5</xref>). These positions have been confirmed to play critical roles in DNA binding and homodimerization (<xref ref-type="bibr" rid="B22">Murphy et al., 2015</xref>). Moreover, and perhaps more significantly, an invariant cysteine residue in the zinc binding module, known to coordinate with zinc atom (<xref ref-type="bibr" rid="B56">Zhu et al., 2000</xref>), is replaced with a histidine only in the Malacostraca <italic>Dsx</italic> (<xref ref-type="fig" rid="F5">Figure 5</xref>). Therefore, we suggest that the Malacostraca <italic>Dsx</italic> may not bind to DNA directly but instead form heterodimers with the <italic>iDmrt1</italic>. Such binding mode also rationalizes their co-expression in AG (<xref ref-type="fig" rid="F2">Figure 2B</xref>), which is similar to the situation observed in vertebrates, where <italic>Dmrt3</italic> heterodimerizes with the co-expressed <italic>Dmrt1</italic> in testis (<xref ref-type="bibr" rid="B23">Murphy et al., 2007</xref>).</p>
<p>Selection analysis (<xref ref-type="table" rid="T2">Table 2</xref>) and ancestral sequence reconstruction (<xref ref-type="fig" rid="F6">Figure 6</xref>) place Malacostraca <italic>Dsx</italic> as an outgroup to Hexapoda and Branchiopoda, which is consistent with previous studies on <italic>Hox</italic> (<xref ref-type="bibr" rid="B2">Averof and Akam, 1995</xref>) and <italic>elongation factor-1 alpha</italic> (<xref ref-type="bibr" rid="B31">Regier and shultz, 1997</xref>) genes. Based on these findings, we incorporated Malacostraca into the stepwise evolutionary history proposed by <xref ref-type="bibr" rid="B45">Wexler et al. (2019)</xref> and <xref ref-type="bibr" rid="B6">Chikami et al. (2022)</xref> (<xref ref-type="fig" rid="F7">Figure 7</xref>) that <italic>Dsx</italic> has become essential for male development in the common ancestor of Pancrustacea; then during the split of Malacostraca and the common ancestor of Branchiopoda and Hexapoda in the late Cambrian, a selective event, which might result from ecological or physiological differences (<xref ref-type="bibr" rid="B10">Glenner et al., 2006</xref>), either builds or reserves the unique regulatory mode of <italic>Dmrt</italic> genes in Malacostraca.</p>
<fig id="F7" position="float">
<label>FIGURE 7</label>
<caption>
<p>Diagram of the evolutionary history of <italic>Dsx</italic> and <italic>iDmrt1</italic> proposed in this study. The upper topological structure and annotations describe possible statuses of <italic>Dmrt</italic> in different common ancestors. The lower part compares the transcriptional regulation of <italic>Dsx</italic> in Malacostraca, Hexapoda and Branchiopoda. Previous research has suggested that heterodimer formation of <italic>Dmrt</italic> proteins may contribute to transcriptional regulation (<xref ref-type="bibr" rid="B23">Murphy et al., 2007</xref>). Mutations in the DM domain and the lack of an oligomerization domain (<xref ref-type="bibr" rid="B57">Zheng et al., 2020</xref>) suggest that Malacostraca <italic>Dsx</italic> may not directly bind to, but rather heterodimerize on DNA with another <italic>Dmrt</italic> protein, which, according to our evolutionary analysis, could be <italic>iDmrt1</italic>. Figure created using BioRender.</p>
</caption>
<graphic xlink:href="fphys-14-1201846-g007.tif"/>
</fig>
<p>In this study, we investigated the <italic>Dmrt</italic> family in a decapod crab and explored its function in AG development, which extend our knowledge of sexual development mechanisms in this species. Besides, we proposed a hypothesis of transcriptional regulation based on the finding of the intriguing <italic>Dsx</italic> and <italic>iDmrt1</italic> groups in Malacostraca. Further investigation would be necessary to test our hypothesis and unravel the evolutionary history of DM domain genes.</p>
</sec>
</body>
<back>
<sec sec-type="data-availability" id="s5">
<title>Data availability statement</title>
<p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/<xref ref-type="sec" rid="s10">Supplementary Material</xref>.</p>
</sec>
<sec id="s6">
<title>Author contributions</title>
<p>PZ: conceptualization, investigation, methodology, drawing, writing&#x2014;original draft. YY: supervision, writing&#x2014;review and editing. YX: methodology and software supporting. ZC: supervision, funding, writing&#x2014;review and editing. All authors contributed to the article and approved the submitted version.</p>
</sec>
<sec id="s7">
<title>Funding</title>
<p>The work was supported by the National Natural Science Foundation of China (32072964), the National Key R and D Program of China (2018YFD0900303), and the Ten Thousand Talents Program.</p>
</sec>
<ack>
<p>We would like to express our sincere gratitude to Prof. David Zarkower for providing valuable insights on the transcriptional regulation of DM domain genes. Our gratitude also extends to Prof. Yilei Wang for generously providing the amino acid sequences of Malacostraca <italic>Dsx</italic>.</p>
</ack>
<sec sec-type="COI-statement" id="s8">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s9">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s10">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fphys.2023.1201846/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fphys.2023.1201846/full&#x23;supplementary-material</ext-link>
</p>
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<supplementary-material xlink:href="Table2.docx" id="SM2" mimetype="application/docx" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Table1.XLSX" id="SM3" mimetype="application/XLSX" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
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