Distribution of estrogen sulfotransferase genes in tunas and their relatives

Distribution of estrogen sulfotransferase genes in tunas and their relatives | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Distribution of estrogen sulfotransferase genes in tunas and their relatives Yoji Nakamura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5525443/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted 6 You are reading this latest preprint version Abstract Objective Estrogen sulfotransferase is an enzyme involved in the inactivation of estrogen, and the encoding gene, sult1st6 , is widely conserved among animal species. Recently, it was reported that a paralog of the estrogen sulfotransferase gene is paternally inherited in Pacific bluefin tuna ( Thunnus orientalis ), suggesting that this gene, sult1st6y , may be involved in sex determination or differentiation of Thunnus species as a suppressor of feminization. However, current knowledge on fish sult1st6 is still limited. This study aims to update the previous findings by thoroughly examining the distribution of sult1st6 in tuna (the species of tribe Thunnini) and their relatives (the species of family Scombridae). Results Based on a survey of the sult1st6 loci in the available chromosome-level genomes of Scombridae species, it was found that tuna species carry two copies of sult1st6 . Based on phylogenetic analysis and genome comparison, these paralogs were identified as sult1st6y and its paralog, namely sult1st6a , respectively, suggesting that sult1st6y may have originated from sult1st6a by duplication prior to the divergence of the Thunnini tribe. In addition, the sult1st6y locus may have undergone more structural changes than has the sult1st6a locus. These results provide insight into the evolutionary scenario of the sex determination system of tunas. estrogen sulfotransferase sex determination Thunnini tribe gene duplication Figures Figure 1 Figure 2 Introduction Estrogens are important sex steroid hormones in animals. In fish, these are considered key factors in sex determination and differentiation [ 1 – 3 ]. Artificial estrogen administration can cause sexual changes in many fish species [ 4 ], and some of the reported sex determination genes are involved in estrogen biosynthesis [ 5 ]. Estrogens can be inactivated by sulfation, which is mediated by one of the cytosolic sulfotransferases (SULTs), that is, estrogen sulfotransferase. In fish estrogen sulfotransferase—first reported in zebrafish in 2005 [ 6 ]—the encoding gene, sult1st6 , was not considered to be related to sexual maturity but to organogenesis, based on its expression during embryogenesis. However, it was recently reported that the male genome of Pacific bluefin tuna ( Thunnus orientalis , Scombridae) encodes two copies of sult1st6 , one of which, named sult1st6y , is absent from females [ 7 ]. Another copy, sult1st6a , was found to be present in both males and females. The male specificity of sult1st6y was indirectly supported by the gonad transcriptome of southern bluefin tuna ( T. maccoyii ) [ 7 ]. Furthermore, a significant level of expression of T. orienatlis sult1st6y was observed in male gonad cells at an early stage of sex differentiation [ 8 ]. In a previous study, three PCR primer sets were developed for sex identification of T. orientalis [ 9 ], and their target regions were linked to the sult1st6y locus. These primer sets were also applicable for sex identification in all other Thunnus species [ 10 ], implying that male-specific amplification might be associated with the male-specificity of sult1st6y . These observations raise the hypothesis that sult1st6y may be a sex-determining gene common to the genus Thunnus as a suppressor of feminization. Mobile element-related genes are found around the sult1st6y locus in the T. orientalis genome, and synteny is not conserved among teleost genomes [ 7 ]. In contrast, the sult1st6a locus was within the conserved synteny of other fish genomes. This observation implies that T. orientalis sult1st6y is evolutionarily unstable compared to the sult1st6a locus, but it is not known if this is also the case with closely related species. Furthermore, it has not yet been determined when the duplication of sult1st6 occurred. In this study, I aimed to update the previous results on sult1st6 reported in T. orientalis . Particularly, the chromosome-level genome assemblies of tunas and mackerels were compared, which have accumulated in recent years, allowing us to compare the syntenies around sult1st6 locus. The presence or absence and gene structures of sult1st6 homologs in tunas and their relative were examined. Materials and Methods Genomic data A total of eight Scombridae genome sequences at the chromosome-level as of August 5, 2024, five of which were those of tunas (tribe Thunnini, namely Thunnus , Euthynnus and Katsuwonus ) and the other three were of mackerels ( Scomber and Scomberomorus ), were downloaded from the GenBank database: southern bluefin tuna ( T. maccoyii ; SBT), yellowfin tuna ( T. albacares ; YFT), Atlantic bluefin tuna ( T. thynnus ; ABT), kawakawa tuna ( E. affinis ), skipjack tuna ( K. pelamis ), chub mackerel ( Scomber japonicus ), Atlantic mackerel ( Scomber scombrus ), and Indo-Pacific king mackerel ( Scomberomorus guttatus ). In addition, four outgroup genomes were downloaded from the GenBank database: Savalai hairtail ( Lepturacanthus savala ) and silver pomfret ( Pampus argenteus ). The accession numbers are listed in Table 1 . The sult1st6 -encoding scaffolds of Pacific bluefin tuna ( T. orientalis ; PBT), namely, M175 and M30, were previously obtained [ 8 ]. Comparison of genomic sequences Nucleotide sequences among the genomes examined were aligned using the nucmer program in MUMmer v4 [ 11 ] with “--minmatch = 9” option and > 70% identity. In the comparison of sult1st6 loci, “--maxmatch” option was added. Alignment dot plots were generated by gnuplot v5.0 ( http://www.gnuplot.info/ ), and genomic synteny was manually checked. Molecular phylogenetic analysis The PBT sult1st6 sequences were obtained previously [ 8 ], and the exon-intron structures were verified using gonad RNA-Seq reads (accession number: DRA016085) [ 8 ] ( Additional file 1 ). The sult1st6 sequences for SBT and YFT were obtained according to annotations in GenBank ( Table 1 ). The sult1st6 loci in the other genomes were predicted by Exonerate v2.4.0 [ 12 ] using the PBT sult1st6 sequences ( Additional file 1 ). The sequences were aligned using MAFFT v7.490 [ 13 ] with genafpair option, where the multiple alignment was constructed using the deduced protein sequences and converted into codon-based nucleotide alignment. A phylogenetic tree based on 1000 bootstrap replicates was constructed using RAxML-NG v.1.1.0 [ 14 ] by HKY + I + G4 model, which was estimated using ModelTest-NG v0.1.7 [ 15 ]. Results and Discussion Identification of sult1st6 loci in tuna and closely related genomes. The sult1st6 loci were explored in 12 chromosome-level genomes of tuna and related species ( Table 1 ). Two sult1st6 copies were newly predicted in the kawakawa, skipjack, and Savalai hairtail genomes, as with the already annotated SBT and YFT genomes. Other genomes, including those of ABT, mackerels, and silver pomfret, encoded one copy. Using the coding-sequences, the phylogenetic tree of fish sult1st6 was constructed (Fig. 1 a, Additional file 2: Figure S1 ). The two sult1st6 paralogs in the SBT male genome were previously identified as sult1st6y on chromosome 13 and sult1st6a on chromosome 20, respectively [ 7 ]. In the present study, the two genes found in the YFT genome were also identified as sult1st6y and sult1st6a based on their phylogenetic relationship, and were located on the counterpart chromosomes to those of SBT ( Table 1 and Fig. 1 b). Only sult1st6a has been identified on chromosome 20 of the ABT genome. Regarding kawakawa and skipjack tuna, one copy was placed near the sult1st6y clade of Thunnus in the phylogenetic tree, and the other was placed near the sult1st6a clade, indicating that these were sult1st6y and sult1st6a , respectively (Fig. 1 a). In particular, sult1st6a was located on chromosome 20 in kawakawa and chromosome 18 in skipjack, both of which are counterparts of Thunnus chromosome 20 ( Table 1 , Fig. 1 b, and Additional file 2: Figure S2 ). The kawakawa sult1st6y was found on chromosome 8, which is not the counterpart of Thunnus chromosome 13. The sult1st6y of skipjack was found on a scaffold (JBFSMI010000026) that was not anchored to any chromosome. In mackerels, a single copy of sult1st6 was close to the clade of tuna sult1st6a in the phylogenetic tree, but was not significantly supported. However, at the genome level, the sult1st6 locus of mackerel was located on the counterpart of Thunnus chromosome 20, which is consistent with sult1st6a synteny ( Additional file 2: Figure S2 ). Thus, the mackerel sult1st6 was identified as sult1st6a . Regarding the tuna and mackerel outgroups, the phylogenetic position of a single sult1st6 of silver pomfret was not significantly supported, but the locus was consistent with sult1st6a synteny ( Additional file 2: Figure S2 ). Two copies of sult1st6 in the Savalai hairtail genome were clustered in the phylogenetic tree and were positioned significantly outside of tuna and mackerel sult1st6 . These genes were tandemly located on chromosome 11, which matched sult1st6a -encoding chromosome 20 of Thunnus . These observations suggest that sult1st6y was duplicated from sult1st6a , which may have predated the divergence of tuna ( Thunnus and kawakawa/skipjack). In addition, at the same time as or after the duplication, at least one transposition of sult1st6y locus may have occurred in either or both Thunnus and kawakawa genomes. The duplication of sult1st6 might further predate the divergence of tuna and mackerel according to the topology shown in Fig. 1 a, by assuming that mackerel might have lost sult1st6y . However, these branching points were not strongly supported by bootstrapping. There remains the possibility that duplication might have occurred after the tuna-mackerel divergence. Regarding the relationship between sult1st6y and sex ( Table 1 ), sult1st6y was identified in the YFT genome but not in the ABT genome; however, their sex is unknown. In the present study, I infer that the sequenced YFT sample may be male and the ABT sample may be female (as will be explained later). The female genome of kawakawa includes sult1st6y , which is not the case in PBT females, and this difference may be associated with a difference in the sex determination system: In kawakawa the sex is possibly determined by the ZZ/ZW system [ 16 ], whereas in PBT, sex is determined by the XX/XY system. This difference suggests that the sex-determination gene may have been altered in one or both of these lineages, which is often observed even between closely related species [ 5 ]. Therefore, in kawakawa, sult1st6y may not be a sex-determination gene: it may be unrelated to sex determination/differentiation, or related to it in a different manner from PBT. As for mackerels, it is reported that chub mackerel has the ZZ/ZW sex-determination system, but blue mackerel ( Scomber australasicus ) has the XX/XY sex-determination system [ 17 ]. Thus, the absence of sult1st6y in mackerels may not be associated with sex determination or differentiation. Comparison of sult1st6 loci among tunas and their relatives. Nucleotide sequences of the PBT sult1st6y and sult1st6a regions were aligned (Fig. 2 a). Here, the sult1st6 locus structures in the PBT genome were verified based on the gonad transcriptome. I concluded that the major transcripts of sult1st6y and sult1st6a were composed of eight and seven exons, respectively ( Additional file 2: Figure S3 ). Alignment revealed that the sult1st6y intron region was larger than that of sult1st6a , and rich in repetitive sequences. In particular, a degenerated sequence of DNA transposon, helentron [ 18 ], was found in the fifth intron of sult1st6y . Next, sequences of the sult1st6 loci of PBT were compared with those of other related genomes (Fig. 2 b, Additional file 2: Figure S4 ). As observed in the PBT genome, repetitive sequences were rich in the introns of SBT and YFT sult1st6y , and a common helentron was found in the fifth intron (Fig. 2 b). In kawakawa and skipjack, no helentron sequences were found in sult1st6y introns. However, the fifth intron of skipjack sult1st6y was very large (861887–1199604 bp; ~340 kb), and many repetitive sequences were found ( Additional file 2: Figure S4 ). Based on these results, one may conclude that skipjack sult1st6y has eventually been split or pseudogenized as not properly transcribed, because introns exceeding 100kb are very rare [ 19 ]. However, this gene was estimated to be still under purifying selection ( Additional file 1, Additional file 2: Figure S5 ). On the other hand, there still remains a possibility of very recent pseudogenization by gene fission. Since there is no experimental information on the function of skipjack sult1st6y , further analysis is necessary for resolving this issue. These observations imply that sult1st6y underwent more evolutionary changes—including transposition from one chromosome to another—than has sult1st6a . Some of these may be involved in the neofunctionalization of sult1st6y in Thunnus ; for example, male specificity or gonad-specific expression. In the platyfish ( Xiphophorus maculatus ), testis-specific expression of a gene may be associated with the insertion of a helentron-like transposon [ 20 ]. Thus, the significance of the Thunnus helentron in sult1st6y needs to be verified in future studies. Lastly, it was found that part of the downstream region of sult1st6y was deleted in the SBT genome but conserved in the YFT genome (Fig. 2 b). This region contains the target sequences of the PCR primers for PBT sex identification [ 9 ], which are male-specifically amplified. The primers showed relatively high sensitivity in the sex identification of all other Thunnus species [ 10 ], which was well explained by the assumption that the linkage of target sequences and sult1st6y locus is conserved among Thunnus species. It should be noted that in the previous study the sensitivity of male identification in SBT was relatively low (i.e., low specificity in female identification). This can be explained by the possibility that the target region is polymorphically degenerated in SBT male population, such as in the individual sequenced. This possibility will be tested by genomic data from multiple SBT samples in the future. Furthermore, YFT males were identified using the primers with 100% specificity [ 10 ], suggesting that the target region was always absent in YFT females. Therefore, the sequenced YFT individual with the target region may be male. No target sequences of the PCR primers were found in the ABT genome. Therefore, the sequenced individual was predicted to be female based on 96% sensitivity and 88% specificity in a previous study [ 10 ]. Limitations In this study, the distribution of sult1st6 in Scombridae species and its evolutionary implications were investigated using chromosome-level genomes. It has been shown that sult1st6y occurred before the divergence of Thunnini lineage, followed by a transposition, but the precise duplication timing was not concluded. This could be resolved in the future with the accumulation of additional genomic data, although these are still limited. The sult1st6 sequences published in this paper should also be useful for PCR primer design in cloning. In particular, the presence or absence of sult1st6y should be examined in other tuna species, such as those in the genera Auxis and Allothunnus . However, this type of research depends on sample metadata and the quality of the genome data. Many of the current genomes are obtained from individuals of unknown sex (five out of 12 genomes in the present study). Because the chromosomal location of the skipjack sult1st6y locus has not yet been determined, the possibility of transposition is unverifiable. Abbreviations PBT Pacific bluefin tuna SBT southern bluefin tuna YFT yellowfin tuna ABT Atlantic bluefin tuna Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material All data supporting the findings of this study are available within the paper and its Supplementary Information. Competing interests The authors declare that they have no competing interests. Funding This work was supported by JSPS KAKENHI Grant Number 21H02275. Author contributions Y.N. conceived the work, collected and analyzed the data, and wrote the manuscript. Acknowledgements The author would like to thank Takashi Yanagimoto, Takao Hayashida and Satoshi Soma for their helpful comments. The author would like to thank Editage (www.editage.jp) for English language editing. References Nagahama Y, Chakraborty T, Paul-Prasanth B, Ohta K, Nakamura M. Sex determination, gonadal sex differentiation, and plasticity in vertebrate species. Physiol Rev. 2021;101(3):1237–308. Li M, Sun L, Wang D. Roles of estrogens in fish sexual plasticity and sex differentiation. Gen Comp Endocrinol. 2019;277:9–16. Yamamoto T. Sex Differentiation. In: Fish Physiology. Edited by Hoar WS, Randall DJ, vol. III: Academic Press; 1969: 117–175. Nakamura M, Kobayashi T, Chang X-T, Nagahama Y. Gonadal sex differentiation in teleost fish. J Exp Zool. 1998;281(5):362–72. Kitano J, Ansai S, Takehana Y, Yamamoto Y. Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish. Annu Rev Anim Biosci. 2024;12:233–59. Yasuda S, Liu CC, Takahashi S, Suiko M, Chen L, Snow R, Liu MC. Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: molecular cloning, expression, characterization, and ontogeny study. Biochem Biophys Res Commun. 2005;330(1):219–25. Nakamura Y, Higuchi K, Kumon K, Yasuike M, Takashi T, Gen K, Fujiwara A. Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes). Int J Genomics. 2021;2021:7226353. Hayashida T, Soma S, Nakamura Y, Higuchi K, Kazeto Y, Gen K. Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel). Sci Rep. 2023;13(1):13867. Suda A, Nishiki I, Iwasaki Y, Matsuura A, Akita T, Suzuki N, Fujiwara A. Improvement of the Pacific bluefin tuna ( Thunnus orientalis ) reference genome and development of male-specific DNA markers. Sci Rep. 2019;9(1):14450. Chiba SN, Ohashi S, Tanaka F, Suda A, Fujiwara A, Snodgrass D, Kiyofuji H, Satoh K, Suzuki N. Effectiveness and potential application of sex-identification DNA markers in tunas. Mar Ecol Prog Ser. 2021;659:175–84. Marcais G, Delcher AL, Phillippy AM, Coston R, Salzberg SL, Zimin A. MUMmer4: A fast and versatile genome alignment system. PLoS Comput Biol. 2018;14(1):e1005944. Slater GS, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005;6:31. Katoh K, Asimenos G, Toh H. Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol. 2009;537:39–64. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35(21):4453–5. Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T. ModelTest-NG: A New and Scalable Tool for the Selection of DNA and Protein Evolutionary Models. Mol Biol Evol. 2020;37(1):291–4. Yazawa R, Takeuchi Y, Machida Y, Amezawa K, Kabeya N, Tani R, Kawamura W, Yoshizaki G. Production of triploid eastern little tuna, Euthynnus affinis (Cantor, 1849). Aquac Res. 2019;50(5):1422–30. Tani R, Kawamura W, Morita T, Klopp C, Milhes M, Guiguen Y, Yoshizaki G, Yazawa R. Development of a polymerase chain reaction (PCR)-based genetic sex identification method in the chub mackerel Scomber japonicus and blue mackerel S. australasicus. Fish Sci. 2021;87(6):785–93. Thomas J, Pritham EJ. Helitrons, the Eukaryotic Rolling-circle Transposable Elements. Microbiol Spectr 2015, 3(4). Piovesan A, Caracausi M, Ricci M, Strippoli P, Vitale L, Pelleri MC. Identification of minimal eukaryotic introns through GeneBase, a user-friendly tool for parsing the NCBI Gene databank. DNA Res. 2015;22(6):495–503. Tomaszkiewicz M, Chalopin D, Schartl M, Galiana D, Volff JN. A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon. BMC Genet. 2014;15:44. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table 1. List of sult1st6 genes in tunas and their relatives. Additionalfile1.docx Additional file 1: Supplementary note (docx). Additionalfile2.pdf Additional file 2: Supplementary figures (Fig. S1, Fig. S2, Fig. S3, Fig. S4, and Fig. S5) (pdf). Fig. S1 Phylogenetic relationship of sult1st6 in fish (uncondensed version of Fig. 1a). Fig. S2 Plots of chromosomal alignments. Fig. S3 Structure of sult1st6 paralogs in T. orientalis. Fig. S4 Plots of nucleotide alignments around sult1st6 locus. Fig. S5 Protein sequence alignment of sult1st6 in tuna species and the orthologs of model organisms (zebrafish, mouse and human). Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2025 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Accepted 08 Jul, 2025 Reviews received at journal 14 Apr, 2025 Reviewers agreed at journal 14 Apr, 2025 Reviewers invited by journal 11 Apr, 2025 Submission checks completed at journal 03 Apr, 2025 First submitted to journal 02 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5525443","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":441639932,"identity":"74ab4600-089f-4340-92ac-e6ee20c3de87","order_by":0,"name":"Yoji Nakamura","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYFACNgTTIMFAQo69AcJhJlJLgY0xzwFStDAwfEhL7DlAwFnm7McSP91suyfPIJF8oOCBweH0HunDDxi/VDCwm+PQYtmTdlg6t63YsEEiLQHol8O5PXxpBswyZxiYLRuwazE4kN4A1JLA2CCRYwDWsp+HwYBZso2B2QCHCw3OP2/+DdRiD9OSzsPD/gG/lhtpx0C2JEK1pCXw8PAYMH7Eq+VZmnXOuYTkBp5nIL/YGPbw8BQcZjgjgdsv59OMb+eUJdg2sCcfM/zxR0Ie6LCND39U2CTjCjE4sL+QwGYA4xzmAYa5AT7lYMB/gPkBjM34g4HBjrCWUTAKRsEoGCEAABm/V4MBaj87AAAAAElFTkSuQmCC","orcid":"","institution":"Japan Fisheries Research and Education Agency","correspondingAuthor":true,"prefix":"","firstName":"Yoji","middleName":"","lastName":"Nakamura","suffix":""}],"badges":[],"createdAt":"2024-11-26 07:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5525443/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5525443/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-025-07372-3","type":"published","date":"2025-07-17T16:05:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80547933,"identity":"2e81a06d-0548-423e-b945-c097bafc1f4f","added_by":"auto","created_at":"2025-04-14 14:20:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein tuna species and close relatives. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Phylogenetic relationship of \u003cem\u003esult1st6\u003c/em\u003e in fish. The other \u003cem\u003esult1st6\u003c/em\u003esequences not predicted in this study were from a previous study [7]. For each of the nodes, the bootstrap support is indicated when it is ≥90%. The species examined in this study were highlighted in bold, and \u003cem\u003eThunnus\u003c/em\u003e, kawakawa/skipjack and mackerels were colored in orange, cyan and green, respectively. Some unrelated clades were condensed for visual convenience, and the full version is shown in \u003cstrong\u003eFigure S1\u003c/strong\u003e (\u003cstrong\u003eAdditional file 2\u003c/strong\u003e). (\u003cstrong\u003eb\u003c/strong\u003e) Plots of chromosomal alignments. The chromosomes 13, 6, and 20 of \u003cem\u003eThunnus maccoyii\u003c/em\u003e were used as the references, respectively (Y-axis). The location of \u003cem\u003esult1st6\u003c/em\u003e is shown in red line.\u003c/p\u003e","description":"","filename":"Fig.1r.png","url":"https://assets-eu.researchsquare.com/files/rs-5525443/v1/e276cc59ea9c4e9d7c831639.png"},{"id":80549584,"identity":"11ed63d2-6abb-49ac-bdf9-4e534bd991b1","added_by":"auto","created_at":"2025-04-14 14:36:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loci among tuna species and close relatives.\u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Plots of nucleotide alignments between the \u003cem\u003esult1st6y\u003c/em\u003e and \u003cem\u003esult1st6a\u003c/em\u003eof \u003cem\u003eThunnus orientalis\u003c/em\u003e. The plots of self-alignment were arranged at the left and bottom sides, respectively. Exonic regions are colored in grey, and a helentron remnant is highlighted in cyan. The region highlighted in yellow is the male-specific PCR target region for PBT sex identification. (\u003cstrong\u003eb\u003c/strong\u003e) Plots of nucleotide alignments around \u003cem\u003esult1st6\u003c/em\u003e locus. (upper) In the case of \u003cem\u003esult1st6y\u003c/em\u003e, scaffold M175 of \u003cem\u003eT. orientalis\u003c/em\u003e was used as the reference. A helentron-inserted region in M175 is highlighted in cyan, and the target region for \u003cem\u003eT. orientalis\u003c/em\u003e sex identification is highlighted in yellow. Note that the plot for \u003cem\u003eKatsuwonus pelamis\u003c/em\u003e is shown divided into two parts, because the predicted fifth intron is too long to display. The full-size plot is shown in \u003cstrong\u003eFigure S4\u003c/strong\u003e (\u003cstrong\u003eAdditional file 2\u003c/strong\u003e). (bottom) Plots of nucleotide alignments around \u003cem\u003esult1st6a\u003c/em\u003e. A scaffold M30 of \u003cem\u003eT. orientalis\u003c/em\u003e was used as the reference.\u003c/p\u003e","description":"","filename":"Fig.2r.png","url":"https://assets-eu.researchsquare.com/files/rs-5525443/v1/f15dcccfb81dfbd5424a99ab.png"},{"id":88506035,"identity":"f26b5c3e-962b-43ce-bd5a-4c954f6a3574","added_by":"auto","created_at":"2025-08-07 07:29:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":914845,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5525443/v1/ad03a09a-4755-4b0b-b4ff-694aa0ad8d32.pdf"},{"id":80549290,"identity":"82d46bb1-fb19-41f1-a0e6-c4a1ee064508","added_by":"auto","created_at":"2025-04-14 14:28:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. List of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in tunas and their relatives.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5525443/v1/4fcbac24220a2ff7ae698406.xlsx"},{"id":80547939,"identity":"50371a7c-d3d4-4149-a41e-4d2fbf8df532","added_by":"auto","created_at":"2025-04-14 14:20:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Supplementary note (docx).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5525443/v1/59b22ab07cbb833efad4de4a.docx"},{"id":80547943,"identity":"6c74cdd0-7642-4b2a-82f8-79197bd96874","added_by":"auto","created_at":"2025-04-14 14:20:03","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3389079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Supplementary figures (Fig. S1, Fig. S2, Fig. S3, Fig. S4, and Fig. S5) (pdf).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1 Phylogenetic relationship of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in fish (uncondensed version of Fig. 1a).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2 Plots of chromosomal alignments.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S3 Structure of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eparalogs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. orientalis.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S4 Plots of nucleotide alignments around \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e locus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S5 Protein sequence alignment of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esult1st6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in tuna species and the orthologs of model organisms (zebrafish, mouse and human).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5525443/v1/e33abdad31b8d78515adac89.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Distribution of estrogen sulfotransferase genes in tunas and their relatives","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEstrogens are important sex steroid hormones in animals. In fish, these are considered key factors in sex determination and differentiation [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Artificial estrogen administration can cause sexual changes in many fish species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and some of the reported sex determination genes are involved in estrogen biosynthesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Estrogens can be inactivated by sulfation, which is mediated by one of the cytosolic sulfotransferases (SULTs), that is, estrogen sulfotransferase. In fish estrogen sulfotransferase\u0026mdash;first reported in zebrafish in 2005 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u0026mdash;the encoding gene, \u003cem\u003esult1st6\u003c/em\u003e, was not considered to be related to sexual maturity but to organogenesis, based on its expression during embryogenesis.\u003c/p\u003e \u003cp\u003eHowever, it was recently reported that the male genome of Pacific bluefin tuna (\u003cem\u003eThunnus orientalis\u003c/em\u003e, Scombridae) encodes two copies of \u003cem\u003esult1st6\u003c/em\u003e, one of which, named \u003cem\u003esult1st6y\u003c/em\u003e, is absent from females [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Another copy, \u003cem\u003esult1st6a\u003c/em\u003e, was found to be present in both males and females. The male specificity of \u003cem\u003esult1st6y\u003c/em\u003e was indirectly supported by the gonad transcriptome of southern bluefin tuna (\u003cem\u003eT. maccoyii\u003c/em\u003e) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, a significant level of expression of \u003cem\u003eT. orienatlis sult1st6y\u003c/em\u003e was observed in male gonad cells at an early stage of sex differentiation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In a previous study, three PCR primer sets were developed for sex identification of \u003cem\u003eT. orientalis\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and their target regions were linked to the \u003cem\u003esult1st6y\u003c/em\u003e locus. These primer sets were also applicable for sex identification in all other \u003cem\u003eThunnus\u003c/em\u003e species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], implying that male-specific amplification might be associated with the male-specificity of \u003cem\u003esult1st6y\u003c/em\u003e. These observations raise the hypothesis that \u003cem\u003esult1st6y\u003c/em\u003e may be a sex-determining gene common to the genus \u003cem\u003eThunnus\u003c/em\u003e as a suppressor of feminization.\u003c/p\u003e \u003cp\u003eMobile element-related genes are found around the \u003cem\u003esult1st6y\u003c/em\u003e locus in the \u003cem\u003eT. orientalis\u003c/em\u003e genome, and synteny is not conserved among teleost genomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, the \u003cem\u003esult1st6a\u003c/em\u003e locus was within the conserved synteny of other fish genomes. This observation implies that \u003cem\u003eT. orientalis sult1st6y\u003c/em\u003e is evolutionarily unstable compared to the \u003cem\u003esult1st6a\u003c/em\u003e locus, but it is not known if this is also the case with closely related species. Furthermore, it has not yet been determined when the duplication of \u003cem\u003esult1st6\u003c/em\u003e occurred. In this study, I aimed to update the previous results on \u003cem\u003esult1st6\u003c/em\u003e reported in \u003cem\u003eT. orientalis\u003c/em\u003e. Particularly, the chromosome-level genome assemblies of tunas and mackerels were compared, which have accumulated in recent years, allowing us to compare the syntenies around \u003cem\u003esult1st6\u003c/em\u003e locus. The presence or absence and gene structures of \u003cem\u003esult1st6\u003c/em\u003e homologs in tunas and their relative were examined.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGenomic data\u003c/h2\u003e \u003cp\u003eA total of eight Scombridae genome sequences at the chromosome-level as of August 5, 2024, five of which were those of tunas (tribe Thunnini, namely \u003cem\u003eThunnus\u003c/em\u003e, \u003cem\u003eEuthynnus\u003c/em\u003e and \u003cem\u003eKatsuwonus\u003c/em\u003e) and the other three were of mackerels (\u003cem\u003eScomber\u003c/em\u003e and \u003cem\u003eScomberomorus\u003c/em\u003e), were downloaded from the GenBank database: southern bluefin tuna (\u003cem\u003eT. maccoyii\u003c/em\u003e; SBT), yellowfin tuna (\u003cem\u003eT. albacares\u003c/em\u003e; YFT), Atlantic bluefin tuna (\u003cem\u003eT. thynnus\u003c/em\u003e; ABT), kawakawa tuna (\u003cem\u003eE. affinis\u003c/em\u003e), skipjack tuna (\u003cem\u003eK. pelamis\u003c/em\u003e), chub mackerel (\u003cem\u003eScomber japonicus\u003c/em\u003e), Atlantic mackerel (\u003cem\u003eScomber scombrus\u003c/em\u003e), and Indo-Pacific king mackerel (\u003cem\u003eScomberomorus guttatus\u003c/em\u003e). In addition, four outgroup genomes were downloaded from the GenBank database: Savalai hairtail (\u003cem\u003eLepturacanthus savala\u003c/em\u003e) and silver pomfret (\u003cem\u003ePampus argenteus\u003c/em\u003e). The accession numbers are listed in \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. The \u003cem\u003esult1st6\u003c/em\u003e-encoding scaffolds of Pacific bluefin tuna (\u003cem\u003eT. orientalis\u003c/em\u003e; PBT), namely, M175 and M30, were previously obtained [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison of genomic sequences\u003c/h3\u003e\n\u003cp\u003eNucleotide sequences among the genomes examined were aligned using the \u003cem\u003enucmer\u003c/em\u003e program in MUMmer v4 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] with \u0026ldquo;--minmatch\u0026thinsp;=\u0026thinsp;9\u0026rdquo; option and \u0026gt;\u0026thinsp;70% identity. In the comparison of \u003cem\u003esult1st6\u003c/em\u003e loci, \u0026ldquo;--maxmatch\u0026rdquo; option was added. Alignment dot plots were generated by gnuplot v5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gnuplot.info/\u003c/span\u003e\u003cspan address=\"http://www.gnuplot.info/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and genomic synteny was manually checked.\u003c/p\u003e\n\u003ch3\u003eMolecular phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eThe PBT \u003cem\u003esult1st6\u003c/em\u003e sequences were obtained previously [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the exon-intron structures were verified using gonad RNA-Seq reads (accession number: DRA016085) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (\u003cb\u003eAdditional file 1\u003c/b\u003e). The \u003cem\u003esult1st6\u003c/em\u003e sequences for SBT and YFT were obtained according to annotations in GenBank (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). The \u003cem\u003esult1st6\u003c/em\u003e loci in the other genomes were predicted by Exonerate v2.4.0 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] using the PBT \u003cem\u003esult1st6\u003c/em\u003e sequences (\u003cb\u003eAdditional file 1\u003c/b\u003e). The sequences were aligned using MAFFT v7.490 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] with \u003cem\u003egenafpair\u003c/em\u003e option, where the multiple alignment was constructed using the deduced protein sequences and converted into codon-based nucleotide alignment. A phylogenetic tree based on 1000 bootstrap replicates was constructed using RAxML-NG v.1.1.0 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] by HKY\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G4 model, which was estimated using ModelTest-NG v0.1.7 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003esult1st6\u003c/b\u003e \u003cb\u003eloci in tuna and closely related genomes.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003esult1st6\u003c/em\u003e loci were explored in 12 chromosome-level genomes of tuna and related species (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Two \u003cem\u003esult1st6\u003c/em\u003e copies were newly predicted in the kawakawa, skipjack, and Savalai hairtail genomes, as with the already annotated SBT and YFT genomes. Other genomes, including those of ABT, mackerels, and silver pomfret, encoded one copy. Using the coding-sequences, the phylogenetic tree of fish \u003cem\u003esult1st6\u003c/em\u003e was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cb\u003eAdditional file 2: Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The two \u003cem\u003esult1st6\u003c/em\u003e paralogs in the SBT male genome were previously identified as \u003cem\u003esult1st6y\u003c/em\u003e on chromosome 13 and \u003cem\u003esult1st6a\u003c/em\u003e on chromosome 20, respectively [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the present study, the two genes found in the YFT genome were also identified as \u003cem\u003esult1st6y\u003c/em\u003e and \u003cem\u003esult1st6a\u003c/em\u003e based on their phylogenetic relationship, and were located on the counterpart chromosomes to those of SBT (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Only \u003cem\u003esult1st6a\u003c/em\u003e has been identified on chromosome 20 of the ABT genome.\u003c/p\u003e \u003cp\u003eRegarding kawakawa and skipjack tuna, one copy was placed near the \u003cem\u003esult1st6y\u003c/em\u003e clade of \u003cem\u003eThunnus\u003c/em\u003e in the phylogenetic tree, and the other was placed near the \u003cem\u003esult1st6a\u003c/em\u003e clade, indicating that these were \u003cem\u003esult1st6y\u003c/em\u003e and \u003cem\u003esult1st6a\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In particular, \u003cem\u003esult1st6a\u003c/em\u003e was located on chromosome 20 in kawakawa and chromosome 18 in skipjack, both of which are counterparts of \u003cem\u003eThunnus\u003c/em\u003e chromosome 20 (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, and \u003cb\u003eAdditional file 2: Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). The kawakawa \u003cem\u003esult1st6y\u003c/em\u003e was found on chromosome 8, which is not the counterpart of \u003cem\u003eThunnus\u003c/em\u003e chromosome 13. The \u003cem\u003esult1st6y\u003c/em\u003e of skipjack was found on a scaffold (JBFSMI010000026) that was not anchored to any chromosome. In mackerels, a single copy of \u003cem\u003esult1st6\u003c/em\u003e was close to the clade of tuna \u003cem\u003esult1st6a\u003c/em\u003e in the phylogenetic tree, but was not significantly supported. However, at the genome level, the \u003cem\u003esult1st6\u003c/em\u003e locus of mackerel was located on the counterpart of \u003cem\u003eThunnus\u003c/em\u003e chromosome 20, which is consistent with \u003cem\u003esult1st6a\u003c/em\u003e synteny (\u003cb\u003eAdditional file 2: Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Thus, the mackerel \u003cem\u003esult1st6\u003c/em\u003e was identified as \u003cem\u003esult1st6a\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRegarding the tuna and mackerel outgroups, the phylogenetic position of a single \u003cem\u003esult1st6\u003c/em\u003e of silver pomfret was not significantly supported, but the locus was consistent with \u003cem\u003esult1st6a\u003c/em\u003e synteny (\u003cb\u003eAdditional file 2: Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Two copies of \u003cem\u003esult1st6\u003c/em\u003e in the Savalai hairtail genome were clustered in the phylogenetic tree and were positioned significantly outside of tuna and mackerel \u003cem\u003esult1st6\u003c/em\u003e. These genes were tandemly located on chromosome 11, which matched \u003cem\u003esult1st6a\u003c/em\u003e-encoding chromosome 20 of \u003cem\u003eThunnus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThese observations suggest that \u003cem\u003esult1st6y\u003c/em\u003e was duplicated from \u003cem\u003esult1st6a\u003c/em\u003e, which may have predated the divergence of tuna (\u003cem\u003eThunnus\u003c/em\u003e and kawakawa/skipjack). In addition, at the same time as or after the duplication, at least one transposition of \u003cem\u003esult1st6y\u003c/em\u003e locus may have occurred in either or both \u003cem\u003eThunnus\u003c/em\u003e and kawakawa genomes. The duplication of \u003cem\u003esult1st6\u003c/em\u003e might further predate the divergence of tuna and mackerel according to the topology shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, by assuming that mackerel might have lost \u003cem\u003esult1st6y\u003c/em\u003e. However, these branching points were not strongly supported by bootstrapping. There remains the possibility that duplication might have occurred after the tuna-mackerel divergence.\u003c/p\u003e \u003cp\u003eRegarding the relationship between \u003cem\u003esult1st6y\u003c/em\u003e and sex (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e), \u003cem\u003esult1st6y\u003c/em\u003e was identified in the YFT genome but not in the ABT genome; however, their sex is unknown. In the present study, I infer that the sequenced YFT sample may be male and the ABT sample may be female (as will be explained later). The female genome of kawakawa includes \u003cem\u003esult1st6y\u003c/em\u003e, which is not the case in PBT females, and this difference may be associated with a difference in the sex determination system: In kawakawa the sex is possibly determined by the ZZ/ZW system [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas in PBT, sex is determined by the XX/XY system. This difference suggests that the sex-determination gene may have been altered in one or both of these lineages, which is often observed even between closely related species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, in kawakawa, \u003cem\u003esult1st6y\u003c/em\u003e may not be a sex-determination gene: it may be unrelated to sex determination/differentiation, or related to it in a different manner from PBT. As for mackerels, it is reported that chub mackerel has the ZZ/ZW sex-determination system, but blue mackerel (\u003cem\u003eScomber australasicus\u003c/em\u003e) has the XX/XY sex-determination system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, the absence of \u003cem\u003esult1st6y\u003c/em\u003e in mackerels may not be associated with sex determination or differentiation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison of\u003c/b\u003e \u003cb\u003esult1st6\u003c/b\u003e \u003cb\u003eloci among tunas and their relatives.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNucleotide sequences of the PBT \u003cem\u003esult1st6y\u003c/em\u003e and \u003cem\u003esult1st6a\u003c/em\u003e regions were aligned (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Here, the \u003cem\u003esult1st6\u003c/em\u003e locus structures in the PBT genome were verified based on the gonad transcriptome. I concluded that the major transcripts of \u003cem\u003esult1st6y\u003c/em\u003e and \u003cem\u003esult1st6a\u003c/em\u003e were composed of eight and seven exons, respectively (\u003cb\u003eAdditional file 2: Figure S3\u003c/b\u003e). Alignment revealed that the \u003cem\u003esult1st6y\u003c/em\u003e intron region was larger than that of \u003cem\u003esult1st6a\u003c/em\u003e, and rich in repetitive sequences. In particular, a degenerated sequence of DNA transposon, helentron [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], was found in the fifth intron of \u003cem\u003esult1st6y\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNext, sequences of the \u003cem\u003esult1st6\u003c/em\u003e loci of PBT were compared with those of other related genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cb\u003eAdditional file 2: Figure S4\u003c/b\u003e). As observed in the PBT genome, repetitive sequences were rich in the introns of SBT and YFT \u003cem\u003esult1st6y\u003c/em\u003e, and a common helentron was found in the fifth intron (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In kawakawa and skipjack, no helentron sequences were found in \u003cem\u003esult1st6y\u003c/em\u003e introns. However, the fifth intron of skipjack \u003cem\u003esult1st6y\u003c/em\u003e was very large (861887\u0026ndash;1199604 bp; ~340 kb), and many repetitive sequences were found (\u003cb\u003eAdditional file 2: Figure S4\u003c/b\u003e). Based on these results, one may conclude that skipjack \u003cem\u003esult1st6y\u003c/em\u003e has eventually been split or pseudogenized as not properly transcribed, because introns exceeding 100kb are very rare [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, this gene was estimated to be still under purifying selection (\u003cb\u003eAdditional file 1, Additional file 2: Figure S5\u003c/b\u003e). On the other hand, there still remains a possibility of very recent pseudogenization by gene fission. Since there is no experimental information on the function of skipjack \u003cem\u003esult1st6y\u003c/em\u003e, further analysis is necessary for resolving this issue.\u003c/p\u003e \u003cp\u003eThese observations imply that \u003cem\u003esult1st6y\u003c/em\u003e underwent more evolutionary changes\u0026mdash;including transposition from one chromosome to another\u0026mdash;than has \u003cem\u003esult1st6a\u003c/em\u003e. Some of these may be involved in the neofunctionalization of \u003cem\u003esult1st6y\u003c/em\u003e in \u003cem\u003eThunnus\u003c/em\u003e; for example, male specificity or gonad-specific expression. In the platyfish (\u003cem\u003eXiphophorus maculatus\u003c/em\u003e), testis-specific expression of a gene may be associated with the insertion of a helentron-like transposon [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, the significance of the \u003cem\u003eThunnus\u003c/em\u003e helentron in \u003cem\u003esult1st6y\u003c/em\u003e needs to be verified in future studies.\u003c/p\u003e \u003cp\u003eLastly, it was found that part of the downstream region of \u003cem\u003esult1st6y\u003c/em\u003e was deleted in the SBT genome but conserved in the YFT genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This region contains the target sequences of the PCR primers for PBT sex identification [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which are male-specifically amplified. The primers showed relatively high sensitivity in the sex identification of all other \u003cem\u003eThunnus\u003c/em\u003e species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which was well explained by the assumption that the linkage of target sequences and \u003cem\u003esult1st6y\u003c/em\u003e locus is conserved among \u003cem\u003eThunnus\u003c/em\u003e species. It should be noted that in the previous study the sensitivity of male identification in SBT was relatively low (i.e., low specificity in female identification). This can be explained by the possibility that the target region is polymorphically degenerated in SBT male population, such as in the individual sequenced. This possibility will be tested by genomic data from multiple SBT samples in the future. Furthermore, YFT males were identified using the primers with 100% specificity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], suggesting that the target region was always absent in YFT females. Therefore, the sequenced YFT individual with the target region may be male. No target sequences of the PCR primers were found in the ABT genome. Therefore, the sequenced individual was predicted to be female based on 96% sensitivity and 88% specificity in a previous study [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eLimitations\u003c/h3\u003e\n\u003cp\u003eIn this study, the distribution of \u003cem\u003esult1st6\u003c/em\u003e in Scombridae species and its evolutionary implications were investigated using chromosome-level genomes. It has been shown that \u003cem\u003esult1st6y\u003c/em\u003e occurred before the divergence of \u003cem\u003eThunnini\u003c/em\u003e lineage, followed by a transposition, but the precise duplication timing was not concluded. This could be resolved in the future with the accumulation of additional genomic data, although these are still limited. The \u003cem\u003esult1st6\u003c/em\u003e sequences published in this paper should also be useful for PCR primer design in cloning. In particular, the presence or absence of \u003cem\u003esult1st6y\u003c/em\u003e should be examined in other tuna species, such as those in the genera \u003cem\u003eAuxis\u003c/em\u003e and \u003cem\u003eAllothunnus\u003c/em\u003e. However, this type of research depends on sample metadata and the quality of the genome data. Many of the current genomes are obtained from individuals of unknown sex (five out of 12 genomes in the present study). Because the chromosomal location of the skipjack \u003cem\u003esult1st6y\u003c/em\u003e locus has not yet been determined, the possibility of transposition is unverifiable.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cp\u003ePBT Pacific bluefin tuna\u003c/p\u003e \u003cp\u003eSBT southern bluefin tuna\u003c/p\u003e \u003cp\u003eYFT yellowfin tuna\u003c/p\u003e \u003cp\u003eABT Atlantic bluefin tuna\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number 21H02275.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.N. conceived the work, collected and analyzed the data, and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author would like to thank Takashi Yanagimoto, Takao Hayashida and Satoshi Soma for their helpful comments. The author would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNagahama Y, Chakraborty T, Paul-Prasanth B, Ohta K, Nakamura M. Sex determination, gonadal sex differentiation, and plasticity in vertebrate species. Physiol Rev. 2021;101(3):1237\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Sun L, Wang D. Roles of estrogens in fish sexual plasticity and sex differentiation. Gen Comp Endocrinol. 2019;277:9\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto T. Sex Differentiation. In: \u003cem\u003eFish Physiology.\u003c/em\u003e Edited by Hoar WS, Randall DJ, vol. III: Academic Press; 1969: 117\u0026ndash;175.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura M, Kobayashi T, Chang X-T, Nagahama Y. Gonadal sex differentiation in teleost fish. J Exp Zool. 1998;281(5):362\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitano J, Ansai S, Takehana Y, Yamamoto Y. Diversity and Convergence of Sex-Determination Mechanisms in Teleost Fish. Annu Rev Anim Biosci. 2024;12:233\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasuda S, Liu CC, Takahashi S, Suiko M, Chen L, Snow R, Liu MC. Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: molecular cloning, expression, characterization, and ontogeny study. Biochem Biophys Res Commun. 2005;330(1):219\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura Y, Higuchi K, Kumon K, Yasuike M, Takashi T, Gen K, Fujiwara A. Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes). Int J Genomics. 2021;2021:7226353.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashida T, Soma S, Nakamura Y, Higuchi K, Kazeto Y, Gen K. Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel). Sci Rep. 2023;13(1):13867.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuda A, Nishiki I, Iwasaki Y, Matsuura A, Akita T, Suzuki N, Fujiwara A. Improvement of the Pacific bluefin tuna (\u003cem\u003eThunnus orientalis\u003c/em\u003e) reference genome and development of male-specific DNA markers. Sci Rep. 2019;9(1):14450.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiba SN, Ohashi S, Tanaka F, Suda A, Fujiwara A, Snodgrass D, Kiyofuji H, Satoh K, Suzuki N. Effectiveness and potential application of sex-identification DNA markers in tunas. Mar Ecol Prog Ser. 2021;659:175\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcais G, Delcher AL, Phillippy AM, Coston R, Salzberg SL, Zimin A. MUMmer4: A fast and versatile genome alignment system. PLoS Comput Biol. 2018;14(1):e1005944.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlater GS, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005;6:31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatoh K, Asimenos G, Toh H. Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol. 2009;537:39\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35(21):4453\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T. ModelTest-NG: A New and Scalable Tool for the Selection of DNA and Protein Evolutionary Models. Mol Biol Evol. 2020;37(1):291\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYazawa R, Takeuchi Y, Machida Y, Amezawa K, Kabeya N, Tani R, Kawamura W, Yoshizaki G. Production of triploid eastern little tuna, \u003cem\u003eEuthynnus affinis\u003c/em\u003e (Cantor, 1849). Aquac Res. 2019;50(5):1422\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTani R, Kawamura W, Morita T, Klopp C, Milhes M, Guiguen Y, Yoshizaki G, Yazawa R. Development of a polymerase chain reaction (PCR)-based genetic sex identification method in the chub mackerel Scomber japonicus and blue mackerel S. australasicus. Fish Sci. 2021;87(6):785\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas J, Pritham EJ. Helitrons, the Eukaryotic Rolling-circle Transposable Elements. Microbiol Spectr 2015, 3(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiovesan A, Caracausi M, Ricci M, Strippoli P, Vitale L, Pelleri MC. Identification of minimal eukaryotic introns through GeneBase, a user-friendly tool for parsing the NCBI Gene databank. DNA Res. 2015;22(6):495\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomaszkiewicz M, Chalopin D, Schartl M, Galiana D, Volff JN. A multicopy Y-chromosomal SGNH hydrolase gene expressed in the testis of the platyfish has been captured and mobilized by a Helitron transposon. BMC Genet. 2014;15:44.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"estrogen sulfotransferase, sex determination, Thunnini tribe, gene duplication","lastPublishedDoi":"10.21203/rs.3.rs-5525443/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5525443/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eObjective\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEstrogen sulfotransferase is an enzyme involved in the inactivation of estrogen, and the encoding gene, \u003cem\u003esult1st6\u003c/em\u003e, is widely conserved among animal species. Recently, it was reported that a paralog of the estrogen sulfotransferase gene is paternally inherited in Pacific bluefin tuna (\u003cem\u003eThunnus orientalis\u003c/em\u003e), suggesting that this gene, \u003cem\u003esult1st6y\u003c/em\u003e, may be involved in sex determination or differentiation of \u003cem\u003eThunnus\u003c/em\u003e species as a suppressor of feminization. However, current knowledge on fish \u003cem\u003esult1st6\u003c/em\u003e is still limited. This study aims to update the previous findings by thoroughly examining the distribution of \u003cem\u003esult1st6\u003c/em\u003e in tuna (the species of tribe Thunnini) and their relatives (the species of family Scombridae).\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on a survey of the \u003cem\u003esult1st6\u003c/em\u003e loci in the available chromosome-level genomes of Scombridae species, it was found that tuna species carry two copies of \u003cem\u003esult1st6\u003c/em\u003e. Based on phylogenetic analysis and genome comparison, these paralogs were identified as \u003cem\u003esult1st6y\u003c/em\u003e and its paralog, namely \u003cem\u003esult1st6a\u003c/em\u003e, respectively, suggesting that \u003cem\u003esult1st6y\u003c/em\u003e may have originated from \u003cem\u003esult1st6a\u003c/em\u003e by duplication prior to the divergence of the Thunnini tribe. In addition, the \u003cem\u003esult1st6y\u003c/em\u003e locus may have undergone more structural changes than has the \u003cem\u003esult1st6a\u003c/em\u003e locus. These results provide insight into the evolutionary scenario of the sex determination system of tunas.\u003c/p\u003e","manuscriptTitle":"Distribution of estrogen sulfotransferase genes in tunas and their relatives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-14 14:19:58","doi":"10.21203/rs.3.rs-5525443/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-07-08T19:29:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T01:17:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165643514196698642584444557194535051201","date":"2025-04-15T00:36:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-11T10:55:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-03T06:36:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-04-02T07:02:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"055a05d6-44f5-469d-971a-dca1d9eca4cd","owner":[],"postedDate":"April 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:12:23+00:00","versionOfRecord":{"articleIdentity":"rs-5525443","link":"https://doi.org/10.1186/s13104-025-07372-3","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2025-07-17 16:05:41","publishedOnDateReadable":"July 17th, 2025"},"versionCreatedAt":"2025-04-14 14:19:58","video":"","vorDoi":"10.1186/s13104-025-07372-3","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07372-3","workflowStages":[]},"version":"v1","identity":"rs-5525443","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5525443","identity":"rs-5525443","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}