Table_3_Protandric Transcriptomes to Uncover Parts of the Crustacean Sex-Differentiation Puzzle.xlsx

Hermaphrodite systems offer unique opportunities to study sexual differentiation, due to their high degree of sexual plasticity and to the fact that, unlike gonochoristic systems, the process is not confined to an early developmental stage. In protandric shrimp species, such as Hippolyte inermis and Pandalus platyceros, male differentiation is followed by transformation to femaleness during adulthood. The mechanisms controlling sexual differentiation have not been fully elucidated in crustaceans, but a key role has been attributed to the insulin-like hormone (IAG) produced by the androgenic gland (AG), a crustacean masculine endocrine organ. To uncover further transcriptomic toolkit elements affecting the sexual differentiation of H. inermis, we constructed eye and whole body RNA libraries of four representative stages during its protandric life cycle (immature, male, young female and mature female). The body libraries contained transcripts related to the reproductive system, among others, while the eye libraries contained transcripts related to the X-organ-sinus gland, a central endocrine complex that regulates crustacean reproduction. Binary pattern analysis, performed to mine for genes expressed differentially between the different life stages, yielded 19,605 and 6,175 transcripts with a specific expression pattern in the eye and body, respectively. Prominent sexually biased transcriptomic patterns were recorded for the IAG and vitellogenin genes, representing, respectively, a key factor within the masculine IAG-switch, and a precursor of the yolk protein, typical of feminine reproductive states. These patterns enabled the discovery of novel putative protein-coding transcripts exhibiting sexually biased expression in the H. inermis body and eye transcriptomes of males and females. Homologs to the above novel genes have been found in other decapod crustaceans, and a comparative study, using previously constructed transcriptomic libraries of another protandric shrimp, P. platyceros, showed similar sexually biased results, supporting the notion that such genes, mined from the H. inermis transcriptome, may be universal factors related to reproduction and sexual differentiation and their control in other crustaceans. This study thus demonstrates the potential of transcriptomic studies in protandric species to uncover unexplored layers of the complex crustacean sex-differentiation puzzle.

雌雄同体系统（Hermaphrodite systems）凭借其高度的性可塑性，且与雌雄异体系统（gonochoristic systems）不同，其性别分化过程并不局限于发育早期阶段，因此为研究性别分化提供了独特的契机。在雄性先熟虾类（protandric shrimp species）中，如细角糠虾（Hippolyte inermis）和扁形对虾（Pandalus platyceros），个体先经历雄性分化阶段，随后在成年期转变为雌性。甲壳类动物的性别分化调控机制尚未完全阐明，但目前认为其雄性内分泌器官——雄性腺（androgenic gland, AG）所分泌的类胰岛素激素（insulin-like hormone, IAG）发挥着关键作用。为进一步挖掘影响细角糠虾（H. inermis）性别分化的转录组工具箱元件，我们构建了其雄性先熟生命周期中4个典型阶段的眼组织与全身体RNA文库，这4个阶段分别为未成熟期、雄性期、年轻雌性期与成熟雌性期。全身体文库包含与生殖系统等相关的转录本，而眼组织文库则包含与调控甲壳类生殖的中枢内分泌复合体——X器官-窦腺（X-organ-sinus gland）相关的转录本。通过对不同生命阶段间的差异表达基因进行二元模式分析，我们分别在眼组织与全身体转录组中筛选得到19605条与6175条具有特异性表达模式的转录本。研究记录到类胰岛素激素（IAG）与卵黄蛋白原（vitellogenin）基因存在显著的性别偏向性转录组表达模式：前者是雄性IAG调控通路中的关键因子，后者则是雌性生殖状态典型的卵黄蛋白前体。上述表达模式帮助我们发现了一批新的潜在蛋白编码转录本，它们在细角糠虾的雄性与雌性个体的体组织与眼组织转录组中呈现性别偏向性表达。我们在其他十足目甲壳动物（decapod crustaceans）中发现了这些新基因的同源序列；此外，通过使用另一雄性先熟虾类——扁形对虾（P. platyceros）此前构建的转录组文库进行比较分析，得到了相似的性别偏向性表达结果，这支持了如下观点：从细角糠虾转录组中筛选得到的这类基因，可能是其他甲壳类动物生殖与性别分化调控相关的通用因子。因此，本研究证实了针对雄性先熟物种开展转录组学研究，有助于揭示复杂甲壳类动物性别分化谜题中尚未被探索的层面。