Sexual fate of the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae) is determined by the complementary sex determination (CSD) mechanism as is the case in honeybees. However, to date, genes involved in sex determination have not been identified in this species. In this study, we attempted to identify orthologs of complementary sex-determiner (csd), feminizer (fem), and doublesex (dsx) from the A. rosae genome, all of which are crucial components of the sex determination cascade in the honeybee. As a result, we identified a sawfly ortholog of dsx (designated as Ardsx). Rapid amplification of cDNA ends (RACE) using total RNA extracted from male and female larvae identified three male-specific variants and three female-specific variants. Comparison between the full-length Ardsx cDNAs and the genomic sequence revealed that exon 5 was differentially spliced between the male- and female-specific variants. RT-PCR analysis demonstrated that Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner at almost all the developmental stages. RNAi-mediated knockdown of Ardsx in males caused severe defects in the reproductive organs and, notably, induced development of the ovipository apparatus containing the dorsal pair of blades and the sheath. These males also showed abnormalities in testes and seminal vesicles and lacked mature sperm. The present study provides the first direct evidence that dsx is essential for sexual development in hymenopteran species.

中文翻译：

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锯蝇性别决定基因 doublesex 的鉴定和功能表征，Athalia rosae（膜翅目：Tenthredinidae）

锯蝇的性命运，Athalia rosae（膜翅目：Tenthredinidae）是由互补性决定（CSD）机制决定的，就像蜜蜂的情况一样。然而，迄今为止，尚未在该物种中鉴定出与性别决定有关的基因。在这项研究中，我们试图从 A. rosae 基因组中鉴定互补性别决定子 (csd)、女性化 (fem) 和双性 (dsx) 的直向同源物，所有这些都是蜜蜂性别决定级联的关键组成部分。结果，我们确定了 dsx 的锯蝇直系同源物（指定为 Ardsx）。使用从雄性和雌性幼虫中提取的总 RNA 的 cDNA 末端快速扩增 (RACE) 鉴定了三种雄性特异性变异和三种雌性特异性变异。全长 Ardsx cDNA 与基因组序列之间的比较表明，外显子 5 在男性和女性特异性变体之间存在差异剪接。RT-PCR 分析表明，Ardsx 前体 mRNA 在几乎所有发育阶段都以性别依赖性方式交替剪接。RNAi 介导的雄性 Ardsx 敲低导致生殖器官的严重缺陷，尤其是诱导包含背对叶片和鞘的产卵器的发育。这些雄性还表现出睾丸和精囊异常，缺乏成熟的精子。本研究提供了第一个直接证据，表明 dsx 对膜翅目物种的性发育至关重要。RT-PCR 分析表明，Ardsx 前体 mRNA 在几乎所有发育阶段都以性别依赖性方式交替剪接。RNAi 介导的雄性 Ardsx 敲低导致生殖器官的严重缺陷，尤其是诱导包含背对叶片和鞘的产卵器的发育。这些雄性还表现出睾丸和精囊异常，缺乏成熟的精子。本研究提供了第一个直接证据，表明 dsx 对膜翅目物种的性发育至关重要。RT-PCR 分析表明，Ardsx 前体 mRNA 在几乎所有发育阶段都以性别依赖性方式交替剪接。RNAi 介导的雄性 Ardsx 敲低导致生殖器官的严重缺陷，尤其是诱导包含背对叶片和鞘的产卵器的发育。这些雄性还表现出睾丸和精囊异常，缺乏成熟的精子。本研究提供了第一个直接证据，表明 dsx 对膜翅目物种的性发育至关重要。这些雄性还表现出睾丸和精囊异常，缺乏成熟的精子。本研究提供了第一个直接证据，表明 dsx 对膜翅目物种的性发育至关重要。这些雄性还表现出睾丸和精囊异常，缺乏成熟的精子。本研究提供了第一个直接证据，表明 dsx 对膜翅目物种的性发育至关重要。