2024, Vol. 31 
2. 福建农林大学动物科学学院，福建 福州 350002
3. 福建农林大学海洋学院，福建省海洋生物技术重点实验室，福建 福州 350002
4. 广东渔泽原生物科技有限公司，广东 珠海 519000
2. College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3. Key Laboratory of Marine Biotechnology of Fujian Province; College of Marine Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
4. Guangdong Yuzeyuan Biotechnology Co., Ltd., Zhuhai 519000, China
十足目甲壳动物种类丰富，有复杂的生活史，性别决定与分化途径也有多种方式。其中，多数十足目水产经济动物表现出性别二态性生长模式或雌雄差异经济性状，如罗氏沼虾(Macrobrachium rosenbergii)[1]、日本沼虾(Macrobrachium nipponense)[2]、凡纳对虾(Penaeus vannamei)[3]、拟穴青蟹(Scylla paramamosain)[4]、中华绒螯蟹(Eriocheir sinensis)[5]、克氏原螯虾(Procambarus clarkii)[6]、红螯光壳螯虾(Cherax quadricarinatus)[7]等。对这些水产经济动物需求的持续增长，推动了其苗种生产与养殖管理技术的不断进步。因此，性别控制与单性(全雄或全雌)苗种培育及其养殖技术研究，在遗传育种和养殖经济效益提高等方面具有重要意义[8-9]，成为十足目水产经济动物的研究热点之一[10-23]。
在十足目甲壳动物的性别控制中，已有三倍体诱导、外源激素或化学物质处理、促雄腺(Androgenic gland, AG)摘除和移植，以及胰岛素样促雄腺激素(insulin-like androgenic gland hormone, IAG)基因的RNA干扰技术等多种方法来建立高度单性化群体的研究报道。这些方法在性别决定与分化的调控机制研究方面具有重要的应用价值。本文综述了十足目甲壳动物性别决定和性别分化的分子调控机制研究进展，阐述在这一过程中发挥作用的关键因素，如眼柄、促雄腺等内分泌组织器官，IAG基因和dmrt[Doublesex and male abnormal-3 (mab-3) related transcription factor]基因家族等关键基因，探讨RNA干扰技术等获得单性群体的性别控制方案，以期为十足目甲壳动物规模化单性苗种生产和养殖研究提供参考和理论依据。
1 十足目甲壳动物性别决定与性别分化机制性别决定和性别分化是动物性别形成的两个主要过程，性别分化则是在性别决定的影响下开始的表型变化。与脊椎动物相比，影响甲壳动物性别决定和分化过程的因素复杂多样，性别的遗传决定差异很大。
十足目甲壳动物的性别决定和性别分化历经多次演变，其分子机制涉及性染色体、性别决定与性别分化的关键因子、表观遗传学修饰以及基因表达调控网络等多个层面。其中的关键基因可能包括tra-2、sxl、fem-1、fruitless、masc、sox基因家族、dmrt基因家族、IAG、CFSH和foxl2等。这些复杂的机制使得十足目甲壳动物在发育早期或特定生长阶段实现性别的决定和分化，从而适应不同的自然环境条件。
1.1 性别决定与性别分化 1.1.1 性别决定十足目甲壳动物的性别具有较大的可塑性，表现出多样的性别决定模式和生殖策略，其中雌雄异体(gonochorism)是最为常见的。大多数情况下雌雄异体的雌雄个体在性别分化后分别保持各自雌性或雄性性别特征。但也有较为复杂的雌雄异体情况，例如，罗氏沼虾存在3种雄性形态[24]，表明其不同的生殖行为和第二性征；红螯光壳螯虾有较为普遍的雌雄间性现象(intersexuality或intersexual phenotype)[25-26]，反映其性别可塑性程度高。而更为复杂的生殖策略是雌雄同体(hermaphroditism)，它分为同时性的(simultaneous hermaphroditism)或顺序性的(sequential hermaphroditism)。同时性雌雄同体指个体可同时为功能性雄性和雌性，如藻虾科鞭藻虾属的锯齿鞭腕虾(Lysmata debelius)、安波鞭腕虾(Lysmata amboinensis)等[27]。顺序性雌雄同体指在生活史中能改变其性功能的个体，分为雄性先熟雌雄同体(protandry)和雌性先熟雌雄同体(protogyny)，存在一个短暂的雌雄间性过渡阶段。两性在功能上是分开的，形态上也有所不同，但个体在不同生活史阶段分别产生雄性和雌性配子。例如，宽角长额虾(Pandalus platyceros)是一种雄性先熟的雌雄同体虾类，其生活史表现为3个连续的生命阶段，即从功能性雄性，经历过渡阶段，最后转变为功能性雌性[28]。而另一种极端的生殖策略是孤雌生殖(parthenogenesis)，大理石纹螯虾[龙纹螯虾(Procambarus fallax)三倍体变异种]通过“无性生殖”策略，即不需要受精就能产生可存活的后代，从而形成一个全雌性的群体[29]。虽然关于性别决定的演化尚缺乏直接证据，但根据现有的研究，认为甲壳动物性别决定最初是以雌雄同体(包括顺序性雌雄同体和同时性雌雄同体)的形式出现，环境型性别决定(environment sex determination, ESD)可能是从顺序性雌雄同体在早期胚胎发育阶段性别转换时的异时性转变进化而来，雌雄同体和ESD都有发展为雌雄异体遗传型性别决定(genetic sex determination, GSD)的潜力，但很少有从GSD转化为ESD的情况[23]。
甲壳动物的性别决定从多基因控制，到具有优势性别决定因素和常染色体控制，再到具有雄性异配子或雌性异配子的高度进化性染色体。在雄性和雌性中，性别决定系统分别使用不同的基因和调节机制，从而来控制性别分化和性腺发育的关键调节基因[30]。雌雄异体动物遗传性别决定的生理过程起始于受精卵形成，由配子贡献的性染色体决定了性别，最终导致性别分化和性成熟。十足目甲壳动物中，同属的物种也可能会采用不同的性别决定模式，包括XX/XY型和ZZ/ZW型等。由于十足目甲壳动物的染色体具有数量多、形态较小等特征，从形态上来辨别性染色体较为困难。根据已研究物种的测交实验和子代性别统计，以及越来越多的分子生物实验证据表明，多数十足目甲壳动物为ZZ/ZW型性别决定模式，但部分蟹类和龙虾中也存在XX/XY性别决定等模式[8,10]。
十足目甲壳动物的性别决定与分化是一个复杂的过程，涉及基因之间以及基因与环境之间的相互作用网络。由于其原始的性别决定系统具有性别可塑性，在一定程度上也受到光照、盐度、温度和激素等环境因素的影响。
1.1.2 性别分化经过性别决定过程后，紧接着是性别分化阶段，这一阶段受到不同激素调节的影响。在脊椎动物中，精巢产生睾酮(17α-methyltestosterone, MT)等激素，而卵巢产生雌激素和孕酮等，这些激素的作用是维持两性的发育。在生命周期的关键阶段，性激素的相对丰度对两性的正常发育非常重要。而在十足目甲壳动物中，调控性别分化的确切机制尚未被阐明。甲壳动物雌性性激素(crustacean female sex hormone, CFSH)和胰岛素样促雄腺激素(IAG)被认为可能是十足目甲壳动物性别分化的两个主要调控因子。CFSH是甲壳动物特有的一种神经激素，存在两个不同的亚型，首次在蓝蟹(Callinectes sapidus)中发现[31]，在甲壳动物性别分化中有重要作用，参与雌性的性别分化和第二性征维持[32-34]。
与雌性不同，在雄性十足目甲壳动物研究中，普遍认为促雄腺分泌的IAG是雄性性别分化的关键调节因子[20]，如雄性性腺分化、第二性征的发育与雄性行为的维持等。在雄性十足目甲壳动物中，CFSH、sxl、sox基因家族、dmrt基因家族、foxl2、fem-1等可能在“眼柄–促雄腺–精巢”内分泌轴上对IAG的调控起重要作用[19]。通过这些性别分化相关基因的差异表达，可能直接或间接地调控促雄腺的发育，调节雄性化发育过程。而眼柄中分泌的甲壳动物高血糖激素(crustacean hyperglycemic hormone, CHH)、性腺抑制激素(gonad- inhibiting hormone, GIH)和蜕皮抑制激素(molt inhibiting hormone, MIH)等多种神经激素可能负向调节IAG的表达，参与“眼柄–促雄腺–精巢”内分泌轴的调节。其中GIH是甲壳动物繁殖最有效的负调节因子，由于其抑制卵子发生和卵黄蛋白原(vitellogenin, VTG)合成的功能而为人熟知，因此，GIH又被称为卵黄生成抑制激素(vitellogenesis inhibiting hormone, VIH)[35]。拟穴青蟹中，发现并验证了vih基因上游调控区域OCT4/SOX9两个转录因子的结合位点，并证明了它们对VIH的正向调控作用[36-38]。通过将Oct4或Sox9的dsRNA注射到眼柄对其进行干扰后，检测到vih的表达显著下调，而vtg在卵巢和肝胰腺中的表达则显著上调[37]。
甲壳动物中类固醇激素主要分为蜕皮类固醇激素和性类固醇激素[39]。在多种甲壳动物的血淋巴、大颚器、卵巢和肝胰腺中均检测到脊椎动物样性类固醇激素，如17β-雌二醇(17β-estradiol, E2)、孕酮和睾酮等，但其来源目前尚不完全清楚，相关理论有待进一步验证[40]。E2参与调控生长、生殖和代谢过程，尚不清楚其调控甲壳动物卵巢发育的分子机制，对雌性甲壳动物的性别相关基因调控机制和相互作用知之甚少。脊椎动物雌激素是通过与雌激素受体(estrogen receptor, ER)结合来行使其功能。目前尚未见ER同源基因在甲壳动物中的报道，但发现一种与脊椎动物ER具有高度序列同源性的雌激素相关受体(estrogen related receptor, ERR)。ERR是基于与ERα结合域序列具有高度相似性，采用低严谨杂交技术进行cDNA文库筛选所发现的，无脊椎动物中仅发现一种ERR基因，目前已在拟穴青蟹[41]、三疣梭子蟹(Portunus trituberculatus)[42]、罗氏沼虾[43]等十足目甲壳动物中报道。
另外，在十足目甲壳动物的中枢神经系统中发现了神经递质如5-羟色胺(5-hydroxytryptamine, 5-HT)、多巴胺(dopamine, DA)[44-45]，以及神经激素如黑化诱导神经肽(corazonin)、促性腺激素释放激素(gonadotropin releasing hormone, GnRH)等[46-47]。有研究报道，5-HT和DA可能影响十足目甲壳动物神经激素的释放，如CHH、GIH、GSH (gonad-stimulating hormone)和MIH[48]。Ohs等[49]报道了DA添加到罗氏沼虾日常投喂中，可以使其雌性化。Siangcham等[50]使用5-HT和LGnRH- III处理罗氏沼虾的雄性小虾(指雄虾3种形态中个体较小，性腺发育不成熟的小雄虾)，观察到促雄腺增生和IAG表达上调。而DA和黑化诱导神经肽对罗氏沼虾的处理则得到相反的结果。以上研究表明，5-HT和DA等可能通过抑制或刺激眼柄神经激素，调控IAG的表达，从而参与到性别分化过程中。
1.2 十足目甲壳动物的促雄腺 1.2.1 促雄腺的形态构造与位置促雄腺是雄性甲壳动物所特有的内分泌腺，促雄腺分泌的激素对雄性分化、维持雄性第二性征以及生长调节起到调控作用[18]。
促雄腺最早在蓝蟹中被发现[51]，十足目甲壳动物的促雄腺位置较为集中但存在略微差异。例如，红螯光壳螯虾和克氏原螯虾的促雄腺常附在输精管近末端的表面[52-53]；罗氏沼虾和日本沼虾的促雄腺则位于输精管末端至壶腹末端表面[54-55]；中华绒螯蟹和锯缘青蟹(Scylla serrata)的促雄腺则附于射精管的表面[56-57]；在凡纳对虾和墨吉对虾(Penaeus merguiensis)中，则位于精荚囊端壶腹外侧的肌肉中[58-59]；而在中国对虾(Penaeus chinensis)[60]中则位于第五步足基部的肌肉中，覆盖在精荚囊和射精管连接处。
促雄腺由许多腺状细胞组成，细胞内粗面内质网、高尔基体和线粒体发达，主要分为两种细胞：I型细胞和II型细胞。I型细胞是新形成的腺体细胞，它们高密度地聚集在一起，细胞体积小，细胞核相对较大，细胞质较少；II型细胞占促雄腺细胞的大多数，在分泌周期中活性最高，相比于I型细胞，它们有更大细胞尺寸，更小的细胞核，更多的细胞质[61]。在腺状细胞周围，由结缔组织连接腺体和周围所附生的生殖系统而形成血窦。邱高峰等[56]将中华绒螯蟹促雄腺发育过程分为增殖、合成、分泌3种时期。叶海辉等[57]将锯缘青蟹促雄腺发育划分为I期(腺体短小)、II期(腺体呈明显索状)、III期(腺体体积达到最大)、IV期(腺体退化)。
1.2.2 促雄腺的功能十足目甲壳动物中，移植促雄腺或注射促雄腺提取物到雌性个体，雌性外部特征退化，卵黄生成受到抑制，发生雌性向雄性的转变；而摘除促雄腺的雄性个体则精巢发育减缓或退化，产生雌性化特征[21,62-64]。红螯光壳螯虾的研究表明，促雄腺移植导致雌虾出现雄性第二性征以及雌性第二性征的退化[65]，比普通雌虾更具好斗性，展示雄性特有的求偶行为，且与正常雌性有配对现象，比普通雌性生长更快，在mRNA水平和蛋白质水平卵黄生成均受到抑制[66]。在其幼虾阶段，移植促雄腺或饲喂促雄腺提取物，使生长性能得到提升[63,67]。Barki等[68]指出，促雄腺对雄虾的行为有重要影响，去除促雄腺会导致雌雄间性个体的行为向雌性方向发生改变。在罗氏沼虾中，摘除促雄腺会导致雄性个体的雄性化程度降低，同时雌性特征会得到发展；如果在早期生长发育阶段摘除促雄腺，会导致雄虾发生性逆转，转化为具有功能性的伪雌虾[69]。例如，Aflalo等[62]在雄性罗氏沼虾性别分化阶段摘除促雄腺，发生了性逆转获得功能性伪雌个体，然后伪雌虾与正常雄性交配，成功获得全雄子代。同样地，若在早期发育阶段移植促雄腺到罗氏沼虾的遗传性别雌性个体，则发生完全性逆转从而获得功能性伪雄虾[70]。进一步地，Levy等[64]将罗氏沼虾促雄腺的细胞悬液注射到幼虾中，导致其个体完全的性逆转，发育为成熟雄性，表现出典型的雄性形态。由此可知，促雄腺是雄性性别分化的关键内分泌腺，促雄腺组织细胞内含有雄性性别分化和性别特征维持的关键因子。在顺序性雌雄同体中的研究，进一步地支持了促雄腺在性别分化中维持雄性特征和雄性化作用的功能。在该性别决定模式中，个体在雄性阶段时，促雄腺是高度活跃的，在两性过渡阶段促雄腺开始退化，并在雌性阶段完全消失[71]。因此认为，促雄腺控制了十足目甲壳动物雌雄异体和雌雄同体的雄性分化。
而促雄腺摘除时所处发育阶段是雌性化成功的重要因素之一。例如，在罗氏沼虾中，早期未分化阶段进行促雄腺摘除时，显示出更高程度的雌性化；然而，在后期发育阶段进行促雄腺摘除时，仅见部分雌性化甚至没有，并检测到了许多异常性腺[72-73]。Aflalo等[62]也指出，在未分化阶段摘除促雄腺的雄性罗氏沼虾，将会自分化为雌性。因此，在进行促雄腺摘除和移植相关生物技术操作或RNA干扰诱导性别分化关键基因沉默时，个体所处发育阶段对于性逆转成功非常重要。
1.2.3 “眼柄-促雄腺-精巢”内分泌轴在十足目甲壳动物中，内分泌通路“眼柄-促雄腺-精巢”调节个体性成熟[52]，眼柄中的X器官-窦腺复合体(X-organ/sinus-gland complex, XO-SG)是其神经内分泌系统的中枢，调节各种生理过程包括新陈代谢、蜕壳、生殖发育等。研究发现，切除眼柄可诱导十足目甲壳动物加速性成熟，并使得促雄腺细胞肥大和IAG基因过表达[52,74-76]。切除雄性凡纳对虾的单侧眼柄后，IAG基因的表达显著上调，对比眼柄切除前后的精巢转录组数据发现多个参与性腺发育和内分泌调控的基因表达显著上调，如dsx (Doublesex)，保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)基因、细胞色素p450酶系基因以及泛素化系统相关基因等[77]。同样地，在墨吉对虾(Penaeus merguiensis)中，切除其单侧眼柄引起IAG转录水平的显著上调[76]。这些研究提示促雄腺及IAG的作用受到眼柄调节，进而调控精巢相关基因的表达。而眼柄中XO-SG复合体分泌多种神经激素，其主要成分包括两个CHH超家族：CHH超家族I包括CHH和离子转运肽(Ion transport peptide, ITP), CHH超家族II包括GIH、MIH和大颚器官抑制激素(mandibular organ-inhibiting hormone, MOIH)。
虽然CHH家族已经在许多十足目甲壳动物中进行了研究，但它调控性别分化和性腺成熟的途径仍不是很清楚。最近的研究表明，罗氏沼虾眼柄切除后，检测到精巢和促雄腺中dsx的表达水平显著上调[78]，以及IAG、胰岛素样促雄腺激素受体(insulin-like androgenic gland hormone receptor, IAGR)基因、胰岛素样促雄腺激素受体结合蛋白(insulin-like androgenic gland hormone- binding protein, IAGBP)基因的表达显著上调[79-80]。RNA干扰引起CHH或GIH基因沉默后，同样检测到IAG、IAGR、IAGBP的表达上调[79-80]。Li等[81]通过RNA干扰，检测了日本沼虾GIH、MIH、CHH和IAG之间的相互调控作用。结果表明，与对照组相比，分别注射GIH dsRNA、MIH dsRNA后，IAG的表达量显著上调。说明由眼柄分泌的CHH、GIH、MIH等多种神经激素可能负向调控IAG的表达，参与到“眼柄-促雄腺-精巢”内分泌轴调节，从而调控性别分化。
1.3 IAG基因的结构与功能 1.3.1 IAG的结构性质促雄腺激素(AGH)首次从鼠妇(Armadillidium vulgare)的促雄腺中纯化获得[82]，其化学结构被确定为具有N-连接聚糖部分的胰岛素样异二聚体肽。在十足目中，Manor等[83]利用抑制消减杂交(suppression subtractive hybridization, SSH)构建了红螯光壳螯虾促雄腺cDNA文库，首次克隆得到IAG基因(Cq IAG)，其序列与等足目AGH相似性较低，但氨基酸结构相似，属于胰岛素样超级家族成员。Cq IAG推测的氨基酸序列与AGH相比较，在A链和C肽之间以及B链和C肽之间都有两个典型的蛋白水解基序(R-X-X-R)，但在基序位置上存在差异，该基因组织表达和原位杂交结果表明其在雄性壶腹末端特异性表达。随后，Ventura等[84]在罗氏沼虾cDNA文库中发现了IAG，与Cq IAG相比，在结构上除了第3个链间二硫键存在差异外，其他方面均表现出了高度的相似性，特别是胰岛素家族中保守的6个半胱氨酸残基存在于所有IAG序列中，目前已从许多十足目甲壳动物中克隆得到[84-87]，这表明IAG介导的性别分化机制在十足目物种间是较保守的。因此，普遍推测IAG对应的成熟多肽可能是十足目甲壳动物的AGH。虽然有相关研究报道[88]，但天然IAG尚未从十足目动物中完全分离纯化，还没有关于IAG多肽功能的直接证据。目前已经通过化学合成或重组蛋白表达系统制备了IAG肽[89-92]。结果表明，IAG的肽构象受二硫键排列的影响。而且，通过体外实验证实，合成的IAG可抑制日本对虾(Penaeus japonicus)雌性特异性基因的表达[92]；东澳岩龙虾(Sagmariasus verreauxi)的重组IAG能提高其精巢中的蛋白质磷酸化作用和受体激活作用[93]。Katayama等[89]合成的龙纹螯虾IAG具有胰岛素样型和鼠妇AGH型两种二硫键排列类型，其CD光谱分析显示，B链上的Asn连接的聚糖部分不影响肽的构象。在体实验结果表明，这两种合成的IAG均能抑制其体内卵母细胞的成熟。
1.3.2 IAG的功能“眼柄-促雄腺-精巢”内分泌轴调节雄性十足目甲壳动物的性别分化和次级雄性特征。而IAG由促雄腺分泌，是雄性甲壳动物的主要性别分化开关[17]。在红螯光壳螯虾的雌雄间性个体中，IAG基因沉默后，精子产生减少，精巢退化，vtg表达上调，并在发育中的卵母细胞中储存卵黄蛋白[94]；雄性墨吉对虾中，dsRNA注射引起IAG基因沉默后，检测到精巢和肝胰腺中vtg的表达[75]。罗氏沼虾中，雄虾的3种不同形态可能与IAG有关，研究发现，橘螯(orange claw, OC)雄虾的IAG表达增强，可以使其向蓝螯(blue claw, BC)的转变数量显著增加[95]。Ventura等[84]通过短期RNA干扰沉默IAG基因后，罗氏沼虾雄性成体第二性征的再生受到抑制；IAG沉默个体表现出非典型的雄性生长模式、精子发生和精原细胞发育停滞的生殖表型，以及促雄腺肥大和增生。之后，Ventura等[96]又通过长期RNA干扰方法使雄性罗氏沼虾仔虾个体的IAG沉默，成功性逆转获得伪雌个体，与正常雄性交配，成功获得全雄子代。
而IAG除了受到眼柄中的CHH、GIH、MIH等激素调控，还受到上游多种转录因子的调控。在中国对虾中，IAG启动子区域发现了一个可能的Dsx结合位点，dsx敲降后，IAG的表达显著下调，这表明Dsx可能是IAG的上游调控因子[97]。在拟穴青蟹中[98]，对IAG基因的启动子区域进行预测发现了Dsx和FOXL2的结合位点，细胞共转染实验结果表明，dsx能显著促进IAG的表达，而foxl2能显著抑制IAG的表达；在体RNA干扰实验表明，分别干扰dsx和foxl2后，IAG的表达分别显著下调和上调。在三疣梭子蟹中，sox9的敲降导致促雄腺和精巢中的IAG表达量显著下调[99]；脊尾白虾中，切除眼柄导致sox9的表达显著下调，而sox9敲降后，IAG的表达显著下调[100]。表明sox9可能在IAG的上游起正向调控作用，且受到眼柄的负调控。在红螯光壳螯虾中，由RNA干扰引起的fruitless-like (果蝇fruitless同源基因)基因沉默，也使得IAG基因的表达显著下调[101]。而在果蝇中，fruitless (fru)是一种多效基因，位于性别决定调节层级的下游，控制着雄性的性行为，被认为在黑腹果蝇的求偶行为和性别决定中具有重要作用[102-103]。Liu等[104]对红条鞭腕虾(Lysmata vittata)的IAG1基因(Lvit-IAG1)研究发现，Lvit- IAG1特异表达于促雄腺，高表达于功能性雄性阶段，并在真雌雄同体阶段显著降低。短期和长期沉默实验均表明，Lvit-IAG1同时负调控眼柄神经节中的性腺抑制激素(Lvit-GIH)和甲壳类雌性性激素(Lvit-CFSH)的表达，这提示IAG可能反馈调节GIH和CFSH。在罗氏沼虾中[105-106], dmrt11E基因敲降导致IAG表达显著降低，在雄性的仔虾阶段，通过RNA长期干扰注射dmrt11E dsRNA，诱导了雄虾完全的功能性性逆转，并成功实现了全雄单性群体的产生。这表明罗氏沼虾dmrt11E可能在IAG基因上游发挥调控作用，并通过直接或间接影响IAG的表达参与了性别分化调控。另一方面，IAG基因沉默使罗氏沼虾两个dmrt基因即idmrt1b和idmrt1c的表达显著下调，提示它们可能在IAG开关的下游参与性别分化调控[107]。
此外，研究结果表明，一些miRNA通过对IAG的表达调控参与到性别分化过程。例如，miR-184通过介导IAG的表达，可能参与罗氏沼虾的性别分化、性腺发育、生长和蜕壳等生理过程[108]。克氏原螯虾中，通过RNA干扰IAG后比较雄性克氏原螯虾mRNA/miRNA的表达谱，发现IAG基因沉默后，卵巢发育相关基因表达量发生上调，精巢发育相关基因表达量发生下调，5个性别相关的miRNA (miR-263a、miR-263b、miR- 2779、miR-133、miR-34)表达发生显著变化且在性腺组织中高表达[109]。
胰岛素样肽(insulin-like peptides, ILPs)在脊椎动物的生长、代谢和繁殖中起着关键作用，IAG被认为是甲壳动物的一种ILPs。然而，关于参与IAG信号级联的关键因子的研究仍然很分散。在罗氏沼虾中，胰岛素样受体基因(insulin-like receptor, IR)在雌雄的大多数组织(包括促雄腺和性腺)中均有表达；幼虾的长期RNA干扰实验表明，IR基因沉默对生长没有显著影响，也没有引起性逆转，但显著引起促雄腺肥大和IAG的表达上调[110]。与上述研究结果稍有不同的是，Tan等[111]研究发现，使用siRNA长期干扰的方法敲降罗氏沼虾IR，发生了性逆转获得伪雌个体，并检测到dmrt11E、dmrt99B、MRPINK、Mrr、Sxl1和Sxl2的表达水平显著下调；而且IR主要在精母细胞、促雄腺细胞和末端壶腹分泌上皮细胞中表达，IR与IAG的共定位证实了IR为IAG的受体。而IAG信号通路可能通过胰岛素样肽与受体的结合而激活，包括IAGR和IAGBP。例如，在罗氏沼虾[112]与日本沼虾中[110]敲降IAGBP，均导致IAG表达下调。而日本沼虾中IAG的敲降也显著降低了IAGBP的转录水平，这表明IAGBP基因可能参与了IAG信号转导[113]。另外，酵母双杂交试验证实IGFBP家族蛋白IGFBP7与中华绒螯蟹IAG存在相互作用，且该蛋白在雄性性腺相关组织中高表达[114]，表明IGFBP可能与IAGBP类似，也作为IAG的受体在十足目甲壳动物性别分化调控中发挥作用。在中国对虾中[115]，克隆鉴定了一种推测的IAG受体FcIAGR，具有受体酪氨酸激酶的保守结构域，主要在促雄腺和精巢中表达，其共定位和酵母双杂交实验证实FcIAGR可与FcIAG1和FcIAG2相互作用。Chen等[116]在凡纳对虾中发现了胰岛素样受体基因，并命名为Pv-IR，主要表达于雄性生殖系统的输精管和壶腹末端。在仔虾期通过RNA干扰对Pv-IR进行基因敲降，结合比较转录组学进行分析，指出差异表达基因中值得关注的两个下调基因——CP蛋白(clottable protein)和胰岛素样肽(insulin-like peptide)基因，和两个上调基因——Flotllin蛋白基因和促甲状腺激素受体基因(thyroid stimulating hormone receptor, TSHR)。
综上，IAG不仅参与性别分化调控和性腺发育，还参与生殖过程中的精子发生等过程。罗氏沼虾中仔虾期的RNA长期干扰引起的IAG基因沉默，能实现由雄到雌的完全性逆转。其上游调控因子主要为GIH、CHH、MIH，以及dmrt11E、sox9、foxl2、dsx和fruitless-like等性别决定基因及其调控因子。下游调控因子方面，则可能有IR、IAGR、IAGBP/IGFBP-IAG受体信号通路等。目前已在多个十足目甲壳动物中发现了IAG，但相关研究还不够深入。了解十足目甲壳动物IAG等基因将有助于规模化性别控制的研究，从而促进其单性养殖技术的发展。本文提及的性别决定与性别分化相关调控因子与基因的调控关系如图1所示。
01-0106-22-F001.jpg "点击查看原图") |
图1 十足目甲壳动物性别决定与性别分化调控因子相互关系简图单向箭头表示直接或间接调控作用. 双向箭头表示两端的调控因子存在相互调控. +表示正调控，−表示负调控. Fig. 1 A simplified view of the relationship of regulatory factors related to sex determination and differentiation in Decapoda crustaceans based on references hereinOne-way arrow indicates the direct or indirect regulation. Double-head arrow denotes mutual regulation of factors at both ends. + indicates positive regulation, and − indicates negative regulation. |
随着分子生物学和相关技术的发展，基于模式生物性别决定与性别分化相关的同源基因，dmrt、sxl、sox、fem-1、fruitless、foxl2、masc等基因已陆续在十足目甲壳动物中鉴定和研究报道。
1.4.1 Dmrt基因家族转录因子dmrt基因家族是一组编码含有DM (doublesex and mab-3)结构域的基因，DM结构域是一个复杂的锌指状DNA结合元件[117-118]。该基因家族参与多种生理过程，特别是性别决定、性别分化和性腺发育，在昆虫中发现的dsx是第一个鉴定的dmrt基因[119]。该基因家族的DM结构域内的序列高度保守，但在结构域之外有大量可变序列[118]。DM结构域的保守性使得人们能够识别与之结合的基因，事实上，在人类和果蝇中已经发现了许多与性别决定相关的基因，但这类研究在甲壳类动物中还较少报道。
虽然dmrt基因不一定是性别决定主效基因，但它们往往直接或间接地与主开关基因相互作用。因此，鉴定dmrt基因将有助于十足目甲壳动物中性别决定位点的定位和机制研究。目前，已在多种十足目甲壳动物中鉴定出dmrt基因家族成员，如中华绒螯蟹[120]、东澳岩龙虾[121]、日本沼虾[122]和罗氏沼虾[106]等。本课题组基于转录组数据和RNA干扰实验等研究，对拟穴青蟹dmrt基因家族7个基因(dmrt-like, dmrt-11E, dmrt-3, dmrt-1, idmrt-1, idmrt-2和dsx)进行了鉴定和初步分析。RNA干扰研究发现，dmrt-like敲降后，foxl2和sox21的表达量显著下调；idmrt-2敲降后，精巢中dmrt-like和foxl2基因的表达显著下调，促雄腺中IAG的表达也显著下调；dsx敲降后，卵巢中vtg和vtgR表达水平显著下调，精巢中dmrt-like和dmrt-1表达水平显著上调，促雄腺中IAG表达水平显著下调[41,123-126]。课题组对dmrt-1和dmrt-3的进一步研究表明(数据暂未公开), dmrt-1敲降导致精巢中foxl-2、dmrt-like、dmrt-3基因和促雄腺中IAG基因的表达水平显著下调，差异表达基因主要富集在Notch信号通路、MAPK信号通路、Hippo信号通路、Calcium信号通路、Apelin信号通路；dmrt-3敲降后，引起精巢中foxl-2、dmrt- like、dmrt-1、dsx基因和促雄腺中IAG基因的表达水平显著下调，差异表达基因主要富集在Hippo信号通路、Calcium信号通路、Apelin信号通路、mTOR信号通路。
Dmrt11E或dsx敲降导致IAG表达显著下调，表明它们可能在“IAG开关”调控信号中发挥上游作用，并直接或间接影响IAG的表达参与性别分化[97,106]；通过dmrt11E的RNA长期干扰，能诱导雄性幼虾完全的功能性性逆转，性成熟后能与正常雄虾配对繁殖产生全雄群体[105]。IAG基因沉默使idmrt1b和idmrt1c的表达显著下调[107]; dmrt99B沉默对IAG表达没有影响[106]，而IAG敲降则使dmrt99B的表达显著下调[127]。综上，dmrt基因家族成员与IAG之间有一个复杂的调控网络，它们在IAG开关上下游调控和性别分化过程中发挥了重要作用。
1.4.2 Fem-1基因Fem-1 (Feminization-1)首次在秀丽隐杆线虫(Caenorhabditis elegans)中被发现，是参与雄性和雌雄同体生殖系精子发生的关键调控基因[128-130]。Fem-1基因包括3个成员，分别是fem-1a、fem-1b和fem-1c，在不同生物中有不同的变体[131]。在十足目甲壳动物中，fem-1基因的鉴定分析与相关分子机制研究已陆续报道。这些研究提示fem-1可能在十足目甲壳动物的早期性别决定以及性腺发育过程中发挥重要作用[41,132-139]。其中，在红螯光壳螯虾中鉴定分析了fem-1a、fem-1b和fem-1c，其中fem-1b在卵巢中表达量显著高于其他组织，并随着卵巢的发育表达量增加。在体RNA干扰敲降fem-1b基因，导致vtg的表达显著下调，这表明fem-1b可能通过调节vtg的表达参与到雌性的生殖调节[140]。中华绒螯蟹中，fem-1在某些组织中表现出一定程度的两性二态表达。在精巢、卵巢、肝胰腺和肌肉中持续检测到较高水平的表达[141]，对fem-1c进行在体RNA干扰，引起雌蟹的眼柄神经节和卵巢中CFSH-1的表达显著下调，以及雄蟹的促雄腺和精巢中IAG的表达显著上调[142]，表明fem-1c可能作为IAG和CFSH-1的上游调控因子，参与性别分化调控。
1.4.3 CFSH基因如前所述，CFSH是甲壳动物特有的一种神经激素，参与雌性的性别分化和第二性征维持。CFSH可能还通过抑制促雄腺中IAG的表达来调控雄性性别分化[33,143-144]，被认为是IAG的上游调控因子。在拟穴青蟹中，CFSH通过抑制STAT (signal transmitters and activators of transcription)表达来抑制IAG的表达[145]；且对拟穴青蟹候选的CFSH受体基因Sp-SEFIR进行RNA干扰后，会诱导促雄腺中IAG和STAT的表达显著上调[146]。在三疣梭子蟹中，雄蟹的CFSH主要表达于眼柄，其次是脑和精巢，研究认为CFSH可能通过“CFSH-IAG-精巢”内分泌轴负调控精子发生和精巢发育，也可能直接作用于精巢，其信号系统类似于MIH的信号通路[147]。因此，CFSH除了在雌性的眼柄和卵巢中表达外，促雄腺和精巢可能也是其重要的靶器官，而最近的证据支持CFSH和IAG之间可能存在负反馈调节[104,143,148]。
1.4.4 Foxl2基因Foxl2 (forkhead box protein L2)是Fox基因家族重要成员之一，其特征是具有高度保守叉头框(forkhead box) DNA结合结构域[149]，是哺乳动物卵巢发育启动的标志性基因之一，在脊椎动物卵巢发育和雌性特征的维持方面有重要作用[150-153]。相比于其他动物，foxl2基因在甲壳动物中的研究报道还较少。在已报道的十足目甲壳动物中，foxl2在多个组织中表达，但表达模式稍有不同。在日本沼虾中，foxl2在促雄腺、眼柄、精巢和卵巢等多个组织中表达，其中精巢中的表达量显著高于卵巢[154]。在罗氏沼虾中，foxl2在精巢、输精管和卵巢中高表达，卵巢发育过程中在卵巢I期表达量最高。foxl2 mRNA原位杂交结果表明，输精管上皮细胞、精母细胞和卵母细胞中都检测到阳性信号[155]。在中华绒螯蟹中，foxl2在多个组织中表达，但卵巢中的表达量显著高于精巢与其他各组织。右侧眼柄切除后，foxl2、ddx20和FTZ-F1 mRNA的表达显著上调。通过免疫共沉淀证实FOXL2与DDX20或FTZ-F1之间存在相互作用[156]。在三疣梭子蟹中，foxl2也在多个组织中表达，但卵巢中的表达量显著高于精巢及其他各组织。切除眼柄后，foxl2的表达出现显著下调；RNA干扰该基因后，卵巢vtg基因的表达显著上调[157]。
拟穴青蟹中[98,158-160], foxl2主要表达于性腺中，且精巢中的表达量显著高于卵巢。此外，在3个早期发育阶段中(溞状幼体V期、大眼幼体和仔蟹I期), foxl2在大眼幼体时期的表达量最高。在拟穴青蟹vtg基因的启动子区域发现了潜在的FOXL2结合位点，foxl2基因敲降后，vtg在卵巢中的表达显著上调。细胞共转染和RNA干扰实验表明，foxl2对IAG也有负调控作用，抑制IAG的表达[98]。另外，首次通过比较卵巢或精巢中RNA干扰(分别注射EGFP和foxl2 siRNA)的转录组数据，分析研究了foxl2在甲壳动物中的功能。卵巢组织中发现645个差异表达基因，包括几个卵巢发育关键基因，如vtg、vtgR (vitellogenin receptor，卵黄蛋白原受体)、AC (adenylate cyclase，腺苷酸环化酶)、cyclinB和cdc2，这些差异表达基因还富集于卵巢发育相关通路，包括松弛素信号通路、卵巢类固醇生成和孕酮介导的卵母细胞成熟相关信号通路；精巢组织中共发现有7892个差异表达基因，其中包括大量参与精巢发育的关键基因，如dmrt基因家族、sox基因家族、caspase基因家族、cdk基因家族、kinesin基因家族等。进一步的分析表明，这些差异表达基因在精子发生的关键通路中富集，如DNA复制、细胞周期、同源重组、减数分裂和细胞分裂细胞凋亡等[161]。
以上研究表明，foxl2受眼柄相关因子的调控，可能是vtg和IAG的上游负调控因子，并在十足目甲壳动物的性别分化和性腺发育中发挥重要作用。
1.4.5 Masc基因Masc (masculinizer gene)位于Z染色体上，编码一个Cys-Cys-Cys-His串联锌指蛋白，控制鳞翅目雄性化和剂量补偿效应，在性别决定中有重要作用[162]。Masc在甲壳动物中的同源基因研究报道还较少。在丰年虫(Artemia franciscana)中，首次鉴定报道甲壳动物masc基因，通过RNA干扰沉默masc基因，导致雌性比例显著提高，表明masc可能参与了雄性性别决定与分化过程[163]。罗氏沼虾中，首次报道了十足目的masc同源基因[164]，该基因高表达于胸神经节、肠道、输精管、精巢和卵巢中。masc基因沉默使foxl2的表达显著下调，表明masc可能对foxl2的表达有正向调控作用；进一步地，在雄性仔虾阶段，长期RNA干扰masc基因，使其性逆转为功能性的伪雌虾，并成功产生全雄子代。此外，通过对masc基因敲降的比较转录组分析，一些显著表达的转录本被富集，并聚焦了关键信号通路的影响，如胰岛素样信号通路、表皮生长因子、IGFBP、Flotillin蛋白、Sxl、Foxl2和HSP蛋白家族等。
本课题组在拟穴青蟹的masc基因(数据未公开)主要研究结果表明，masc基因在精巢中的表达量远高于卵巢；原位杂交结果显示，masc mRNA阳性杂交信号在精原细胞、精母细胞、精细胞和精子中均有分布；RNA干扰敲降masc基因后的精巢转录组测序和qRT-PCR结果表明，dsx和DDX5等雄性相关基因表达量下调。此外，通过RNA干扰敲降DDX5基因后，发现精巢组织中foxl2、dmrt-like和idmrt-2表达显著下调，dsx表达显著上调[165]。
这些研究表明，masc基因在甲壳动物复杂的内分泌轴或性别分化调控网络中位于上游，可能参与调控性别决定与分化。
2 十足目甲壳动物性别控制与性别鉴定在沼虾、小龙虾、龙虾、蟹类等，雄性个体具有明显的生长优势，而在凡纳对虾和斑节对虾(P. monodon)中，雌性个体则具有更显著的生长优势。具有雄性生长优势的物种如罗氏沼虾和红螯光壳螯虾，在高密度养殖条件下，全雌养殖群体可能比全雄养殖群体更实用，因为雌性不具备强的攻击性和领地行为，有更高的成活率和规格均匀度，这更适宜高密度养殖模式。又如长久以来，人们对不同生长、生理阶段的青蟹都有消费需求，如肉蟹、奄仔蟹、重壳蟹、软壳蟹、膏蟹以及黄油蟹，已形成了丰富的消费文化，其中奄仔蟹(指尚未交配的雌蟹)因其味道滑嫩、甘香鲜美而广受消费者追捧，若有单性雌蟹群体进行养殖则更具规模化优势，从而提高养殖经济效益。因此，无论是全雄养殖还是全雌养殖，都有潜在的经济价值，对其需求的增加，推动了基于性别控制的相关生物技术的研究发展。在鱼类养殖中，通过培养全雄或全雌个体获得更高产量的技术已成功得到应用。已经明确在早期发育阶段，性别分化尚未开始时，个体对性激素等处理非常敏感，是实现性逆转稳定获得单性群体的关键时期。
直接性反转获得单性群体的方法需用大量的激素或生物化学物质处理，而间接干预方法是最有益的，虽然亲本通常仍涉及激素或药物处理，但子代群体并没有处理，相较于直接性反转的方法，更为安全高效，可用于规模化单性群体制种。无论是直接或间接的方法，处理时期是性逆转成功的关键。而间接雄性化或雌性化在理论上是可能的，但在不同性别决定系统(ZW和XY)中，间接雌性化/雄性化的表现和经济效率的可行性有待深入研究。此外，类固醇激素(MT和17β-雌二醇等)或其他化学物质(DA和5-HT等)参与甲壳动物的性别控制机制尚不完全清楚，需要进一步研究。
2.1 性别控制获得单性群体 2.1.1 激素诱导性逆转外源性激素处理是性别控制的常用方法之一，通过激素诱导的雌性化或雄性化方法，不需要考虑是何种雌雄异配型的性别决定模式，可应用于大多数物种。研究表明，脊椎动物的性激素也可以影响十足目甲壳动物性别分化，甚至造成性逆转。E2能促进小龙虾的卵黄发生[166]，并诱导甲壳动物的雌性化[167]。在罗氏沼虾中，通过投喂不同含量MT处理的卤虫无节幼体50 d，雄性幼虾的性别比例显著提高[168]。在日本沼虾中，在日龄25 d仔虾的饲料中E2添加量200 mg/kg，养殖投喂40 d，实验组获得了相比于对照组雌性率高的群体[169]；通过在饲料中添加不同浓度(分别为50、100、200 mg/kg)的MT，雄性比例随MT浓度的提升而提高[170]。在200 mg/kg时雄雌性别比例为2.61∶1，性腺组织切片显示该浓度处理下存在部分精巢-卵巢共存的个体，而在雄虾中dmrt11E、foxl2和soxE1性别分化相关基因的表达量分别是未处理对照组的8.65倍、3.75倍和3.45倍。同样在日本沼虾中[171], E2的处理抑制了IAG的表达，500 mg/kg浓度的E2和MT处理可分别显著提高雄、雌幼虾的生长性能；浓度≥500 mg/kg的E2和MT分别处理雄虾和雌虾，则阻碍了精巢和卵巢的发育。
红螯光壳螯虾中，通过虾苗口服含MT (50 mg/kg)的饲料来提高雄性率，结果表明，相比于对照组24.93%的雄性率，实验组提高到59.96%，且对成活率没有显著影响[172]；通过花刺参(Stichopus variegatus)类固醇提取物(sea cucumber steroid extract, SCSE) 2 mg/L浓度浸泡18 h，养殖50 d，雄性率从31.03% (对照组)提高至79.86%，浓度50 mg/kg的SCSE口服饲喂50 d，雄性率为75.16%[173]；利用SCSE以及蜂蜜对红螯光壳螯虾进行雄性化实验处理40 d，当SCSE添加量为2 mg/L、蜂蜜剂量为20 mL/L时，雄性率最高达到83.75%[174]；进一步研究表明，SCSE具有剂量依赖性，能促进幼虾雄性生殖系统的形成，提高精巢的MT水平，SCSE剂量和幼虾不同浸泡时间的组合对其生长和MT水平有显著影响[175]。
以上研究为外源类固醇激素诱导十足目甲壳动物性逆转建立单性群体提供了参考依据，但还需进一步开展相关研究，从而确定使用剂量和处理时间，确保性逆转的高效性和生物安全性，为控制定向性别分化、揭示诱导十足目甲壳动物性逆转的调控机制提供理论基础与实践基础。若激素诱导的方法再结合性别特异分子标记，对性转群体的遗传性别进行鉴定，制备候选伪雄/伪雌亲本，就可用于单性群体的规模化制种。
2.1.2 多倍体诱导与性别控制通过抑制减数分裂I或II期诱导形成三倍体，可使凡纳对虾、中国对虾和日本对虾的雌性性别比例提高[176-180]。其中温度休克法所需的条件与设施简单，操作简便易行，处理量大，具备应用于规模化生产单性群体的潜力。
2.1.3 IAG及相关基因沉默与规模化制种促雄腺和IAG是十足目甲壳动物性别控制研究的重点。通过干预促雄腺和RNA干扰技术诱导IAG等性别决定与分化相关基因沉默，结合可靠的性别特异分子标记，在罗氏沼虾中陆续实现了功能完整的性逆转[96,105,111,164,181-183]，并应用于全雄群体生产研究，这在水产养殖中具有重要的应用价值。而将促雄腺的细胞悬液注射到幼虾中，可导致个体完全的性逆转，发育为成熟雄性[64]。通过罗氏沼虾血细胞原代细胞培养开展规模化生产IAG的研究，以及通过化学合成或重组蛋白表达系统制备IAG肽的研究，为取代促雄腺细胞悬液的传统制备方法以及全雌群体的规模化生产奠定了技术基础[184]。但无论是幼虾时期的dsRNA/ siRNA或细胞悬液注射，还是促雄腺的摘除和移植，实际应用起来都较为困难，不能做到性激素拌料投喂诱导性逆转那样简便。研究者们尝试通过注射、口服、转染、浸泡等各种RNA分子传递方式，不断研究和开发新的RNA给药方法，以提高给药方法的效率，并最大限度地减少其不良副作用或局限性，而通过脂质体、微藻和细菌等作为核酸分子载体的口服饲喂方式，为十足目甲壳动物通过RNA干扰方法诱导性逆转提供了新的研究思路与方向，具有极大的应用价值[185]。基于RNA干扰的生物技术方法原理，注射7 d后dsRNA或siRNA会逐渐消失，因此被认为是安全的，但也有可能会触发机体免疫反应和产生脱靶效应[185-186]。因此，RNA干扰技术在水产养殖中有一定局限性，而且体外制备RNA干扰的核酸分子成本高，这进一步限制其应用推广。而RNA干扰在性别控制和抗病等多个方面具有广泛的应用前景[187]，利用细菌、酵母和微藻等通过生物体内合成获得大量dsRNA分子的研究逐渐受到关注[188-190]，为其规模化应用提供了可能。
理论上，对于雌性异配ZW型性别决定的物种，全雄群体可以通过两步程序获得：第一步雄性(♂ ZZ)性逆转为伪雌虾(♀ ZZ)，第二步与正常雄性(♂ ZZ)交配，获得遗传性别为全雄(♂ ZZ)的群体；全雌群体可以通过三步程序获得：第一步雌性(♀ ZW)性逆转为伪雄虾(♂ ZW)，第二步与正常雌虾(♀ ZW)交配获得超雌个体(♀ WW)，第三步WW个体再与正常雄虾(♂ ZZ)交配，获得遗传性别为全雌(♀ ZW)的群体。对于雄性异配XY型性别决定的物种，全雄群体可以通过三步程序获得：第一步雄性(♂ XY)性逆转为伪雌虾(♀ XY)，第二步与正常雄虾交配，获得超雄个体(♂ YY)，第三步超雄个体与正常雌虾(♀ XX)交配，获得遗传性别为全雄(♂ XY)的群体；全雌群体则可以通过两步程序获得：第一步雌性(♀ XX)性逆转为伪雄虾(♂ XX)，第二步与正常雌虾(♀ XX)交配，获得遗传性别为全雌(♀ XX)的群体。
基于IAG及其上游调控基因和促雄腺的生物技术获得全雄或全雌个体的方法，目前仅在雌性异配ZW性别决定模式的物种中成功开展了相关研究，相比于鱼类单性育种研究较为滞后。一方面，十足目甲壳动物的遗传基础研究滞后，相关理论基础缺乏，激素诱导性逆转的方法也尚未取得实质性突破，十足目甲壳动物需要较为繁琐的手术操作方法才能获得伪雄性(♂ ZW或♂ XX)或伪雌性(♀ ZZ或♀ XY)；另一方面，大多数十足目甲壳动物缺乏可靠的性别特异分子标记，超雌个体(♀ WW)和超雄个体(♂ YY)的筛选鉴定以及后裔测定，目前并没有高效的方法。
2.2 性别分子标记开发与遗传性别鉴定随着分子生物学和高通量测序技术的发展，各种遗传技术已越来越多地应用于鉴定十足目甲壳动物的性别特异DNA序列和标记。三疣梭子蟹中，基于高密度遗传连锁图谱的性别QTL定位(quantitative trait locus mapping)的鉴定与分析，以及简化基因组测序获得性别连锁SNP位点，证实其为XX/XY (雄性异配型)性别决定系统，并挖掘到可靠的性别特异分子标记[191-193]。同样地，在花蟹(Charybdis feriatus)中发现了5个雄性特异性单核苷酸多态性(single Nucleotide Polymorphism, SNP)标记，为花蟹的XX/XY性别决定模式提供了可靠的分子遗传证据[194]。SNP性别特异分子标记和高密度遗传连锁图谱为拟穴青蟹、紫螯青蟹(S.tranquebarica)、锯缘青蟹、红螯光壳螯虾、中华绒螯蟹等的雌性异配ZW型性别决定模式提供了分子证据[195-198]。Shi等[195]根据雌性特异性核苷酸成功设计了雌性特异性引物，该引物可以扩增来自雌性的预期条带，而不能扩增来自雄性的预期条带。由此，建立了一种快速有效的拟穴青蟹分子性别鉴定方法，同时，该方法可以成功地鉴定紫螯青蟹和锯缘青蟹的性别。在中华绒螯蟹中，Liu等[199]构建了中华绒螯蟹深覆盖基因组BAC文库(bacterial artificial chromosome library)，首次鉴定出中华绒螯蟹性别特异分子标记，并据此建立了基于PCR的遗传性别鉴定方法。在红螯光壳螯虾中，开发了5个性别特异标记，其中一对Z/W-76567性别标记通过PCR反应和凝胶电泳分析，能够区分ZZ和ZW遗传性别，即ZZ在电泳结果中为一条特异条带，ZW为两条，这些为红螯光壳螯虾的单性育种奠定了基础[197]。
稳定的性别特异分子标记对十足目甲壳动物早期遗传性别鉴定及单性养殖研究至关重要。随着甲壳动物基因组测序和组装的陆续完成，在构建了高质量的基因组图谱的基础上，加快性别特异分子标记筛选与验证工作，将有助于十足目甲壳动物性别控制与单性育种研究。
3 展望本文综述了十足目甲壳动物性别决定和分化分子调控机制的研究进展，总结了在其中发挥重要作用的关键因素。同时，还探讨了获得十足目甲壳动物单性群体的性别控制方案。这些研究对于实现十足目甲壳动物规模化单性苗种生产和养殖具有一定参考价值。随着CRISPR/Cas9基因编辑技术的广泛研究与应用，十足目甲壳动物中也取得了一定的进展。Gui等[200]开发了脊尾白虾(Exopalaemon carinicauda)的显微注射方法，成功地将CRISPR/ Cas9技术应用于脊尾白虾的基因编辑，结果表明获得的这些突变可以稳定遗传给下一代。Song[201]通过CRISPR/Cas9基因编辑成功构建MIH敲除系，为深入研究MIH基因的功能奠定了良好基础。同样在脊尾白虾中，Gao等[202]通过CRISPR/Cas9基因编辑技术，获得了一种胰岛素样肽(ILP)的敲除系，其生长抑制特性和死亡率显著高于正常对照组，而RNA干扰敲降ILP组也表现为生长速度缓慢，死亡率较高的特征，从侧面验证了基因编辑实验结果的准确性。而IAG作为一种胰岛素样肽，使用CRISPR/ Cas9基因编辑方法，应也能成功构建IAG敲除系，从而对其进行功能分析和应用研究。利用基因编辑技术进行性别控制是未来十足目甲壳动物单性育种研究的重要方向之一，也是研究性别决定和分化相关基因的重要技术手段。
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