Use of cytoplasmic incompatibility to generate male sterility in insects.

细胞内母系遗传的管状沃尔巴克氏体可以影响昆虫的繁殖。最常见的操作是细胞质不相容（CI），在某些杂交中表现为不育，这可以使沃尔巴克氏体迅速入侵昆虫种群。感染沃尔巴克氏体的雄性精子在成熟过程中被修饰，如果它们与未感染的卵子受精，就会发生早期发育停滞。然而，当父母双方都携带沃尔巴克氏体时，可存活的后代被“拯救”，因此受感染的雌性具有选择优势。了解CI的分子机制可能是沃尔巴克氏体生物学中尚未解决的主要问题。最近在Sinkins实验室对沃尔巴克氏体存在时宿主基因表达的研究（1,2）表明，在雄性和雌性以及蚊子和果蝇中，一种基因在D. melanogaster中参与卵子发生和控制雌性减数分裂细胞周期，这种基因在雄性和雌性中都一致上调。我们的假设是，在沃尔巴克氏体存在的精子发生过程中，该基因的不适当表达水平可能是CI的原因，从细胞生物学研究中得知，CI涉及雄性和雌性原核之间的异步细胞周期事件。沃尔巴克氏体转录调节基因是CI生成的有力候选基因，也已通过比较基因组研究确定(3)。埃及伊蚊是一种传播登革热和其他病毒的热带蚊子，迫切需要新的控制方法；释放不育雄虫是实现这一目标的一个有希望的途径(4)。CI提供了一种无需辐照的潜在灭菌方法，既可以使用沃尔巴克氏体本身，也可以复制沃尔巴克氏体使用的生化/遗传方法。因此，CI的研究可以为Oxitec提供具有商业意义的新控制方法，并且具有相当大的基础兴趣。Ae的种系转化。使用已建立的胚胎显微注射技术（如先前在Sinkins实验室和Oxitec进行的那样），将使用表达a)宿主细胞周期调节基因和b)沃尔巴克氏体转录调节基因的构建体，在生殖系特异性启动子的条件控制下（Oxitec已经开发了条件生殖系表达系统的原型），并使用DsRed标记。转化系的雄性将与野生型群体的雌性进行杂交。埃及伊蚊自然未感染沃尔巴克氏体)，也与来自沃尔巴克氏体感染群体的雌性一起，以检查是否发生类似ci的不育，以及这种不育是否可以在沃尔巴克氏体存在的情况下获救。我们将使用Oxitec在埃及伊蚊（例如5）中广泛使用的两部分表达系统。很可能需要进行几轮改造和结构优化，每一轮都是根据前一轮的数据以及Oxitec和Sinkins实验室的背景数据进行的。然后将对改造后的品系进行适应性和竞争性实验。如果候选基因没有表现出预期的表型，我们将使用替代效应剂通过破坏其他已知的精子发生特异性途径来提供不育。具有良好无菌性的结构将被重建为单插入系；考虑到这一点，初始构建将被设计为便于使用phageC31系统在体内进行修饰(6)。Kambris Z等人。在准备中；Kambris Z等人。科学326:134-6。3. Sinkins等。在准备中。（2009）动物病媒传播，动物疾病，10,295-311。傅刚等。（2010）中国科学院学报（自然科学版）：37 - 41。Nimmo D et al. (2006)；摩1:1:129-136

The intracellular maternally inherited bacterium Wolbachia pipientis can influence the reproduction of insects to its own advantage. The most common manipulation is cytoplasmic incompatibility (CI), manifested as sterility in certain crosses, which can allow Wolbachia to rapidly invade insect populations. Sperm from Wolbachia-infected males is modified during maturation such that early developmental arrest occurs if they fertilize uninfected eggs. However, viable progeny are 'rescued' when both parents carry Wolbachia, and thus infected females have a selective advantage. Understanding the molecular mechanism of CI is perhaps the major unanswered question in Wolbachia biology. Recent work on host gene expression in the presence of Wolbachia in the Sinkins lab (1, 2) has shown a consistent upregulation, in both males and females and in both mosquitoes and Drosophila, of a gene that in D. melanogaster is involved in oogenesis and control of the female meiotic cell cycle. Our hypothesis is that inappropriate levels of expression of this gene in the presence of Wolbachia during spermatogenesis may be the cause of CI, which is known from cell biology studies to involve asynchronous cell cycle events between the male and female pronuclei. A Wolbachia transcriptional regulator gene that is a strong candidate for CI generation has also been identified using comparative genomic studies (3). Aedes aegypti is a tropical mosquito species that transmits Dengue and other viruses, and new control methods are much needed; the release of sterile males is a promising route to this end (4). CI provides a potential method for sterilisation without the need for irradiation, either using Wolbachia themselves or by replicating the biochemical/genetic method used by the Wolbachia. Thus studies on CI could provide new control methodologies of commercial significance to Oxitec as well as being of considerable basic interest. Germline transformation of Ae. aegypti using established embryo microinjection techniques (as previously performed in both the Sinkins lab and Oxitec) will be undertaken using a construct that expresses a) the host cell cycle regulator gene and b) the Wolbachia transcriptional regulator gene, under the conditional control of a germline-specific promoter (prototype conditional germline expression systems have been developed at Oxitec) and using a DsRed marker. Males from the transformed line will be crossed with females from the wild-type colony (Ae. aegypti is naturally uninfected with Wolbachia) and also with females from a Wolbachia-transinfected colony, to examine whether CI-like sterility occurs, and whether this sterility can be rescued in the presence of Wolbachia. We will use a bipartite expression system that has been extensively used by Oxitec in Aedes aegypti (e.g. 5). It is likely that several rounds of transformation and construct optimisation will be required, each informed by data from the previous round and by background data from Oxitec and the Sinkins lab. Fitness and competitiveness experiments with the transformed lines will then be conducted. As a fall-back if the candidates do not show the expected phenotypes, we will use alternative effectors to provide sterility by disrupting other known spermatogenesis-specific pathways. Constructs giving good sterility will be rebuilt as single-insertion lines; with this in mind the initial constructs will be designed to facilitate modification in vivo using the phageC31 system (6). 1. Kambris Z et al. In prep. 2. Kambris Z et al. Science 326:134-6. 3. Sinkins S et al. In prep. 4. Alphey L et al. (2009) Vector Borne Zoo Dis 10, 295-311 5. Fu G et al. (2010) PNAS 107:4550-4554 6. Nimmo D et al. (2006) Ins. Mol. Biol 15:129-136