Use of cytoplasmic incompatibility to generate male sterility in insects.

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