Expanding the toolbox for genetic modification in medfly

Ceratitis capitata, commonly known as the Mediterranean Fruit Fly or medfly, is an invasive Tephritid pest with a wide geographic spread and potential to expand further due to climate change. With the ability to damage over 300 species of fruit and vegetable crops, this agricultural pest is of major economic importance. Control of medfly has previously relied upon pesticides and Sterile Insect Technique (SIT). With the advent of CRIPSR/Cas9 new and improved methods of genetic pest control are possible. To develop new technologies such as gene drives (drivers of a transgene through inheritance bias) and improve existing ones, the toolbox for genetic modification in medfly must be expanded. Here I develop and validate several endogenous, germline specific Cas9 promotors, one of which was subsequently used for the development of the first homing drive (drives which utilise homology-directed repair) in medfly. The sex determination pathway in medfly provides several potential targets for sex conversion, including the conserved genes transformer (tra) and doublesex (dsx) and the masculinising signal of Maleness-on-the-Y (MoY). Guide cassettes were designed to target both tra and dsx, with two tra guide constructs being synthesised and used to generate transgenic lines. Crossing these with Cas9 lines gave no evidence of cutting, and subsequent investigations revealed an error in the guide designs, prompting a redesign of the guides for future use. The use of MoY to induce masculinisation is another promising method of sex conversion in medfly, though the lethal effects of overexpression of MoY need to be overcome. A construct was designed to package MoY into gfp-tagged sperm cells by placing it under the control of the spermatogenesis specific promotor of s2-tubulin. This aimed to create a transgenic line that could induce masculinisation through MoY-carrying sperm and reduce the temporal expression of MoY to a smaller window to avoid lethality. The results revealed post-microinjection embryonic death, indicating overexpression of MoY was causing lethality. A further Cre-Lox construct was designed for future usage which should allow for the transgenic line to be established without lethality during microinjection. The final part of the genetic toolbox focused on essential genes. Potential essential genes were identified and sequenced across two wildtype, laboratory-maintained populations to check for sequence conservation. Based on these results, guides targeting these genes were designed, alongside rescue versions of these genes the guides should be unable to target. In this research, several important toolbox parts were designed, synthesised and tested in medfly which can contribute to the future development of genetic pest control strategies

The misapplication of defensive realism: The security dilemma and rising powers of East Asia

Although generally a useful explanatory and analytical tool for understanding the dynamics of the East Asian security environment, the fundamental utility of security dilemma theory is dependent upon whether the system contains only security-seekers, or whether there are ‘greedy’ states. In the case of ‘greedy’ states, the role of the security dilemma is diminished and competitive policies are more likely to avoid conflict. United States foreign policy makers have failed to recognise this distinction rooted in state motivations, and continue to apply excessively cautious policies based upon defensive realist prescriptions in the face of a rising and revisionist China. This misapplication could lead to potentially disastrous effects for East Asian stability in the twenty-first century

Manipulating insect sex determination pathways for genetic pest management: Opportunities and challenges

Sex determination pathways in insects are generally characterised by an upstream primary signal, which is highly variable across species, and that regulates the splicing of a suite of downstream but highly-conserved genes (transformer, doublesex and fruitless). In turn, these downstream genes then regulate the expression of sex-specific characteristics in males and females. Identification of sex determination pathways has and continues to be, a critical component of insect population suppression technologies. For example, “first-generation” transgenic technologies such as fsRIDL (Female-Specific Release of Insects carrying Dominant Lethals) enabled efficient selective removal of females from a target population as a significant improvement on the sterile insect technique (SIT). Second-generation technologies such as CRISPR/Cas9 homing gene drives and precision-guided SIT (pgSIT) have used gene editing technologies to manipulate sex determination genes in vivo. The development of future, third-generation control technologies, such as Y-linked drives, (female to male) sex-reversal, or X-shredding, will require additional knowledge of aspects of sexual development, including a deeper understanding of the nature of primary signals and dosage compensation. This review shows how knowledge of sex determination in target pest species is fundamental to all phases of the development of control technologies

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata

Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate two different mechanisms by which CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way