How to produce male-only progeny in pest insects for SIT: a biotech approach.

Insects represent the most abundant group of animals on earth, comprising about 800,000 described species, and approximately 10,000 of these species can be actually destructive for human activities. Pest control interventions, alternative to pesticides, are increasingly being implemented within the concept of Integrated Pest Management, involving the biological control to eradicate a pest from the area of interest. This concept has been recently extended also to those hemathophagous insects transmitting human diseases, such as for example mosquitoes. The Sterile Insect Technique (SIT) is a species-specific and environmentally friendly method for insect biological control, based on release of sterilized insects within reasonable proximity of all native females to decrease the next progeny, due to the reduced mating between their fertile wild counterparts. Release of insects of both sexes is less effective than male-only release in reducing the wild population, while removing females from the production and release procedures, would produce considerable advantages in economic and effectiveness terms. Hence a genetic sexing system to produce male-only progeny, would improve the effectiveness of SIT. My PhD project has been focused on novel biotechnological approaches to control insect species, such as the agricultural pest Ceratitis capitata (Diptera; Tephritidae) and the mosquito disease vector Aedes aegypti (Diptera; Culicidae). The first part of my Ph.D. experimental work has been focused on the molecular and functional studies of the C. capitata transgenic strain Cc5.3, which is a first prototype of a novel sexing system, based on masculinization of XX individuals. This strain can produce male-only progeny in specific crosses, by a maternal transgene-mediated RNAi against a key gene for female sex determination. The male only progeny obtained by this method could be used, in future, for release programs in SIT, after sterilization. I asked some questions to which I partially answered during my PhD thesis: 1) are the XX males competitive in mating with wild type females? 2) is the masculinization observed at phenotypic level, also fully penetrant at molecular level? 3) which are the regulatory elements influencing the peculiar maternal effect in this transgenic strain? Moreover, I have also investigated additional novel genes, such as Drosophila orthologues of doa and fru genes, potentially involved in sex determination and sexual differentiation of C. capitata (doa gene, a kinase involved in splicing control) and Ae. aegypti (fru gene, a transcriptional factor involved in courtship behaviour) which can be possible targets for novel transgene-mediated RNAi and hence the development of improved sexing strains. Interestingly, I have found similar to Drosophila, sex-biased isoforms of Ceratitis DOA encoding transcripts, which could be involved in controlling sex-specific alternative splicing of target genes. Furthermore I have contributed to clarify the molecular differences underlying the sex-specific splicing of the fru gene in Aedes with the respect of other dipteran species, proposing a model for sex determination in this mosquitoes species. In addition to RNAi, novel reverse genetic tools are available. I've tested the TALENs' action in Ae. aegypti and C. capitata and more recently the CRISP-CAS9 method in Ceratitis