De genes à fenótipos: um estudo de genômica funcional na Mosca-da-bicheira, Cochliomyia hominivorax (Diptera: Calliphoridae)

Author(s)

Daniel Fernando Paulo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-12-11

Examining board members

Ana Maria Lima de Azeredo Espin; Marcelo Mendes Brandão; Carla Cristina Judice; Fernando Luis Cônsoli; Reinaldo Otávio Alvarenga Alves de Brito

• Advisor: Ana Carolina Martins Junqueira; Ana Maria Lima de Azeredo Espin

Abstract

Despite their destructive nature, insect pests provide us with an unparalleled opportunity to investigate how species evolve new behaviors and adapt to novel ecological niches. Especially, identifying the genetic bases underlying their lifestyle seems to be the key to unlock the evolutionary routes leading to their ecological adaptation. The New World Screwworm fly, Cochliomyia hominivorax (Coquerel, 1858), is a promising model for studying shifts in host-preference in blowflies (Diptera: Calliphoridae). While most closely related species lay their eggs on decaying organic matter, including carcasses, C. hominivorax oviposits on the dry borders of wounds and orifices of living warm-blooded vertebrates. Surprisingly, and in opposition to its associated economical and veterinary importance, the genetic basis of C. hominivorax¿s preference towards living hosts has largely been neglected. To overcome this constraint, we developed highly efficient protocols for mutagenesis and generation of Screwworm knockouts (KOs) using CRISPR/Cas9 genome editing (Chapter 1). Site-directed mutations were introduced in the C. hominivorax yellow gene (ChY), resulting in the brown body phenotype (bwb), a visual trait to score for Cas9-induced mutations. High rates of somatic and germline mutagenesis were achieved when delivering Cas9-sgRNA pre-assembled complexes at high concentrations into Screwworm embryos. We next targeted transformer (Chtra), a major gene required for normal female development in blowflies. Experiments resulted in the masculinization of C. hominivorax females (intersex XX flies), suggesting that Chtra is a potential target for future control strategies in this species. Due to these outcomes, we anticipate that CRISPR technology will have a tremendous implication for the implementation of new genetic-based strategies to manage this important insect pest. However, and most importantly, the data described here provide the foundation for functional investigations on the evolution of natural traits in C. hominivorax. Signaling and reception have been shown to evolve in synchrony to shape olfactory landscape, thus chemosensory-mediated behaviors, in many dipterans. Like in other insects, the Screwworm relies on volatiles (odors) to find suitable hosts for oviposition. Therefore, we hypothesized that olfaction may have played a critical role in the shift of niche-preference in the C. hominivorax lineage. To test this assumption, we functionally examined the Odorant Receptor Co-receptor gene (Orco) in Screwworm (Chapter 2). Insects detect odors through the expression of Odorant Receptors (ORs) and Ionotropic Receptors (IRs). Orco is an obligatory co-receptor for all ORs but not the IRs function, which in turn allows the screening of olfactory-mediated behaviors through the manipulation of a single gene. We found that C. hominivorax Orco (ChomOrco) is highly conserved within Diptera, as a consequence of a strong purifying selection regime. Expression of ChomOrco is linked to many morphological, developmental and behavioral aspects of the Screwworm, and is broadly represented in olfactory appendages of the adult flies. We next utilized this molecular information to knocking-out ChomOrco using the CRISPR/Cas9 methods previously described. Two-choice behavioral bioassays revealed that mutants for ChomOrco exhibit disrupted flight orientation, no longer responding to floral-like and host-associated volatiles. These results suggest that the OR-pathway is linked to host preference in Screwworm, and implies that evolution might have worked on the OR-pathway to architect the specific olfactory landscape in C. hominivorax. Overall, these results have several implications for functional investigations on blowflies, and will be a great addition to the current literature on Screwworm and other related blowflies. Yet, it is important to keep in mind that, the present study is just the beginning of a way longer investigation regarding the genetic basis of such extraordinary ecological adaptation: From a free-living to an obligatory parasitic fly (AU)

• FAPESP's process: 14/15042-3 - Expression profile analysis of microRNAs during different development stages of the New World Screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae)
• Grantee: Daniel Fernando Paulo
• Support Opportunities: Scholarships in Brazil - Doctorate