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Postdoctoral research: Genetics of adaptation to toxins, natural host plant toxins and synthetic insecticides, across ecological and phylogenetic timescales

1. Genetics of adaptation to host plant toxins in the monarch butterfly and other specialist insects

Insects across six orders, including the monarch butterfly, have evolved resistance to toxic cardiac glycosides produced by their host plants as defense compounds. Cardiac glycosides inhibit the sodium pump (Na+/K+-ATPase) by binding to the cardiac glycoside -binding site in its α subunit (ATPα), and convergent amino acid replacements at this site appear to have rendered the sodium pump insensitive to the toxin, a phenomenon known as target site insensitivity (TSI).
To examine whether these mutations on their own can explain the resistance observed in nature and to begin to understand how the mutations occurred, I co-led with Dr Niels Groen an international collaboration of four labs with complementary expertise. Using ancestral state reconstruction, we first identified potential mutational pathways to resistance involving three ATPα mutations at positions 111,119 and 122 in independent lineages of specialist insects resistant to cardiac glycosides. By using CRISPR/Cas9 genome engineering in Drosophila melanogaster, we recreated the mutational pathway observed in the monarch butterfly lineage. For this, we generated Atpα knock-in Drosophila lines carrying substitutions at sites 111, 119 and 122 of four consecutive Atpα genotypes in the monarch lineage. In feeding assays with ouabain, a cardiac glycoside and enzymatic assays for sodium pump activity,

we found that the mutations, when they appeared in the same order and in the same combinations as in the monarch butterfly lineage, increased resistance to cardiac glycosides through TSI. In fact, flies carrying the monarch genotype were as insensitive to cardiac glycosides as the monarch butterflies. These results were groundbreaking because they showed, for the first time in multicellular organisms, that CRISPR/Cas9 genome engineering can be used to retrace a multi-step adaptive mutational pathway.

         We then investigated whether pleiotropic costs associated with the resistance ATPα mutations might explain the order in which the mutations evolved along the adaptive pathway in the monarch lineage. The sodium pump is essential for the function of the nervous system. We found that all the monarch lineage knock-in ATPα Drosophila lines displayed neuronal defects, which were less severe in knock-in lines carrying the ATPα mutation at 119 in combination with mutations at 111 and/or 122. We also generated additional knock-in lines and found that the mutation at 119 alone conferred low resistance with no neuronal defects, while the mutation at 122 alone displayed high resistance as well as severe neuronal defects. These results may explain why position 119 mutated before or together with position 122 in the monarch and all other insect lineages of specialist insects resistant to cardiac glycosides. These results may illustrate in detail how antagonistic pleiotropy has constrained the mutational pathway to cardiac glycoside resistance.

2. Genetics of adaptation to synthetic insecticides in Drosophila and other insect pests

in preparation

Doctoral Research: Multistep evolution of sensory ecology in a Drosophila pest

How species evolve new behaviors to adapt to novel environments remains largely unknown. During my graduate research, I analyzed novel egg-laying behavior in the fruitfly pest Drosophila suzukii as a model for understanding how a novel adaptive behavior emerges and evolves in nature. D. suzukii is an invasive species that causes significant economic damage to fruit industries in Europe and the US. This work was the first, to our knowledge, to apply neurogenetic tools and CRISPR/Cas9 gene editing to D. suzukii and to compare its egg-laying behavior to that of other Drosophila species.
I first investigated the shifts in the sensory ecology that have enabled D. suzukii to turn into a pest. Through a comparative behavioral analysis between D. suzukii and close relatives, including the genetic model D. melanogaster, I first established that D. suzukii has shifted its reproductive niche from rotting to ripe fruit. Insects are known to choose egg-laying sites based mainly on chemosensory cues, and I demonstrated that D. suzukii females use sensory information to select ripe fruit. By comparative cross-species analysis, we showed that D. suzukii acquired the ability to integrate mechanosensation, gustation and olfaction in a stepwise manner, leading us to propose a multi-step mechanism by which the species shifted its reproductive niche (Karageorgi et al., 2017).
To help lay the foundations for comprehensive molecular studies of D. suzukii egg-laying behavior, I performed RNA sequencing of all sensory organs and these results were used to provide the first high-quality annotation of the D. suzukii genome (Paris et al., 2020). The insights from this work will help us understand how multiple sensory systems co-evolve to lead to adaptive behaviors. In the more immediate term, the detailed knowledge about D. suzukii gleaned from this work may help the agricultural industry control it.

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