Investigating the dual role of mate choice genes in behavioural isolation and hybridization

Natural and human-induced changes in species' distributions lead to novel interactions between species and increase the opportunity for interbreeding between closely related species (hybridization). Hybridization may have a profound effect on the fate of species, although whether two species will fuse, divide or subdivide upon secondary contact is difficult to predict. Whether two species will interbreed depends, in large part, upon whether the species recognize each other as mates. Sensory cues, such as courtship songs and pheromones that are produced in one sex, and perception and preferences for these cues in the opposite sex promote assortative (within species) mating. The genetic control and function of the genes that determine mate choice will either facilitate or constrain hybridization between species. Yet little is currently known of how such genes shape species boundaries, largely because most studies of hybridization between species have been conducted in non-model organisms for which genetic manipulation is challenging.
D. simulans and D. sechellia are sibling species that diverged on alternate islands in the Indian Ocean about 250,000 years ago. Our colleagues recently discovered D. simulans-D. sechellia hybrids in the Seychelles Archipelago, and this provides an opportunity to study how mate sorting genes will shape the outcomes of hybridization. Behavioral barriers prevent mating between D. simulans males and D. sechellia females, but D. sechellia males are more likely to mate with D. simulans females than with females from their own species by a margin of 2:1.
Our long-term goal is to understand how the genes that confer mate choice will shape the outcome of hybridization. We take an important step toward that goal in this proposal by identifying genes that govern mate choice between D. simulans and D. sechellia. Our preliminary work finds two major effect loci in the male genome and two major effect loci in the female genome influence whether copulation will occur with heterospecific flies of the opposite sex. We have successfully verified the role of two genes that confer production of a species- specific pheromone, 7,11-heptacosadiene (7,11-HD), and our mapping data implicates a second pheromone that is produced in males. We propose that assortative mating in the D. sechellia - D. simulans species pair is conferred by a set of alternate alleles that determine differences in the perception and production of two distinct pheromones, and that particular combinations of these pheromone alleles will promote or reduce hybridization.
In our first activity we combine CRISPR-assisted fine mapping with molecular genetics to reveal the alternate alleles that confer attraction in D. sechellia males, or aversion in D. simulans males, to 7,11-HD. Next, we will isolate the genes that likely confer production of an uncharacterized pheromone produced in D. sechellia males, and investigate the role of alternate alleles of a candidate pheromone receptor in conferring differences in female response to the male pheromone. In the final objective, we will recombine alternate alleles in the same genome to test whether we can alter assortative mating in ways that are relevant to speciation. Essentially, we are mimicking the effects of recombination between D. simulans and D. sechellia that occur in the Seychelles hybrid zone. Once we understand how pheromone transmission/reception alleles affect hybridization in the lab, we will be able to make predictions about the direction of gene flow in the field. Future studies, that will follow completion of this grant, will track the fate of the alleles in wild populations in the Seychelles as hybridization progresses.

The impact of this research program could benefit the United Kingdom by: 1) informing policy on invasive species that interbreed with endemics, 2) through development of alternative control methods of related dipteran pests and disease vectors, 3) training and career development, and 4) public outreach.

The UK is home to many species that are distinct from those of continental Europe. As global shipping imports invasive species from their native range it becomes increasingly important to predict how hybridization will impact native species and to incorporate the likely outcomes of hybridization into public policy. While interbreeding between native and invasive species has had a large impact upon the British Isles in some cases, not all invasions result in substantial mixing between species. A greater general understanding of the genetics of mate choice could help place resources where they are most needed.

The outcome of the proposed work could potentially contribute to development of more targeted insect control methods for disease vectors and invasive pests. Drosophila belongs to the Diptera, an insect order that encompasses 80% of disease vectors, including mosquitos, which kill about one million persons each year. A number of Diptera are also crop pests. Drosophila suzukii, for instance, is an invasive species that destroys soft and stone fruits, first detected in the U.K. six year ago. The most effective tools to combat dipteran pests and disease vectors are neurotoxic insecticides. Yet such broad-spectrum insecticides have unintended environmental and health effects, and more targeted insect control tools are urgently needed. Sex pheromones have been successfully used in biological control of some agricultural pests by luring males from females, thereby reducing the pest population. Contact pheromones (like 7,11-HD) have been found in many dipterans, including mosquitos. Ionotropic receptors (like IR67a) are also found throughout the Diptera. Yet outside of Drosophila, very little is known of what they are tuned to detect, how they direct behavior, or how their functions evolve. Further research on these molecules within the Drosophila genus, and their extension to studies of other dipteran insects, could potentially lead to targeted pest vector control programs that have fewer off-target effects upon humans and the environment.

Finally, this proposal has the added value of potential benefits to the public and private sector more directly through training and career development, and to wider society through engaging members of the general public. The Researcher Co-Investigator (RCI) will receive career development training and participate in public outreach. The PI has a strong track record of contribution of public dissemination of research achievements, for example via the Oxfordshire Science Festival, Wellcome initiatives and the general media. The topic of speciation and the evolution of sexual behaviour will provide many opportunities for further public engagement.