It's good to be picky

Seeking a romantic partner poses a unique set of challenges. One typically considers age, common interests, physical attractiveness, and similar life goals when looking for someone to form a relationship with. But have you ever considered how much more difficult it would be if we were forced to also determine if our love interest was the same species as us? Thankfully, humans are able to do this subconsciously. Male Drosophila melanogaster and Drosophila simulans, closely related fly species, on the other hand, cannot distinguish the species of females simply by sight. While the two are physically able mate, it is not beneficial to either species as the offspring that are produced are often inviable or sterile. So how do they detect the proper female species to court? Dr. Laura Seeholzer, Dr. Vanessa Ruta and their team at The Rockefeller University in New York, New York, investigate how these two species utilize species-specific pheromones to detect proper mates.

D. melanogaster females produce a pheromone known as 7,11-heptacosadiene (7,11-HD) while D. melanogaster males and D. simulans males and females produce 7-tricosene (7-T). Males from both species are able to detect 7,11-HD, but demonstrate opposing behaviors. D. melanogaster males receive an excitatory input from 7,11-HD while D. simulans receive inhibitory inputs from the pheromone. Dr. Seeholzer and her colleagues aimed to discover whether these species-specific pheromone responses were due to evolution of peripheral detection mechanisms or the central circuits that process pheromone signals to regulate courting. The team was able to discover that the responses to 7,11-HD come from the reweighting of excitatory and inhibitory inputs in the courtship circuitry.

To rule out the possibility that detection mechanisms affect conspecific female selection, the team examined the forelegs of both fly species as they are known to be a crucial part of the courting process. They amputated the forelegs of the male flies to prove their importance in proper mating and found that the flies still courted, but were not choosy. This shows that the detection of pheromones are not necessary for mating, but are essential for species differentiation. Furthermore, they looked at the pickpocket23 (ppk23) neurons on the male fly foreleg that allow the fly to determine the gender of the potential mate. It was previously discovered that D. melanogaster utilizes ppk23 to detect female pheromones, including 7,11-HD, to promote courtship. Also, Gr32a receptors in D. melanogaster males’ legs are able to detect 7-T and suppress courting of D. simulans females and D. melanogaster males. However, the role of ppk23 and Gr32a was relatively unknown in D. simulans males. When Ppk23 and Gr32a mutants were created in D. simulans using genome editing, it was discovered that both types of mutant males still mated. Gr32a mutants failed to mate with D. melanogaster females which suggests that Gr32a does not influence mate choices in D. simulans. Contrarily, ppk23 mutants could not distinguish between the females’ species and did court D. melanogaster females. This finding supports the idea that males of both species rely on ppk23 to detect 7,11-HD to determine proper partners, but that detection of the pheromone initiates opposing behaviors. Also, being that both species utilize the same neurons for determining conspecific females, the team is able to show that evolution of peripheral detection mechanisms is not the cause of the species-specific pheromone responses.

To further verify their findings, Dr. Seeholzer and her colleagues went on to develop genetic tools to examine the sensory neurons, f-cells (where ppk23 is expressed), and the circuits that they activate that further contribute to the species-specific responses. Their experiments demonstrated that ppk23 sensory neurons in both D. simulans and D. melanogaster males are qualitatively and quantitatively similar in relation to 7,11-HD. Activation of these neurons was sufficient to drive opposite behavioral responses in the two species and suggests that the adaptational/evolutionary differences must exist downstream of the f-cells.

D. melanogaster and D. simulans possess vAB3 neurons that receive sensory inputs from several pathways, including from the f-cells on the male fly foreleg, and send outputs to descending neurons that drive behavior of male courtship. These outputs include excitatory routes to the P1 (primary courtship promoting neurons) and mAL (GABAergic, inhibitory neurons) neurons in the fly brain. And, while the responses to 7,11-HD detection are equal in strength, their pathways appear to be activated by diverging P1 neuronal excitation. Being that both species have similar activation of vAB3 and mAL pathways when they come in contact with D. melanogaster females, it suggests that alterations in the strength of their signaling to P1 might underlie the species-specific actions. When vAB3 was directly stimulated in males of both species, vAB3 and mAL activation was equivalent, but P1 neurons were excited in D. melanogaster but not in D. simulans. This experiment verified that vAB3 neurons mediate the divergent activity between the two species.

In summary, D. melanogaster and D. simulans are very similar fly species that have diverging pathways when the pheromone 7,11-HD, located only on D. melanogaster females, is detected. Both species utilize ppk23 sensory neurons (f-cells) to detect the pheromone, but detection triggers opposing behaviors in the two species. D. melanogaster males have activation of both P1 and mAL neurons, but have net excitation and initiate courtship with D. melanogaster females. Oppositely, D. simulans males also have activation of P1 and mAL neurons, but have net inhibition and refrain from courting the opposing species. The difference in activation is controlled by the vAB3 neurons and the degree to which they excite P1. While there are possibly other inputs working on vAB3, mAL, and/or P1, the findings of Dr. Seeholzer and her colleagues suggest that the differing pheromone-processing pathways play an important role in the evolution of mate preferences in D. melanogaster and D. simulans when presented with the same sensory signals.

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