Fruit Fly Dating and Mating

Today, many people meet their significant others through dating websites by electing to initiate a conversation after glancing at the biography and pictures posted by the other user. Without realizing it, humans are able to determine the sex of their potential partners from these short descriptions...but how are other organisms able to make the same judgments? While humans typically use physical cues such as size, mannerisms, genitalia, etc., fruit flies like Drosophila melanogaster use pheromones.

Flies are coated with waxy pheromones that differ between males and females. When a male fly first courts a female, he taps the female’s abdomen with his foreleg. The male’s foreleg has tiny hairs on them called bristles, some of which house gustatory, or taste, sensory neurons that can detect the pheromones on another fly. As naturally heterosexual animals, tapping and tasting the other fly’s body is imperative for the male to decide if the fly he’s courting is suitable; that is, one with which he can successfully mate.

In a recent paper published in eLife, Dr. Kristin Scott and her team at the University of California, Berkeley, studied the neural circuitry between the male’s foreleg and the neurons in the fly brain that influence the decision to court a female or not court a male. Their findings offered a surprising twist on how the neural circuits that control courtship behavior are organized.

In a previous study, Dr. Scott’s team examined the foreleg of male flies and identified two types of pheromone-sensing, sensory neurons that receive gustatory cues when the male taps another fly’s body. One set of cells they called M-cells (male cells) and the other they called F-cells (female cells). These sensory neurons express different genes, and hence detect different pheromones. The F-cells are unique in that they express a gene called pickpocket-25 (or ppk25) among other genes and respond to pheromones on the female’s body. The M-cells, however, express ppk25 and another pickpocket gene called ppk23. Using this information, Dr. Scott’s lab set out to determine the neurons that were connected to the F- and M-cells that influence male’s ability to court appropriate partners.

Using GCaMP, an experimental procedure that reveals which neurons are active in a behaving animal, it was found that activation of M-cells could activate a group of neurons in the male’s brain called the mAL neurons (Figure 1).

Figure 1 shows brains of adult flies stained with anti-GFP (green) to show the mAL neurons and a projected sketch of these neurons in both males (e) and females (f).  Kimura, Ken-Ichi, et al. “Fruitless Specifies Sexually Dimorphic Neural Circuitry in the Drosophila Brain.” Nature, vol. 438, 10 Nov. 2005, pp. 229–233., doi:10.1038.
Figure 1

The mAL neurons are GABAergic, meaning they produce the neurotransmitter GABA, which can inhibit the activity of the neurons that they are connected to. The mAL neurons are in turn connected to a group of neurons called P1 which, when active, drive the male’s courtship behavior. So, the pathway from M-cells to the P1 neurons is simple: when a male encounters another male and taps his abdomen, the taste sensory neurons on his foreleg detect the male pheromones via the ppk23/ppk25-expressing M-cells. The M-cells activate the mAL neurons, which then inhibit the activity of the courtship-promoting P1neurons. The male decides not to court.

In order to verify the function of the mAL neurons, Dr. Scott’s team expressed tetanus neurotoxin in the mAL neurons of the male’s brain and examined how his behavior was affected. Amazingly, when a group of males with inactivated mAL neurons were grouped together in a vial, the males started to court each other, creating long chains of males, each one courting the male in front of him. This confirmed that the role of the mAL cluster is to inhibit courtship. And because the mAL neurons are connected to the M-cella, male flies are blocked from courting other males. To further confirm these findings, the team tested for courtship frequency when the M-cells were inhibited in the male fly’s foreleg. The inability of M-cells to detect male pheromones prevented the activation of the mAL cluster and allowed the P1 neurons to become active. This resulted in male flies becoming bi-sexual and verified the connectivity between M-cells and the mAL cluster.

What about the F-cells? What are they connected to and what effect do they have on P1 neurons? When Dr. Scott’s team activated the F-cells, another type of neuron, the PPN1 neurons or Pheromone Projection Neuron class 1, became active. Like the mAL neurons, the PPN1 neurons are connected to the P1 neurons (Figure 2), but unlike the mAL neurons, PPN1 is an excitatory neuron—meaning it activates neurons it is connected to.

Figure 2 displays (a) complete, (b) dendritic, and (c) axial views of the PPN1 neurons in the brain of an adult, male fly.  By identifying these crucial areas and activating (e) PPK25 cells and (f) P1 neurons, Dr. Scott’s team was able to visualize the connectivity between these neurons.  Kallman, Benjamin R, et al. “Excitation and Inhibition onto Central Courtship Neurons Biases Drosophila Mate Choice.” ELife, vol. 4, 14 Nov. 2015, pp. 1–18., doi:10.7554/elife.11188.
Figure 2

The PPN1 neurons are located in the ventral nerve cord of the fly, similar to the spinal cord in humans. Downstream of the F-cells, PPN1 acts as a link between the pheromone-sensing cells and the courtship promoting neurons, P1. This then encouraged male flies to court. But the real surprise of this study was yet to come.

Curiously, it was discovered that the F-cells also activate the mAL cluster. Why would the courtship-promoting pathway also activate a pathway that would inhibit courtship? Dr. Scott and her team suggest that when deciding whether or not to court, the balance between excitation and inhibition is really important. If the F-cells only activated the P1 neurons, then they run the risk of over-activating the P1 neurons, which would drive the male to court uncontrollably. If the fly the male started to court was accidentally a male or a female from another species, it would be a waste of time and energy for him to keep courting. By activating a parallel inhibitory pathway, the F-cells could activate the P1 neurons in a more controlled way and allow the male to better determine if he should or should not continue courting the other fly.

Distinguishing between males and females is an essential part of the reproductive process for the majority of earthly organisms. Dr. Scott and her team used the M- and F-cells located in the taste bristles on the forelegs of Drosophila melanogaster males to discover the neuronal pathways that initiate courtship. By tracing the activation of neurons by activating or inhibiting these gustatory cells, the circuitry shown below (Figure 3) was discovered. M-cells inform the fly that the potential mate it is tapping is a male, activates the mAL cluster which inhibits P1, and prevents the male from courting. Oppositely, when F-cells are activated by female pheromones, which activate the PPN1 neurons, which then activate the P1 neurons and courtship is encouraged. However, the mAL cluster is also slightly activated by PPN1 so that the urge to court does not overcome the fly and he is still able to consider other factors.

Figure 3 displays the circuitry discovered by Dr. Scott’s team that connects the F- and M-cells to the PPN1 in the VNC and the mAL neurons and P1 neurons in the brain.
Figure 3

Although this work revealed a lot, many questions remain unanswered. Primarily, do females also possess the ability to distinguish the sex of their partners? How do their brains react to the possibility of courting? Future studies will need to be done in order to discover the neuronal circuitry of the female brain and their role in courtship decisions.