Art Courtesy of Court Johnson.
From evoking long-forgotten memories through nostalgic scents to detecting imminent danger through noxious odors, smells hold undeniable power. Our sense of smell has served as a prime mechanism for survival since the beginning of time. Much of the work in the lab of John Carlson, a Yale professor of Molecular, Cellular, and Developmental Biology, is dedicated to studying the intricate mechanisms of Drosophila (fruit fly) olfaction: how do these insects detect and behave in the presence of volatile pheromones? An example of chemosensation, these pungent odorants—chemical compounds that have a smell—produced by organisms facilitate sexual attraction and mating by affecting behavior. The lab is interested in studying chemosensation as a method to control populations of insects that spread disease—such as the tsetse flies, insects that are responsible for spreading African trypanosomes, the causative agents of African sleeping sickness in Sub-Saharan regions.
In a paper published in Science earlier this year, a team of researchers conducted a study to find natural odorants that may control the behavior of tsetse flies. Led by Shimaa Ebrahim, a postdoctoral researcher in the Carlson lab, the project was conducted in collaboration with Hany Dweck, an associate scientist in the Carlson lab, and Brian Weiss, a senior research scientist at the Yale School of Public Health. “I fell in love with the [tsetse fly],” Ebrahim, who studies the courtship behavior of Drosophila, said. Weiss echoed Ebrahim’s fascination, pointing out behaviors unique to the tsetse flies, such as the live birth and nurturing of their young, in contrast to the majority of insects, which lay eggs.
Importantly, tsetse flies feed exclusively on vertebrate blood, leading to the transmission of Trypanosoma brucei (trypanosomes), a classification of protozoan parasites that cause African sleeping sickness and nagana in humans and domesticated animals, respectively. These diseases have devastating effects: nagana is responsible for an average loss of 4.5 billion dollars in resources every year in Africa. If left untreated, African sleeping sickness has a one hundred percent mortality rate. Unfortunately, access to treatment is not available everywhere. “Most of the drugs used to treat sleeping sickness are arsenic-based and horribly toxic. Very recently, there’s been some newly-developed medications, and they’re much less toxic—but they’re much more expensive,” said Weiss, who studies tsetse flies and their associated microorganisms.
To find alternative solutions, the researchers turned their attention to preventative measures. According to Weiss, the best solution to reduce fly populations is to use traps, as they are cheap and effective. However, not much is understood about the chemical signaling of tsetse flies, which may be key to luring the flies to traps. “If you want to control any insect that could cause disease or damage to any crop, you have to study their behavior to find the way to control this insect,” Ebrahim said.
The role of smell in mating
The researchers utilized the tsetse fly species Glossina morsitans. While Glossina fuscipes is the most abundant species in Kenya, they are difficult to rear in the insectary, according to Weiss. To examine possible odorants in sexual attraction, the researchers first studied tsetse fly mating behaviors. When paired together, G. morsitans males initiate mating with G. morsitans virgin females within seconds, and copulation continues on average for an hour. However, this reaction was not observed when G. morsitans males were paired with mated females, suggesting that there may be a difference in chemical signaling that results in the male’s behavioral responses.
To determine whether differences in mating are driven by olfaction, pheromone extracts were obtained from the exoskeleton, also called the cuticle (the layer that covers the extracellular surface of the fly), of male and female flies that were soaked and gently shaken in hexane for ten minutes. Dummy tsetse flies made out of yarn were sprayed with these extracts and placed in a container with male G. morsitans, and a measure of attraction was determined by the percentage of males that initiated mating and stayed attached to the decoy flies for more than five minutes. However, the males were not attracted at all, suggesting that any odorants associated with mating are stored beyond the cuticles.
After soaking another set of tsetse flies in hexane for twenty-four hours, it was observed that males were attracted sixty percent of the time to dummies sprayed with extracts from virgin females and twenty-seven percent of the time with extracts from mated females, with no response observed with male extracts. This observation indicates that there may be a difference in the composition of the extracts that made male flies more attracted to virgin than mated female G. morsitans. The longer soaking time may also explain why these compounds were not detected as potential pheromones in earlier research.
Discovery of a key pheromone
To determine if differences in compound composition were responsible for mating behaviors, the researchers obtained the chemical profile of the extractions via gas chromatography-mass spectrometry (GC-MS), which separates and detects different compounds. GC-MS identified three fatty acids and three fatty acid methyl esters that were not present in the ten-minute hexane immersion, which suggests the pheromones stored in internal glands may play a part in facilitating attraction.
To test this hypothesis, the researchers repeated the previous experiment by spraying the dummy flies with each of these six compounds. They found that the male flies were strongly attracted to certain compounds that are present in higher levels in virgin females, such as methyl palmitoleate (MPO)—which attracted G. morsitans males eighty-seven percent of the time even when diluted—methyl oleate (MO), and methyl palmitate (MP). The male fly stayed attached to the dummy for a prolonged period of time, suggesting that these compounds also act as arrestants—halting all motion—to prevent premature interruption of mating. Clearly, smell seemed to play a powerful role in mating!
Next, the researchers sought to investigate cellular mechanisms that may facilitate the observed behavioral responses. Removing G. morsitans males’ antennae eliminated their attraction response, suggesting that the observed behavioral responses in mating are facilitated by scent. Furthermore, research has suggested that volatile odors are detected via the trichoid sensilla, a sensory organ located in the antenna of the fly.
In a method called single-sensillum electrophysiology, the researchers obtained recordings of trichoid sensilla response to odors detected in the air by antenna. Initially, only MPO elicited excitatory responses from both sexes, but particularly from males. After reducing the distance from which the odor was delivered, activation in neurons was seen with MP, MO, and MPO, correlating with the behavioral results observed in earlier experiments. However, there
was little to no response in olfactory neurons to these six compounds in G. fuscipes, another
tsetse species that is the prominent vector of trypanosomes in east Africa. This finding suggested
that these pheromones are specific to G. morsitans mating mechanisms. Indeed, it was found that G. morsitans males made no attempt to mate with untreated G. fuscipes females; however, when G. fuscipes females were sprayed with MPO, the males began to engage, suggesting that MPO may act as an aphrodisiac, a stimulant for sexual desire, for G. morsitans males.
Could infection change mating?
The last study examined if these findings held for tsetse flies infected with trypanosomes, which is the ultimate target for traps to prevent the spread of African sleeping sickness. There were no changes in single-sensillum electrophysiology, meaning there was no change in neuronal response. However, there were significant behavioral changes observed in mating.
When paired together, uninfected virgin female G. morsitans mated with infected and uninfected males at the same frequency. However, uninfected virgin male G. morsitans mated with uninfected females one hundred percent of the time over infected females, suggesting that there may be a compound that lowers the sexual receptivity of infected females and acts as a repellent against G. morsitans males. The group hopes to further study twenty-one compounds that were identified in GC-MS as specific to infected flies. “I’m interested to study if this compound is produced as a defense against the parasites,” Ebrahim said.“We don’t know if those compounds were produced by the tsetse fly in response to an infection, or maybe they were produced by the parasites themselves,” Weiss said.
Harnessing the power of pheromones
The results of the study suggest that MPO acts specifically as an attractant, aphrodisiac, and arrestant on G. morsitans males to activate circuits that mediate olfactory attraction, sexual desire, and the halting of movement, respectively. The usage of MPO in traps holds great promise from both an environmental and economic perspective. “Compounds from the fly itself […] will be less toxic if we want to use it in the field compared to other compounds like DEET,” Ebrahim explained. These natural compounds are also much less expensive than DEET, which makes implementation of tsetse fly control more realistic in developing countries.
In the future, the researchers hope to test MPO in Kenya with collaborators, who are currently using tsetse fly host odors such as cow urine as attractants. “If we combine MPO with natural host odors, it might increase the efficiency of control for a trap,” Ebrahim said. “A more specific odor might attract more flies and reduce the number of cases of infection by trypanosomes.”
However, there are challenges validating field work with lab work, since the flies in the experiment are different from those found in a typical African savanna. In addition, in the real world, there are many fluctuating and uncertain factors, according to Ebrahim. Despite these challenges, the group remains undeterred, and their passion stays vibrant. “You will have to be optimistic and creative for the biggest experiments you design,” Ebrahim said.