The Story of Depressed Single Drosophila Males

Hearing our friend sobbing out their singlehood frustration over a beer bottle is a common thing amongst us. But surprisingly, this is also social behavior amongst fruit flies too! Research says that sexually deprived Drosophila males drink four times more alcohol than mated males.

TQR drosophila image_Soumi

While working with Drosophila, a group of scientists at the University of California, San Francisco got this bizarre idea of testing whether there is a molecular mechanism in the brain related to sexual rejection which can lead to depression. So they carried out a whole experiment showing that sexually deprived males develop more affinity towards alcohol due to a depression in the level of neuropeptide F (NPF) in their brain. It was seen that alcohol acted as a reward to stimulate the flies’ brains to balance sexual deprivation and rejection. A reward is a stimulus delivered in the form of neural structures to alter a behavior required for species survival, social interaction, and food consumption. It was observed that flies exhibited addiction-like behaviors like getting intoxicated with alcohol and consuming more food containing ethanol. Scientists tried to link this observation with NPF-NPF system activity.

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The experiment was conducted using two cohorts of male flies. One was the rejected-isolated males who were exposed to one hour sessions of sexual rejection by mated females, 3 times a day for 4 days. This continuous exposure to rejection of their courtship behavior affects their future courtship behavior too, also towards virgin females. They seem to lose interest and show much-suppressed courtship behavior in the future. In the other set, they took mated-grouped male flies and let them mate with many virgin female flies for 6-hours sessions for 4 days (male: female ratio- 1:5). Then they tested the preference of the two groups of male flies towards food with or without 15% ethanol supplementation. The results portrayed that the rejected-isolated males had consistent higher preference for food with ethanol supplement. In the absence of ethanol, there was no preference for food.

But these two groups of males also differed in a few more aspects along with sexual deprivation, the two groups experienced certain housing conditions-individual versus group housing. The sex-deprived group experienced social rejection and also got exposed to chemosensory cues found on mated females. So the scientists needed to eliminate all these possibilities and pinpoint out the exact reason for the affinity of rejected males towards ethanol. First, they compared males differing in a sexual experience but not in housing conditions. Virgin and mated males were both group-housed -they were kept in the same vial so that the rejected males don’t experience isolation. The virgin male flies still showed higher ethanol preference than the mated flies but not as much as the virgin isolated male flies. Hence isolation could not be considered as the major explanation for higher ethanol preference. The social experience of rejection from mated female flies also had a possibility of inducing this specific kind of behavior into the male flies. To test this out, the male flies were individually exposed to decapitated(headless) virgin females. Thus this group of males faced neither rejection nor copulation but still showed a higher affinity towards alcohol than the mated ones. This proved that the social experience of rejection did not affect this behavior of inclination towards ethanol supplemented food.

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Now, an obvious question that pops up is why do the female mated flies reject further mating. In Drosophila melanogaster females have some post-mating responses (PMR) which include reduction of sexual receptivity and increase in oviposition. This change in the mating behavior of the female flies has a contribution from the males as they inject sex peptide into virgin females during copulation through the seminal fluid. The sex peptide is a 36-amino-acid long peptide that stimulates PMR and its overexpression in the females reduces sexual receptivity and stimulates oviposition. If a male fails to inject the female with sex peptide during copulation, the female doesn’t develop PMR and has no reduction of sexual receptivity. There is another interesting molecule, cis-vaccenyl acetate (cVA) which is a male-specific lipid molecule synthesized in the ejaculatory bulb of the male. This molecule has several functions in intraspecies communication in Drosophila melanogaster. This unique lipid although enhances female copulation receptivity but inhibits the courtship of a male with a mated female as the cVA repels away the males from the mated females. And this explains the behavior of females of rejecting copulation 1week just after their previous mating. The cVA also gets transferred from the male to the female during copulation. It was also found out that cVA has an anti-aphrodisiac effect; male extract or synthetic cVA inhibited the vibration of the male wing which is an important courtship behavior of the males. It also reduced, in general, most of the courtship activities of males towards the mated females and decreased copulation success.

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Multiple functions of cis-vaccenyl acetate. A male-specific lipid (Z)-11-octadecen-1-yl acetate, so-called cis-vaccenyl acetate (cVA) has multiple functions in social communication. From distance, cVA works as an aggregation pheromone, attracting other flies. In close proximity, for a male, presence of cVA on the other male promotes male–male aggression behavior, while absence of cVA indicates that the nearby fly is a female and stimulates courtship. Meanwhile, for a female, presence of cVA on the courting male enhances copulation receptivity. Presence of little amount of cVA on the mated female, transferred from the male during copulation, works as an anti-aphrodisiac pheromone, decreasing sexual motivation of a male

So it had to be verified whether there was any role of the chemosensory cue cVA, for this specific behavior. Some of the rejected-isolated groups of males were kept with mated females and some were kept with virgin decapitated females. So some males got exposed to the cVA (mated females) and some were not (virgin decapitated females). But the results didn’t show any difference in the behavior between the two differently trained males. Finally, all the outcomes implied that sexual deprivation mainly contributed to the ethanol preference, not any other factors! Again the effects of sexual deprivation could be reversed by copulation.

Now looking into the neural part of this behavior, it was investigated that neuropeptide F is the mediator of the sexual experience and regulated ethanol sensitivity in Drosophila. They tested the NPF transcript level in the brains of the differently trained Drosophila males and found out that the rejected-isolated males showed the lowest transcript levels of NPF, the virgin-grouped males showed higher levels, and the mated males showed the highest transcript levels of NPF in the brains. The inverse correlation between NPF levels and ethanol preference was also tested and it was seen that the ethanol preference got reduced while NPF levels increased in the mated males by the manipulation, but this did not happen in virgin males. All these experiments and results summarized the relation of NPF, ethanol preference, and sexual activity. Interestingly, mammals also have a neuropeptide, called neuropeptide Y which has similar mediatory functions in different regions of the brain as the Drosophila. NPY’s roles include sleep regulation, feeding habits, anxiety, stress, sexual motivation, and ethanol preference. If NPY is injected into the nucleus accumbens (tested in rats) it gives a rewarding effect and counters the drug withdrawal and depression. So it is quite evident that a faint line of correlation may be drawn between the effects NPF and NPY to understand the behavior of humans too.

This research work and paper tried to explain that ethanol intoxication and mating are rewarding experiences for male flies. They also confirmed NPF as the main molecular transducer between social experience and drug reward and showed relatable results with the mode of regulation of mammalian NPY too.

By Soumi Bhattacharyya, Department of Biological Sciences, IISER Kolkata.



About the author:

Soumi pic

I am Soumi Bhattacharyya from IISER Kolkata. I’m interested in evolutionary and behavioural biology and also synthetic biology and have already worked in the behavioural biology lab, working upon Drosophila and hope for more work opportunities in synthetic biology field to explore my interests more. In my free time, I spend time in music and play the piano.

This is Soumi’s first blog for TQR.

Author: Soumi Bhattacharyya

Hi! I am Soumi Bhattacharyya, currently a 3rd-year BS-MS undergraduate at IISER Kolkata majoring in Biology. I’m highly enthusiastic about evolutionary and behavioural biology and am exploring the world of synthetic biology through iGEM. Interning in an EB Lab working on fruitflies I've grown to become a huge fan of Drosophila and hope for more work opportunities in the synthetic biology field to explore my interests more. In my leisure, I indulge in music and play the piano.

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