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Alcohol potentiates a pheromone signal in flies

  1. Annie Park  Is a corresponding author
  2. Tracy Tran
  3. Elizabeth A Scheuermann
  4. Dean P Smith
  5. Nigel S Atkinson  Is a corresponding author
  1. Department of Neuroscience and Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, United States
  2. Department of Pharmacology and Neuroscience, University of Texas Southwestern Medical Center, United States
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Cite this article as: eLife 2020;9:e59853 doi: 10.7554/eLife.59853

Abstract

For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. In this study, we find that when male flies inhabit ethanol-containing food substrates they become more aggressive. We identify a possible mechanism for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, we observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors.

Introduction

Conflict that results in aggression occurs across the animal kingdom. Aggression in Drosophila melanogaster is a well-documented behavior; studies have identified several aggression-regulating pheromones, circuits, and genes (Wang and Anderson, 2010; Vrontou et al., 2006; Nilsen et al., 2004; Asahina et al., 2014; Dow and von Schilcher, 1975). The most well-studied pheromone, 11-cis-Vaccenyl acetate (cVa) is produced by males and has been shown to increase aggression in male flies (Wang and Anderson, 2010). Most studies of cVa use this pheromone in isolation by adding it to a behavioral arena or painting flies with it. However, in the wild, flies will encounter cVa when they aggregate on fermenting fruits where they will experience cVa in combination with volatile compounds produced by fermentation (Zhu et al., 2003; Keesey et al., 2016). Despite the ecological complexity of the fruit fly niche, little is understood about how ethologically relevant combinations of odors influence the underlying neurobiology of behavior.

Ethanol is one of the products of fermentation and fruit flies in particular are attracted to alcohol-containing fruits (<7% ethanol by volume) (McKenzie and McKechnie, 1979). Remarkably, there is no identified canonical ethanol olfactory receptor despite the fact that a wide range of ethanol-related behaviors have been identified in flies (reviewed in Park et al., 2017). Kim et al., 1998 identified an odorant-binding protein known as LUSH that mediates aversion to high concentrations of alcohol. LUSH was the first protein shown to directly bind ethanol (Kruse et al., 2003). In the fly antennae, the only neurons known to respond to cVa are also those that express LUSH (Xu et al., 2005). However, these neurons do not show any electrophysiological responses to ethanol alone (Figure 1a and Kim et al., 1998). We hypothesized that these neurons could respond to the combination of ethanol and cVa. Here, we ask if ethanol potentiates the cVa signal to enhance inter-male aggression.

Alcohol odor increases aggression in male flies.

(a) Traces of T1 sensilla recordings with a 300 ms exposure to air or vapor from 30% ethanol. (b) Hemolymph ethanol concentration (mg/mL) in flies in aggression arenas for 30 mins show no significant increases except with 20% ethanol (one-way ANOVA with Dunnett’s p<0.0001, n = 11–12). (c) Time spent fighting on ethanol-containing food (Control vs. 5% p=0.038 Kruskal-Wallis test with Dunn’s correction, n = 10–20). (d) Number of fights on ethanol-containing food (Control vs. 5% p=0.0012, statistical tests as in c). (e) Lunges on ethanol-containing food (Control vs. 5% p=0.0225, statistical tests as in c). (f) Latency to lunge (p=0.009, Log-rank Mantel-Cox with Bonferroni correction). (g) Cumulative latency of flies that lunged during the test (Control vs. 10% p=0.009, Log-rank Mantel-Cox with Bonferroni correction). (h) Locomotion as measured by line crossings during the test (Control vs. 20% p=0.0048, statistical tests as in 1 c). p<0.05 *; p<0.01 **; p<0.001 ***. Error bars denote SEM.

Using fly aggression arenas, we added ethanol to fly food in amounts of 5%, 10%, and 20% by volume (Mundiyanapurath et al., 2006). To determine the fly’s level of aggression we video recorded two wild-type (Canton-S) males in the arena for 30 min and scored fighting events manually. We catalogued fencing, shoving, boxing, tussling, and lunging as aggressive behaviors (described in Chen et al., 2002 as offensive actions). We measured hemolymph or ‘blood’ ethanol concentration (BEC), after the flies were in the arena for 30 min (Figure 1b), to determine if the flies were receiving an intoxicating dose of ethanol. Neither the 5% nor the 10% ethanol-containing foods caused any detectable rise in ethanol in the flies.

Males that fought on 5% ethanol-containing food exhibited an increase in time spent fighting, number of fights they engaged in, and number of lunges executed (Figure 1c,d and e). Although, males on 10% ethanol food exhibited an increase in the proportion that lunged and decreased latency to lunge, they did not show an increase in total number of lunges executed compared to control animals (Figure 1e,f & g). These data demonstrate that when flies occupy food patches with ethologically relevant concentrations of ethanol, they display elevated levels of aggression that are not due to increased locomotion (Figure 1h). One possible explanation for the reduction of aggression in flies that fought on 20% ethanol compared to those on 5% ethanol can be explained by Wang and Anderson, 2010 observation that males respond to cVa in a dose-dependent manner with low concentrations of cVa promoting aggregation and aggression, whereas high concentrations of cVa cause dispersion (Wang and Anderson, 2010). Twenty-percent ethanol may have potentiated the cVa signal so that it mimics a high cVa concentration causing males to disperse and spend less time fighting.

We sought to determine if ethanol influences the neuronal responses to cVa pheromone by monitoring activity of the T1 cVa sensing neurons in the presence of mixtures of these two odorants. We recorded from cVa-responsive neurons while exposing them to 1% cVa and 5% or 30% ethanol (cVA concentration chosen for comparison to prior literature; ethanol concentrations chosen because they were commonly used in other experiments; Figure 2a). Although ethanol does not acutely activate these neurons, we found that it substantially enhanced their cVa responses (Figure 2b). We found that 30% ethanol substantially increased cVa-evoked activity (Δ Spikes), while 5% ethanol increased activity to a lesser extent (Figure 2c,e,f and g). One possible explanation for why we did not observe an increase in Δ Spikes with cVa and 5% ethanol even though 5% ethanol increased aggression is because the duration of exposure was considerably shorter (1 min) compared to the behavioral assay (30 min), which may not have allowed for sufficient accumulation of alcohol. These T1 neurons have an unstimulated spontaneous firing rate of approximately 1 Hz and increase their firing in response to cVa (Xu et al., 2005). Spontaneous responses consistently increased when we applied 5% or 30% ethanol (Figure 2d). Finally, the time constant to deactivation increased with 30% ethanol application indicating that neurons had greater sustained activity following cVa treatment (Figure 2h). These data are consistent with the notion that ethanol increases inter-male aggression by potentiating responses to cVa.

Alcohol odor potentiates the response to cVa.

(a) Experimental timeline and diagram of recording site on fly antenna. (b) Traces of cVa-sensing T1 neurons. The red bar denotes 300 ms cVa exposure. (c) Δ Spikes calculated as cVa-induced activity (1 s during and after cVa) – spontaneous activity (paired two-tailed t-test, p=0.002, n = 7,15,15). (d) Spontaneous activity before, during, and after ethanol exposure. Spontaneous activity calculated as the total number of spikes 10 s prior to cVa delivery/10 s (Pre- vs. Post-5% p=0.002, Pre- vs. Post-30% p<0.0001, During vs. Post-30% p=0.0152, Kruskal-Wallis test with Dunn’s correction). (e), (f), (g) Averaged spikes over time for air, 5% ethanol, and 30% ethanol, respectively. Red bar denotes 300 ms cVa exposure (h) Time constant (τ) of decay of the cVa-induced spikes (Mann-Whitney test, p=0.0043). p>0.05, n.s. (not significant); p<0.05 *; p<0.01 **; p<0.0001 ****. Error bars denote SEM.

In the wild, ethanol is almost always present with other fruit volatiles and fermentation odorants. Farnesol is an odorant present in the rinds of citrus fruits, which are known to be attractive to flies (Ronderos et al., 2014; Dweck et al., 2013). We asked if ethanol would potentiate the electrophysiological response to farnesol and attraction to farnesol. We chose farnesol because Or83c neurons are not broadly tuned and display strong activation to farnesol (similar to how cVa-sensing neurons are only tuned to cVa) and do not show a response to ethanol alone (Ronderos et al., 2014). Farnesol also conveys an entirely different behavioral response from cVa. We performed single-sensillum recordings (SSR) of the ai2 sensilla while exposing them to 30% ethanol and recorded their responses to farnesol (Figure 3a). We found that both evoked activity and spontaneous activity increased following ethanol treatment (Figure 3b-e). To determine if ethanol can augment attraction to farnesol we used two-choice olfactory trap assay (pictured Figure 3f). We used a dilution of farnesol that elicited no attraction when used alone (10−5). However, when combined with 30% ethanol the odor from a 10−5 dilution of farnesol displayed much greater attractiveness than either the odor of farnesol or ethanol alone (Figure 3g). Flies preferred the mixed farnesol and ethanol odor over farnesol alone or ethanol alone suggesting ethanol potentiates attraction to farnesol.

Ethanol increases attraction to and potentiates the neuronal response of a food related odor.

(a) Paradigm used to evaluate farnesol responses pre- and post-ethanol treatment. Responses are shown in panels b-e. (b) Δ Spikes of farnesol-induced activity in ai2 sensilla, calculated as in 2b (paired two-tailed t-test, p=0.0059, n = 9–10). (c) Spontaneous activity before, during, and after ethanol exposure, calculated as in 2 c (Pre- vs. During Ethanol p=0.017, Pre- vs. Post-Ethanol p<0.0001, Kruskal-Wallis test with Dunn’s correction). (d, e) Traces from ai2 farnesol-sensing neurons. The red bar denotes a 300 ms farnesol exposure. (f) Graphic of the two-choice olfactory trap assay used to measure relative attraction to odors. (g) Preference Indices calculated as Number of flies in Choice 1-Number of flies in Choice 2/Total Number of flies. p>0.05, n.s. (not significant); p<0.05 *; p<0.01 **; p<0.0001 ****. Error bars denote SEM.

Previous studies on behavioral responses of Drosophila to ethanol have focused on the systemic effects of ethanol. These studies demonstrated that flies, like mammals, become hyperactive with low doses of ethanol, sedate at high doses of ethanol, acquire tolerance to ethanol, display a withdrawal response, and seek ethanol despite negative consequence (reviewed in Park et al., 2017). However, ethanol responses described in this paper are fundamentally distinct in that they are most likely olfactory (non-systemic) and are ethologically important to the life of a fly. Fischer et al., 2017 demonstrated that flies are more attracted to natural mixtures of microbial by-products than the individual components of the mixture. We find evidence that ethanol potentiates two fundamentally distinct odorants and behaviors in Drosophila. First, ethanol odor increases cVa signaling, which in turn increases aggression. Second, ethanol augments farnesol signaling, resulting in increased attraction to this food odorant. Female flies are thought to prefer to consume ethanol-laden food and lay their eggs in ethanol-containing food because ethanol has caloric value, antimicrobial properties, and provides a protected niche by suppressing competition from other Drosophila species that find the ethanol to be toxic (Kacsoh et al., 2013; Park et al., 2018; Azanchi et al., 2013). When female flies accumulate on the food substrate, males eventually follow, and fight one another for a chance to mate with the female. Interestingly, male flies are known to deposit cVa directly onto food substrates and will spend more time around the marked area (Keesey et al., 2016; Mercier et al., 2018). Both the cVa pheromone and farnesol odorant are naturally encountered by flies in the wild and evoke behaviors believed to provide selective advantages. The odor of ethanol combined with a food odor could enhance the perceived value of the food as a reproductive resource, while the combination of the scent of ethanol and cVA could increase the male drive to fight for control of this resource. We documented evidence for potentiated responses to two odors, but it seems likely that ethanol could increase attraction to other food odors and perhaps other fly pheromones as well.

Materials and methods

Fly handling

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All flies were raised on corn meal malt extract food (7.6%) CH Guenther and Son Pioneer Corn Meal (Walmart, Inc), 7.6% Karo syrup (Walmart, Inc), 1.8% Brewer’s yeast (SAF, Milwaukee WI), 0.9% Gelidium agar (Mooragar, Inc, Rocklin, CA), 0.1% nipagin (Fisher Scientific, Inc) in 0.5% ethanol, 11.1% #5888 amber malt extract (Austin Homebrew, Austin, Tx) and 0.5% proprionic acid (Fisher Scientific, Inc). Solids are weight/volume and liquids are volume/volume. Flies were housed a 12:12 light:dark cycle. Flies used in aggression and courtship receptivity behavioral assays were all taken from group housed bottles as pupae and individually raised in vials. Flies used in imaging, immunohistochemistry, and qPCR were group housed.

Behavioral tests

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Aggression chambers were assembled based on description by Mundiyanapurath et al., 2006. using a fly vial cut one inch high and glued to one petri dish. The top of the chamber has two holes; one large hole is used for loading flies and one other smaller hole is in the center of the top and is used for circulation. Food wells were made by cutting 1.5 mL microfuge tube tops. Fly food was melted and pipetted into the microfuge tube tops. Ethanol was added into the fly food once the food cooled down to roughly 35°C. We added sucrose to the top of each fly food surface and a decapitated virgin female fly. Flies were loaded into the chamber by gentle aspiration and the video camera began recording 5 min after the flies were in the chamber. Aggression tests were conducted between the hours of 9 AM and 4 PM (Lights on 8 AM – 8PM). Flies tested for aggression were between 4 and 6 days old and Canton S.

Line crossing assays were performed in the aggression chambers. Flies were aspirated into the chamber in pairs and we recorded the total number of line crossings within a 5-min time period. We recorded video from the top of the chambers and drew a line bisecting the chamber. We used Canton S males between 4 and 6 days old.

Olfactory Traps were based on the protocol developed by Woodard et al., 1989 and were constructed using a 1.5-microfuge tube with a hole drilled on the cap. A yellow-tip pipette was then cut to fit in the hole so that the tops were flush against the cap of the microfuge and cut on the bottom so that flies would be able to enter. For the odorants, we cut pieces of Fisherbrand medium porosity filter paper (Hampton, NH, Catalog No. 09-801E) into 2.5 × 2 cm squares. We used Sigma-Aldrich 95% Farnesol (St. Louis, MO) diluted in Paraffin Oil. For the low concentration Farnesol we used 0.1% and for the high concentration of Farnesol we used 10%. For ethanol, we used a 5% (w/v) solution in water. We pipetted 35 µL of each odorant onto the filter paper squares and folded them up into the microfuge tubes. The paper was pushed to the bottom of the tube to prevent obstruction of the yellow tip pipettes. For the tubes with single odorants, we added the solvent in the opposite tube (e.g. Farnesol in Paraffin oil + Ethanol in water vs. Ethanol in water and Paraffin oil). For the no odorant tubes we used both paraffin oil and water. For each test, we used 20 male Canton S flies that were 4–5 days old. We aspirated the flies into the testing arenas and left them for about 12 hr overnight then placed the whole arena in −20°C kill the flies prior to counting the number of flies in each trap.

Ethanol assay kit

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We used a Megazyme Ethanol Assay Kit Cat # K-ETOH (Megazyme, Bray, CO) to measure BACs (limit of detection 0.093 mg/L). About 40 flies were treated with ethanol or air, then homogenized in ddH2O and centrifuged at 10,000 xg for 10 min. The supernatant was taken and used to measure ethanol concentrations. A negative control without fly homogenate was also used. For concentration calculations, all flies were estimated to contain 1 µL of water (calculated from previous data in Park et al., 2018).

Single sensillum recording electrophysiology

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Single Sensillum extracellular electrophysiology was conducted according to de Bruyne et al., 1999 using 3–5 days old w1118 flies. Flies were assayed under a constant stream of charcoal filtered air (36 ml/min, 22–25°C) to prevent any contamination from environmental odors. cVa was diluted in paraffin oil (1% dilution); 35 μl was applied to filter paper and inserted into a Pasteur pipette; air was passed over the filter and presented as the stimulus. We used 1% cVa because the responses it evokes is most functionally relevant, as the magnitude of response it evokes is similar to exposing a virgin female to a male fly. Signals were amplified 1000x, fed into a computer via a 16-bit analog-to-digital converter (USB-IDAC system; Syntech), and analyzed offline with AUTOSPIKE software. The low cut-off filter setting was 200 Hz and the high cut-off setting was 3 kHz. Action potentials were recorded by inserting a glass electrode in the base of a sensillum. Data analysis was performed as reported by Xu et al., 2005. Signals were recorded starting 10 s before odorant stimulation. cVa-evoked action potentials were counted by subtracting the number of spikes 1 s before cVa stimulation from the spike number 1 s after cVa stimulation (Spikes/sec). The recordings were performed from separate sensilla with a maximum of two sensilla recorded from any single fly.

Ethanol was delivered by adding it into the conical flask that feeds into the IDAC. The ethanol was made from 200 proof ethanol and diluted in ddH2O to make either 30% or 5% ethanol (w/v) and always covered with parafilm. For acute treatments, the flow rate was roughly 36 ml/min, 22–25°C at roughly 2.5 L /min.

Spontaneous Activity = Total number of spikes 10 s prior to cVa delivery/10 s and ΔSpikes = Evoked activity (1 s during and after cVa delivery) – Spontaneous Activity.

Statistical methods

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For data with multiple comparisons we used a One-way ANOVA with Dunnett’s correction for multiple comparisons. To test for normality of the data we used a Shapiro-Wilk’s test. If one of the datasets contained non-normally distributed data we used a Kruskal-Wallis test with Dunn’s correction.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

    1. Kim MS
    2. Repp A
    3. Smith DP
    (1998)
    LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster
    Genetics 150:711–721.
    1. Woodard C
    2. Huang T
    3. Sun H
    4. Helfand SL
    5. Carlson J
    (1989)
    Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota mutants
    Genetics 123:315–326.

Decision letter

  1. Michael B Eisen
    Senior and Reviewing Editor; University of California, Berkeley, United States
  2. Karla R Kaun
    Reviewer; Brown University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Park et al. demonstrate a relationship between alcohol and aggression that may have some relevance both to the ecology of flies, and, perhaps, humans. It is a creative careful study that mixes detailed behavioral assessment with electrophysiological findings to explore a novel topic. This work is an important launching point for what we expect to be many future studies that build on this initial observation.

Decision letter after peer review:

Thank you for submitting your article "Alcohol potentiates a pheromone signal in flies" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by EIC Michael Eisen acting as the Senior and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Karla R Kaun (Reviewer #1), Fred W Wolf (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Park et al. provide a short report describing the phenomenon that exposure to alcohol present in food in a small chamber with decapitated virgins increases male-male aggressive behaviors. They also found that wafting alcohol vapor over the antenna of a fly prior to exposure to cVa increases activity of T1 cVa sensing neurons. Alcohol vapor also increased activity of ai2 farnesol-sensitive sensilla, and when presented in combination with farnesol in a two-choice odor test, flies preferred the alcohol+farnesol combination over farnesol or alcohol alone. The authors conclude that alcohol potentiates cVa signaling, and allude to this being the cause of increased aggression. They also conclude that alcohol increases attractiveness of a food-odor which would be relevant to flies because a food substrate may where flies can find mates. The results are interesting and of potential ecological relevance. The data is tight and presented in a transparent matter which provides confidence in the rigor of the study.

However, as written, the paper is focused on the ecological consequences of low dose ethanol on aggression, and while the data are very interesting and are an important contribution to the literature, the authors make conclusions about the ecological significance of their study that are not fully established here. With minor revisions to the text, we believe the study with the current data as is, will provide a strong addition to our understanding of how ecological concentrations of ethanol affect the physiology of olfactory response and aggressive behavior.

We recommend the following changes:

1) Reframing the manuscript so that speculation about the role of alcohol on aggression in the wild emerges in the Discussion.

2) Incorporating a more comprehensive perspective on how ethanol, or other food odors, can alter behavior and preference in Drosophila.

3) Add information from the literature of the role of aggression in the ecological niche (ie fermenting fruit).

4) Avoid conflating low and high dose ethanol experiments as they may induce different physiological and thus behavioral responses.

Comments and questions that emerged in review:

1) The data support the conclusions of the study. Although a direct connection between cVA and aggression wasn't presented in the current study, it has been previously published, so it makes sense the authors would conclude that alcohol increases aggression via altering cVA sensitivity. The data showing it also enhances sensitivity to food odors provides an important ecological context and suggests that alcohol may play a greater role in altering sensitivity to odors in general.

A question that arises from this, is whether this response is unique to alcohol or if it is the result of a combination or series of odors presented. Would a similar effect arise if the flies were presented with a different appetitive food odor (ethyl acetate for example) instead of alcohol? If the authors found similar results, then the effect they characterize here might be a much more general mechanism through which flies find and complete for appetitive resources (food, mates, etc). Although I think that this would be a relevant experiment to add and would strengthen the merit and general interest of the paper, the authors should not be obligated to perform this experiment since the paper stands on its own merit as an important addition to the literature. However, if the authors chose not to add this experiment, they may want to consider this possibility in the Discussion.

2) There is a relatively broad literature of the effects of natural food odors on behavioral choice in Drosophila, but the authors don't incorporate this into their paper. I recommend incorporating a more comprehensive perspective on how ethanol, or other food odors, can alter behavior and preference in Drosophila. If the authors agree, I think this would strengthen their argument substantially. Note that since some of these papers are older and some in ecologically-oriented journals, they are likely not in PubMed.

Similarly, is anything known about aggression in the ecological niche (fermenting fruit)? Zhu, Park and Baker, 2003 does not address this. Can cVA be detected in the occupied niche? How much does this affect the interpretation of the results.

Finally, in the realm of context, how do the results tie in with what is known about the broader literature on alcohol's neuronal effects. For instance, are these findings consistent with alcohol's known stimulant and depressant activities? e.g. Smoothy, R., Berry, M.S. Time course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice. Psychopharmacology 85, 57-61 (1985). https://doi.org/10.1007/BF00427322 Presumably the field has come a long way since 1985. What is currently known? Please clarify the novel insights from the present study.

3) The manuscript presupposes that LUSH is the connection between alcohol and aggression: why not test LUSH mutants? Does low concentration alcohol still promote aggression when the flies are unable to smell (a simple and classic experiment is to surgically remove the antennae)?

4) Many experiments are done at alcohol concentrations well above what flies encounter in nature. Outside of Figure 1, with interesting effects of low alcohol concentration on fighting, there are two data points in 2D that drive an effect at the low concentration. It's more like there is one paper in Figure 1, and a different one in Figures 2 and 3. It's not clear that there is any connection between the parts of the paper. This disconnect should be addressed in a revised manuscript.

5) Please address the following questions about In Figure 2: Why are cVA and alcohol presented in serial? In the wild, they would be encountered simultaneously? Why do the authors use an even higher concentration of alcohol instead of a longer presentation of 5% alcohol? Could you comment on why it appears that alcohol is increasing baseline activity of the recorded neurons instead of spike properties?

6) Figure 3 begs the question about the generalizability of alcohol exposure on all olfactory tuning. If it is general, then the arguments for niche specific amplification of food and fighting signals falls apart.

https://doi.org/10.7554/eLife.59853.sa1

Author response

Park et al. provide a short report describing the phenomenon that exposure to alcohol present in food in a small chamber with decapitated virgins increases male-male aggressive behaviors. They also found that wafting alcohol vapor over the antenna of a fly prior to exposure to cVA increases activity of T1 cVA sensing neurons. Alcohol vapor also increased activity of ai2 farnesol-sensitive sensilla, and when presented in combination with farnesol in a two-choice odor test, flies preferred the alcohol+farnesol combination over farnesol or alcohol alone. The authors conclude that alcohol potentiates cVA signaling, and allude to this being the cause of increased aggression. They also conclude that alcohol increases attractiveness of a food-odor which would be relevant to flies because a food substrate may where flies can find mates. The results are interesting and of potential ecological relevance. The data is tight and presented in a transparent matter which provides confidence in the rigor of the study.

However, as written, the paper is focused on the ecological consequences of low dose ethanol on aggression, and while the data are very interesting and are an important contribution to the literature, the authors make conclusions about the ecological significance of their study that are not fully established here. With minor revisions to the text, we believe the study with the current data as is, will provide a strong addition to our understanding of how ecological concentrations of ethanol affect the physiology of olfactory response and aggressive behavior.

We recommend the following changes:

1) Reframing the manuscript so that speculation about the role of alcohol on aggression in the wild emerges in the Discussion.

The reviewers indicate that stated ecological consequences and conclusions are speculative and should be restricted to the Discussion. We agree that in this paper there are no experiments explicitly testing these behaviors in the wild. However, the mention of these points in the Abstract and introductory text meant to provide the intellectual motivation for performing these studies and were not originally meant to be conclusions. We have tried to present these motivating ideas in a less muddled way. To address the concerns of the reviewers, we reworded the Abstract and the last paragraph of the paper.

2) Incorporating a more comprehensive perspective on how ethanol, or other food odors, can alter behavior and preference in Drosophila.

In the last paragraph of the manuscript we summarize what is known about alcohol’s systemic effects on fly behavior. We also describe the interaction between cVa, ethanol, and food to acknowledge the possibility that these effects could extend to other food odors and pheromones.

3) Add information from the literature of the role of aggression in the ecological niche (ie fermenting fruit).

Unfortunately, we are not aware of any studies that demonstrate the role of aggression in a natural ecological niche. The one study we do know of is Soto-Yéber, L., Soto-Ortiz, J., Godoy, P., and Godoy-Herrera, R. (2018). The behavior of adult Drosophila in the wild. PloS one, 13(12), e0209917. Soto-Yéber et al. examined how flies behave on a few different niches and noted they did not observe aggression on the grape leaves or grains of grape. The researchers did not record their observations on video and based on the images there are too many flies to keep track of (Figures 2-3). However, the absence of evidence is not the evidence of absence, and the authors note that there are very few studies that examine fly behavior in a natural setting.

4) Avoid conflating low and high dose ethanol experiments as they may induce different physiological and thus behavioral responses.

We have specified low and high alcohol more carefully.

Comments and questions that emerged in review:

1) The data support the conclusions of the study. Although a direct connection between cVA and aggression wasn't presented in the current study, it has been previously published, so it makes sense the authors would conclude that alcohol increases aggression via altering cVA sensitivity. The data showing it also enhances sensitivity to food odors provides an important ecological context and suggests that alcohol may play a greater role in altering sensitivity to odors in general.

A question that arises from this, is whether this response is unique to alcohol or if it is the result of a combination or series of odors presented. Would a similar effect arise if the flies were presented with a different appetitive food odor (ethyl acetate for example) instead of alcohol? If the authors found similar results, then the effect they characterize here might be a much more general mechanism through which flies find and complete for appetitive resources (food, mates, etc). Although I think that this would be a relevant experiment to add and would strengthen the merit and general interest of the paper, the authors should not be obligated to perform this experiment since the paper stands on its own merit as an important addition to the literature. However, if the authors chose not to add this experiment, they may want to consider this possibility in the Discussion.

There is a possibility that alcohol acts as a general positive modulator of other odorants encountered by flies. However, if true this would not weaken the importance of this report. Flies are innately attracted to alcohol at low to moderate concentrations. When flies are on the fermenting food patch, they will encounter other odors that could be potentiated by alcohol and accelerate relevant behaviors such as feeding, fighting, and egg-laying.

One experiment we elected to omit from this paper was a courtship assay in which we measured courtship behavior (unilateral wing extensions) of male flies on ethanol laden food. Male courtship and copulation behavior are regulated by female pheromones methyl laurate (ML), methyl myristate (MM), and methyl palmitate (MP) Dweck et al., 2015. However, when we measured male courtship on ethanol food, there was no change in courtship or copulation behavior as measured by Unilateral Wing Extensions (UWEs), suggesting that 5% ethanol does not potentiate all odors and behaviors (Author response image 1).

We modified the last paragraph of the paper to account for the idea this response might generalize to other food odors.

Author response image 1

2) There is a relatively broad literature of the effects of natural food odors on behavioral choice in Drosophila, but the authors don't incorporate this into their paper. I recommend incorporating a more comprehensive perspective on how ethanol, or other food odors, can alter behavior and preference in Drosophila. If the authors agree, I think this would strengthen their argument substantially. Note that since some of these papers are older and some in ecologically-oriented journals, they are likely not in PubMed.

It is difficult to review literature in such a short report format. However, we have modified the last paragraph to discuss this idea.

Similarly, is anything known about aggression in the ecological niche (fermenting fruit)? Zhu, Park and Baker, 2003 does not address this. Can cVA be detected in the occupied niche? How much does this affect the interpretation of the results.

There is not as much known about aggression in an ecological niche (See point 3 above). But there is some literature that describes the natural deposition of cVa in the environment. In Keesey et al., 2016, the researchers found that deposits made on blueberries include ML, MM, MP, cVa and other cuticular hydrocarbons using GC-MS. In another paper by Mercier et al., 2018. Researchers found that male flies will deposit cVa in a particular spot on the arena and spend more time around the deposit they made (<16 mins). We have included this information in the manuscript. However, there is no literature to our knowledge about detecting cVa deposits on fermented fruit patches specifically.

Finally, in the realm of context, how do the results tie in with what is known about the broader literature on alcohol's neuronal effects. For instance, are these findings consistent with alcohol's known stimulant and depressant activities? e.g. Smoothy, R., Berry, M.S. Time course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice. Psychopharmacology 85, 57-61 (1985). https://doi.org/10.1007/BF00427322 Presumably the field has come a long way since 1985. What is currently known? Please clarify the novel insights from the present study.

We believe the reviewers are suggesting that alcohol odor could also increase locomotor activity in flies, which could inflate the number of aggressive encounters. Stimulatory effects of low doses of alcohol have been reported in flies (Wolf et al., 2002). To address this, we added a line-crossing experiment to the paper, which provides a measure of locomotor activity (Figure 1H). We performed the line crossing assays within the aggression chambers with alcohol added into the food and found no correlation between locomotor activity and aggression. Flies exposed to 5% alcohol food did not increase their locomotor activity and they also did not have an increase in BAC, but they did show an increase in multiple aggressive behaviors. We did see a very slight decrease in locomotion in flies on 20% alcohol food. However, at this concentration aggressive behaviors are not changed. Thus, it appears that our alcohol treatments are not increasing aggression simply by acting as stimulants.

3) The manuscript presupposes that LUSH is the connection between alcohol and aggression: why not test LUSH mutants? Does low concentration alcohol still promote aggression when the flies are unable to smell (a simple and classic experiment is to surgically remove the antennae)?

We were dissuaded from doing this experiment because the cVa pathway itself is necessary for aggression (Wang et al., 2010). Although we did not test LUSH mutants, we did attempt to assay Or67d mutants and saw they were not aggressive (Author response image 2 shows lunges of Or67d receptor mutants with CantonS controls). Or67d is the receptor that hetero-dimerizes with ORCO and SNMP, which binds to ligand-bound LUSH. Although there is no increase in aggression following exposure to 5% alcohol food in Or67d -/- flies, the flies are not aggressive in the first place and so this cannot be interpreted to mean that the cVa sensing pathway is necessary for alcohol-induced aggression. It was this Or67d receptor mutant experiment that made the LUSH experiment appear uninformative.

One approach to confirm that LUSH mediates alcohol-induced aggression would be to mutate the alcohol-binding sites of LUSH (identified in Kruse et al., 2003). Ideally, the mutagenesis would only affect alcohol binding and leave cVa binding unimpaired. However, we believe this is outside the scope of this short reports paper and is not of critical importance for this study.

Author response image 2

4) Many experiments are done at alcohol concentrations well above what flies encounter in nature. Outside of Figure 1, with interesting effects of low alcohol concentration on fighting, there are two data points in 2D that drive an effect at the low concentration. It's more like there is one paper in Figure 1, and a different one in Figures 2 and 3. It's not clear that there is any connection between the parts of the paper. This disconnect should be addressed in a revised manuscript.

Figure 1 and 2 both share a low concentration of alcohol (5%) that can occur in nature. However, as detailed in the manuscript the concentration of alcohol in the air in Figure 1 and Figures 2 and 3 is probably never identical. The reason for this is that in the behavioral assays in Figure 1 the alcohol vapor is generated by passive evaporation from the food dish (% alcohol refers to concentration in the food) and was never indended to exactly equal the alcohol in the ephys experiments in which the alcohol vapor was generated using a bubbler (in the ephys, the % alcohol refers to the concentration in the solution through which air is bubbled). While this was described in the original document, we have made a small adjustment that we think helps make this clearer. We think that this addresses the apparent disconnect between Figure 1 and Figures 2 and 3.

In Figure 2D post 5% Alc the two largest value data points do not meet the formal definition of outliers. Furthermore, even if these two data points are removed, the pre- and post-treatment statistical significance is still maintained. Therefore, they are not driving the effect (raw data in the Source data 1).

However, in re-examining these data we realized that the largest value in Figure 2D post 30% Alc is actually a statistical outlier. Thank you for helping us find this. Removing it does not eliminate the significant difference between post-30% and pre or during treatment. Therefore, we have removed this outlier. This also has the beneficial effect of making the relationship between all of the datapoints easier to see in the graph.

5) Please address the following questions about In Figure 2: Why are cVA and alcohol presented in serial? In the wild, they would be encountered simultaneously? Why do the authors use an even higher concentration of alcohol instead of a longer presentation of 5% alcohol? Could you comment on why it appears that alcohol is increasing baseline activity of the recorded neurons instead of spike properties?

Serial presentation occurred because the rig was designed for serial presentation. However, we do not think that this limitation invalidates the work because on approach to a food patch alcohol would more likely be encountered before cVa, as cVa has a relatively low range of detection (about 2.5 mm from Figure 2F in Mercier et al., 2018).

Examining the responses after a longer odor presentation might have been a good idea and might have revealed other aspects of the response. At the time, the value of a longer exposure was not apparent to us. However, it is not just the baseline that is altered, as spike duration changes as well.

Thirty percent was chosen because other work on the systemic effects of alcohol had used 30%. While the other work ended up being irrelevant to this paper we had completed a body of work using 30% ethanol. Having said this, one should keep in mind that 5% and 30% bubbler ethanol in the ephys experiments should be viewed as low and high concentrations when compared to the behavioral experiments that used 5% and 20% ethanol in the food. It is unlikely that identical concentrations of ethanol in a bubbler and in the food result in the exact same concentration in the air. While this lack of a one-to-one relationship is not what one would want in a perfect world, we still think that we have documented behavioral responses and ephys responses to low concentration alcohol that support a single unambiguous interpretation.

We do not have any data that allow us to do anything other than speculate on the origins of the shift in activity with alcohol. We might imagine that mechanistically alcohol is either (1) binding to LUSH and stabilizing it in its active confirmation causing it to activate the Or67d/Orco complex in the absence of cVa OR (2) ethanol could also be directly acting on Orco as an allosteric modulator to increase its spontaneous openings. Because the farnesol-sensing neurons do not have an identified OBP, it is difficult to say if (1) is true for all alcohol-sensitive OSNs. However, both farnesol and cVa-sensing neurons (and most OSNs) have Orco which would mean that the behavioral responses to olfactory alcohol are broadly tuned. Examination of these hypotheses require future experiments to elucidate how alcohol is potentiating these pathways.

6) Figure 3 begs the question about the generalizability of alcohol exposure on all olfactory tuning. If it is general, then the arguments for niche specific amplification of food and fighting signals falls apart.

Perhaps, alcohol does augment all odor detection. But we disagree with the premise that such a phenomenon would negate our niche-specific arguments. Even if olfaction was enhanced across the board it could still result in niche-specific changes in behavior such as increased attraction to reproductive resources and increased aggressive responses to other males. Also see 1) in which we find that courtship behavior in males on 5% ethanol food does not change.

https://doi.org/10.7554/eLife.59853.sa2

Article and author information

Author details

  1. Annie Park

    Department of Neuroscience and Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, United States
    Present address
    Centre for Neural Circuits and Behaviour, The University of Oxford, Oxford, United Kingdom
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    annie.park@dpag.ox.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5618-2286
  2. Tracy Tran

    Department of Neuroscience and Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, United States
    Contribution
    Data curation, Formal analysis, Validation, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Elizabeth A Scheuermann

    Department of Pharmacology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Data curation, Formal analysis, Validation
    Competing interests
    No competing interests declared
  4. Dean P Smith

    Department of Pharmacology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Resources, Formal analysis, Supervision, Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4271-0436
  5. Nigel S Atkinson

    Department of Neuroscience and Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, United States
    Contribution
    Conceptualization, Resources, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    nigela@utexas.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8963-7478

Funding

National Institute on Alcohol Abuse and Alcoholism (2R01AA01803706A1)

  • Nigel S Atkinson

National Institute on Alcohol Abuse and Alcoholism (F31AA027160)

  • Annie Park

National Institute on Alcohol Abuse and Alcoholism (T32AA07471)

  • Annie Park

National Institutes of Health (R01DC015230)

  • Dean P Smith

National Institutes of Health (5T32GM008203)

  • Elizabeth A Scheuermann

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Senior and Reviewing Editor

  1. Michael B Eisen, University of California, Berkeley, United States

Reviewer

  1. Karla R Kaun, Brown University, United States

Publication history

  1. Received: June 10, 2020
  2. Accepted: November 1, 2020
  3. Accepted Manuscript published: November 3, 2020 (version 1)
  4. Version of Record published: November 17, 2020 (version 2)

Copyright

© 2020, Park et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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