Plants and fungi often produce toxic secondary metabolites that limit their consumption [], but herbivores and fungivores that evolve resistance gain access to these resources and can also gain protection against nonresistant predators and parasites []. Given that Drosophila melanogaster fruit fly larvae consume yeasts growing on rotting fruit and have evolved resistance to fermentation products [], we decided to test whether alcohol protects flies from one of their common natural parasites, endoparasitoid wasps []. Here, we show that exposure to ethanol reduces wasp oviposition into fruit fly larvae. Furthermore, if infected, ethanol consumption by fruit fly larvae causes increased death of wasp larvae growing in the hemocoel and increased fly survival without need of the stereotypical antiwasp immune response. This multifaceted protection afforded to fly larvae by ethanol is significantly more effective against a generalist wasp than a wasp that specializes on D. melanogaster. Finally, fly larvae seek out ethanol-containing food when infected, indicating that they use alcohol as an antiwasp medicine. Although the high resistance of D. melanogaster may make it uniquely suited to exploit curative properties of alcohol, it is possible that alcohol consumption may have similar protective effects in other organisms.

The raison d'ětre of secondary plant substances; these odd chemicals arose as a means of protecting plants from insects and now guide insects to food.

Results and Discussion

14 Gibson J.B.

May T.W.

Wilks A.V. Genetic variation at the alcohol dehydrogenase locus in Drosophila melanogaster in relation to environmental variation: Ethanol levels in breeding sites and allozyme frequencies. 15 McKechnie S.W.

Morgan P. Alcohol dehydrogenase polymorphism of Drosophila melanogaster: Aspects of alcohol and temperature variation in the larval environment. 16 Chawla S.S.

Perron J.M.

Radoucothomas C. Effects of ingested ethanol on adult Drosophila melanogaster (Diptera: Drosophilidae). 17 Geer B.W.

Langevin M.L.

McKechnie S.W. Dietary ethanol and lipid synthesis in Drosophila melanogaster. 18 Parsons P.A.

Stanley S.M.

Spence G.E. Environmental ethanol at low concentrations: Longevity and development in the sibling species Drosophila melanogaster and D. simulans. 18 Parsons P.A.

Stanley S.M.

Spence G.E. Environmental ethanol at low concentrations: Longevity and development in the sibling species Drosophila melanogaster and D. simulans. 19 McKechnie S.W.

Geer B.W. Regulation of alcohol dehydrogenase in Drosophila melanogaster by dietary alcohol and carbohydrate. 20 McKenzie J.A.

Parsons P.A. Alcohol tolerance: An ecological parameter in the relative success of Drosophila melanogaster and Drosophila simulans. 3 Price P.W.

Bouton C.E.

Gross P.

McPheron B.A.

Thompson J.N.

Weis A.E. Interactions among three trophic levels: Influence of plants on interactions between insect herbivores and natural enemies. 7 Ode P.J. Plant chemistry and natural enemy fitness: effects on herbivore and natural enemy interactions. 21 Barbosa P. Natural enemies and herbivore-plant interactions: Influence of plant allelochemicals and host specificity. 22 Flanders S.E. Abortive development in parasitic hymenonoptera, induced by the food-plant of the insect hosts. 23 Owen R.E. Utilization and tolerance of ethanol, acetic acid and acetaldehyde vapor by Asobara persimilis, a parasitoid of Drosophila. 24 Bouletreau M.

David J.R. Sexually dimorphic response to host habitat toxicity in Drosophila parasitic wasps. 25 Carton Y. Attraction de Cothonaspis sp (Hymenoptera: Cynipidae) par le milieu trophique de son hote: Drosophila melanogaster. 26 Fleury F.

Gibert P.

Ris N.

Allemand R. Ecology and life history evolution of frugivorous Drosophila parasitoids. 25 Carton Y. Attraction de Cothonaspis sp (Hymenoptera: Cynipidae) par le milieu trophique de son hote: Drosophila melanogaster. 27 Dicke M.

van Lenteren J.C.

Boskamp G.J.F.

van Dongen-van Leeuwen E. Chemical stimuli in host-habitat location by Leptopilina heterotoma (Thomson) (Hymenoptera: Eucoilidae), a parasite of Drosophila. 28 Schlenke T.A.

Morales J.

Govind S.

Clark A.G. Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. Figure 1 The Effect of Ethanol on Wasp Knockdown and Oviposition Show full caption Survival curves were generated for adult insects living in petri dishes with 6% ethanol food (A). Error bars indicate 95% confidence intervals. The number of wasp eggs laid per host (B) was measured by dissecting fly larvae grown on food containing 0% or 6% ethanol and exposed to wasps for 2 hr. Error bars indicate SD. Dm, D. melanogaster; Lb, L. boulardi; Lh, L. heterotoma. There were five dish replicates for all treatments. See also Figure S1 Ethanol levels found in natural D. melanogaster habitats range up to 6% ethanol by volume in rotting fruits, and 11% in wine seepages found at wineries []. Fly consumption of food with moderate levels of ethanol (i.e., less than 4% by volume) results in increased fitness [], but consumption of higher ethanol concentrations (i.e., greater than 4%) causes increasing fly mortality []. Given that secondary metabolites were shown to harm endoparasitoid wasps in other systems [], and the suggestion that D. melanogaster living in fruits with high ethanol concentrations might experience less wasp parasitism [], we decided to test whether natural levels of ethanol could act as a protective toxin in fly interactions with two wasp species: Leptopilina boulardi is a specialist parasite of D. melanogaster and its close relatives that was previously shown to have relatively high ethanol knockdown resistance, whereas L. heterotoma is a generalist parasite that infects a diversity of Drosophila species living in fermenting fruits, decaying plant materials, and sap fluxes []. Both wasp species are attracted to the odor of fermentation products such as ethanol, presumably as a means to locate hosts [], and they are each highly infectious in D. melanogaster lab strains []. We compared ethanol knockdown resistance of adult female flies and wasps over a 24 hr period using Drosophila food mixed with concentrations of ethanol ranging from 4% to 10% by volume ( Figure 1 A ; see also Figure S1 available online). At 6% ethanol, D. melanogaster adults and adults of the specialist wasp L. boulardi both showed significantly greater knockdown survival than adults of the generalist wasp L. heterotoma ( Figure 1 A). Considering all ethanol concentrations used, D. melanogaster is most ethanol resistant, followed by the specialist wasp L. boulardi, followed by the generalist wasp L. heterotoma ( Figure S1 ).

Given that wasps suffer knockdown by natural levels of environmental ethanol, we tested whether wasps also show a reduction in oviposition when presented with host fly larvae grown in 6% ethanol food ( Figure 1 B). There was a significant effect of ethanol in reducing oviposition of both wasp species. A significant ethanol-by-wasp interaction effect also indicated that ethanol had a stronger effect in reducing oviposition by the generalist L. heterotoma than the specialist L. boulardi. This difference is not explained by a difference in wasp mortality, because there was no wasp death over the course of the 2 hr trial. Wasps may lay fewer eggs because they are sickened by ethanol fumes and attack less, but it is also possible that they insert their ovipositors into fly larvae growing on ethanol food at a normal level and limit oviposition because they detect a hostile environment for their offspring. Given that wasp oviposition was not reduced in fly larvae briefly removed from ethanol (data not shown), we favor the former hypothesis. Thus, ethanol can provide protection to fly larvae from being attacked by endoparasitoid wasps.

29 Cowmeadow R.B.

Krishnan H.R.

Atkinson N.S. The slowpoke gene is necessary for rapid ethanol tolerance in Drosophila. 30 Scholz H.

Ramond J.

Singh C.M.

Heberlein U. Functional ethanol tolerance in Drosophila. 31 Bozic J.

DiCesare J.

Wells H.

Abramson C.I. Ethanol levels in honeybee hemolymph resulting from alcohol ingestion. 32 Maze I.S.

Wright G.A.

Mustard J.A. Acute ethanol ingestion produces dose-dependent effects on motor behavior in the honey bee (Apis mellifera). Figure 2 Increased Hemolymph Ethanol Is Associated with Wasp Death and Fly Survival Show full caption Hemolymph ethanol concentration was compared between 72 hr old fly larvae grown on food with or without 6% ethanol (A). Error bars indicate SD across five dish replicates. Infected fly larvae grown on control or ethanol food were dissected to determine the viability of wasp larvae growing within them (B). Error bars indicate 95% confidence intervals across five dish replicates. The proportion of infected fly larvae resulting in each of the three infection outcomes (fly eclosion, wasp eclosion, and death of both fly and wasp) was compared across ethanol and wasp treatments (C). Error bars represent 95% confidence intervals across three dish replicates. See also Figure S2 We next considered whether ethanol can help flies kill wasp parasites in the hemocoel once flies are infected. First, we measured the hemolymph ethanol concentration of D. melanogaster larvae grown in 6% ethanol food and found that fly hemolymph ethanol concentration was significantly higher in flies grown on food containing ethanol, with concentrations reaching approximately 6 mM (0.02% hemolymph ethanol content by volume) ( Figure 2 A ). This ethanol concentration is low relative to those found in adult flies and honeybees [], suggesting that D. melanogaster larvae may be particularly resistant to passage of ethanol across the gut wall or cuticle into the hemolymph and/or may have very efficient ethanol detoxification mechanisms. Fly hemolymph ethanol content returned to baseline level within 24 hr after larvae were removed from ethanol food, and wasp infection did not result in increased fly hemolymph ethanol concentration or prolong the presence of ethanol in the hemolymph ( Figures S2 A and S2B). Altogether, these data show that wasp eggs and larvae living in fly hemolymph are exposed to a moderate level of ethanol (and presumably to ethanol breakdown products such as acetaldehyde) when flies live in or consume ethanol. Any protective effect ethanol might have for infected flies is likely passive, because infected flies do not appear to purposefully increase hemolymph ethanol levels, for example by downregulating ethanol breakdown enzymes.

To determine whether host ethanol consumption affects wasp larval development, we briefly removed D. melanogaster larvae from the 6% ethanol food for attack by wasps before being returned to the food. There was a significant effect of host ethanol consumption on wasp larval mortality ( Figure 2 B). There was also a significant effect of wasp species and a significant interaction between ethanol treatment and wasp species, indicating that the increase in wasp larval mortality due to host consumption of ethanol was significantly greater for the generalist L. heterotoma than the specialist L. boulardi. To determine whether wasp larval mortality was an effect of ethanol experienced by the host fly larvae before or after attack, we performed a similar infection experiment in which food treatments were switched after the fly larvae were attacked ( Figure S2 C). Although there was no overall effect of different ethanol treatments on wasp larval mortality, in a regression analysis stratified by wasp type there was a significant increase in death of L. boulardi larvae in hosts grown on ethanol food postinfection compared to preinfection (p = 0.003), whereas L. heterotoma larvae suffered high mortality regardless of ethanol consumption timing (p = 0.623). Larval wasp death resulted in a decreased proportion of wasps surviving through eclosion and a significant increase in the proportion of flies that eclosed, despite an overall increase in ethanol-mediated fly mortality ( Figure 2 C). There were significant ethanol-by-wasp interaction effects on the proportion of flies and wasps eclosed, again indicating that ethanol has a stronger protective effect in flies infected by the generalist L. heterotoma. Altogether, these results indicate that ethanol consumption enhances fitness of wasp-infected flies and that flies can receive maximal therapeutic benefit by consuming ethanol postinfection.

33 Carton Y.

Poirie M.

Nappi A.J. Insect immune resistance to parasitoids. 34 Lochmiller R.L.

Deerenberg C. Trade-offs in evolutionary immunology: Just what is the cost of immunity?. Wasp larvae dissected from singly infected control hosts invariably had defined internal organs and moved vigorously ( Figure S2 D). However, wasp larvae dissected from fly larvae grown on 6% ethanol food often did not move, showed amorphous internal organ structure, and had everted tissues, in many cases in close proximity to their anuses ( Figure S2 E), suggesting that ethanol causes defects in wasp organ development or maintenance. Normally, flies attempt to kill wasps in a process termed encapsulation, and the increased mortality of wasps growing in ethanol-fed host flies might be the result of a heightened fly encapsulation response. Encapsulation involves constitutively produced plasmatocytes recognizing a wasp egg or larva as foreign and signaling to induce differentiation of lamellocytes, which spread over the wasp in a multilayered capsule, leading to wasp death []. The wasp strains used here are highly virulent in D. melanogaster hosts and normally completely suppress the encapsulation response, but no wasp eggs or larvae dissected from ethanol-consuming fly larvae were found to be encapsulated by host hemocytes either. Although ethanol consumption was associated with a significant increase in fly plasmatocyte numbers, ethanol consumption was associated with a significant decrease in the number of lamellocytes, the hemocyte type specifically induced to mount the encapsulation response ( Figures S2 F and S2G). Lack of induction and/or death of host lamellocytes could be the result of ethanol toxicity, but it may be adaptive for hosts to purposefully suppress induction of an immune response that is unneeded in the presence of an antiparasite toxin, given the presumed energetic cost of mounting an immune response [].

35 Clayton D.H.

Wolfe N.D. The adaptive significance of self-medication. 36 Singer M.S.

Mace K.C.

Bernays E.A. Self-medication as adaptive plasticity: increased ingestion of plant toxins by parasitized caterpillars. Figure 3 Choice of Ethanol Food by Wasp-Infected Fly Larvae Show full caption Preference for food containing 6% ethanol was compared between infected and uninfected flies over time using bisected petri dishes, with fly larvae initially placed on the control food side (A) or ethanol food side (B) of the dish. Error bars indicate 95% confidence intervals across three dish replicates. EtOH, ethanol. See also Figure S3 Use of toxic secondary metabolites in defense against enemies is usually preventative, i.e., organisms consume a toxic food source as part of their normal diet and the presence of toxin in their bodies results in internal host conditions that limit subsequent predation and infection. However, parasitized organisms can also therapeutically self-medicate, whereby they actively seek out compounds that help cure preexisting infections []. The fact that fly consumption of ethanol postinfection has strong protective effects ( Figure S2 C) led us to consider the possibility that D. melanogaster might self-medicate. To test this idea, we placed infected and uninfected fly larvae in bisected petri dishes containing half control food and half 6% ethanol food, and the proportions of fly larvae that moved to ( Figure 3 A ) or remained on ( Figure 3 B) the ethanol food side of the dish were measured over time. Fly larvae initially placed on control food showed a significant effect of wasp treatment at 24 hr, with fly larvae infected by each wasp species significantly more likely to have moved to the ethanol food side of the dishes ( Figure 3 A). Infected fly larvae initially placed on ethanol food moved off the ethanol food faster than uninfected fly larvae but returned to the ethanol food in significantly greater numbers than uninfected fly larvae by 24 hr ( Figure 3 B).

18 Parsons P.A.

Stanley S.M.

Spence G.E. Environmental ethanol at low concentrations: Longevity and development in the sibling species Drosophila melanogaster and D. simulans. 19 McKechnie S.W.

Geer B.W. Regulation of alcohol dehydrogenase in Drosophila melanogaster by dietary alcohol and carbohydrate. 20 McKenzie J.A.

Parsons P.A. Alcohol tolerance: An ecological parameter in the relative success of Drosophila melanogaster and Drosophila simulans. These results are not caused by an increased sensitivity to ethanol sedation in infected fly larvae, which might cause the ethanol half of the dishes to act as an “absorbing state” for these flies, because infected larvae were highly mobile and vigorously masticated the food once they were settled on the ethanol side of the dishes. Instead, these results show that infected flies self-medicate by actively sampling their environment for a food source containing levels of ethanol most suitable for fighting off wasp infection, despite the otherwise toxic effects of ethanol consumption on fly developmental rate and survival found by us ( Figure S3 ) and others []. Interestingly, in both choice experiments, fly larvae infected by the generalist L. heterotoma showed a significantly stronger preference for ethanol food than fly larvae infected by the specialist L. boulardi ( Figure 3 ). These data suggest that fly larvae can distinguish between endoparasitoids with different levels of ethanol resistance or that the specialist L. boulardi can better manipulate the ethanol seeking behavioral immune response of D. melanogaster.

Figure 4 The Option of Ethanol Food Enhances Fitness of Wasp-Infected Flies Show full caption Larvae were placed in bisected petri dishes with either 0% or 6% ethanol food on each side of the dish. The proportion of wasp-infected fly larvae resulting in each of the three infection outcomes (fly eclosion, wasp eclosion, and death of both fly and wasp) was compared across wasp and ethanol choice treatments. Error bars represent 95% confidence intervals across three dish replicates. Finally, we tested the eclosion success of infected flies allowed to self-medicate by giving them the option of 0% or 6% ethanol food in bisected petri dishes ( Figure 4 ). Survival of self-medicating flies was significantly greater than that of flies given no ethanol and equivalent to that of flies grown in dishes where both sides contained ethanol. Death of infected flies given a choice between control and ethanol food was significantly greater than that of flies given no ethanol, indicating the choice of ethanol food results in ethanol-mediated death, but death was significantly lower than for flies grown in dishes where both sides contained ethanol. Altogether, these data show that flies not only choose to consume ethanol as self-medication against wasp infection, but also balance their ethanol intake to limit toxic effects on themselves. Furthermore, there were significant effects of wasp species on infection outcomes, where flies infected by the generalist wasp L. heterotoma achieved a relatively greater increase in eclosion success due to self-medication.