Fruit Flies Medicate Offspring After Seeing Parasites

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Science  22 Feb 2013:
Vol. 339, Issue 6122, pp. 947-950
DOI: 10.1126/science.1229625


Hosts have numerous defenses against parasites, of which behavioral immune responses are an important but underappreciated component. Here we describe a behavioral immune response that Drosophila melanogaster uses against endoparasitoid wasps. We found that when flies see wasps, they switch to laying eggs in alcohol-laden food sources that protect hatched larvae from infection. This change in oviposition behavior, mediated by neuropeptide F, is retained long after wasps are removed. Flies respond to diverse female larval endoparasitoids but not to males or pupal endoparasitoids, showing that they maintain specific wasp search images. Furthermore, the response evolved multiple times across the genus Drosophila. Our data reveal a behavioral immune response based on anticipatory medication of offspring and outline a nonassociative memory paradigm based on innate parasite recognition by the host.

Although immune systems are often thought of as a set of infection-responsive molecules and cells within a host, they comprise a much more diverse array of biological structures and processes that collectively protect an organism from infection. Medication, the prophylactic (pre-infection) or therapeutic (post-infection) use of substances found in the environment to combat infection, is a type of behavioral immune mechanism (1). Medication requires the recognition of infection, or infection risk, by the host, leading to the use of a substance directed against the identified parasite (2, 3). Endoparasitoid wasps are a serious threat to flies in nature (4), and we recently showed that infected Drosophila melanogaster larvae preferentially consume toxic levels of alcohol because the benefit of alcohol-mediated wasp death outweighs the cost to flies of alcohol consumption, an example of therapeutic self-medication (5). In the present study, we tested whether adult fruit flies choose to lay their eggs in food containing toxic levels of alcohol when wasps are present in the environment as a means of prophylactically medicating their offspring against infection.

We tested the oviposition preferences of adult female D. melanogaster by placing 300 flies in population cages containing two food dishes, one of which contained 6% ethanol by volume (Fig. 1A). Flies were housed with or without 50 female wasps, and fly eggs were counted from separate sets of dishes 24 and 48 hours later. Control flies preferred to oviposit on dishes containing no ethanol; but in the presence of female Leptopilina heterotoma, a common wasp parasite of D. melanogaster larvae in nature (6), flies laid a significantly greater proportion of eggs on ethanol dishes at both time points (Fig. 1B and table S1). The flies displayed no such alcohol preference in the presence of male wasps. To determine the extent of fly preference for alcohol-laden oviposition sites in the presence of female wasps, flies were given a choice of various concentrations of ethanol. Control flies preferred to oviposit on dishes containing 3% ethanol (Fig. 1C and table S2), which is consistent with the known benefits to fly larvae of low-level alcohol consumption and the costs of higher-level consumption (5, 79). In the presence of wasps, however, flies overwhelmingly preferred to oviposit on dishes containing ethanol concentrations corresponding to the highest levels found in nature (12 and 15%) (Fig. 1C and table S2) (10).

Fig. 1

D. melanogaster medicates offspring with alcohol after exposure to wasps. (A) Standard oviposition preference setup. (B) Proportion of eggs laid on 6% ethanol dishes for three wasp treatments, at two time points. ***P < 0.001. (C) Proportion of eggs laid on dishes with increasing ethanol (EtOH) concentrations, depending on wasp presence. P < 0.001 for distribution comparisons at both time points. (D) Proportion of offspring of unexposed fly parents that eclose when laid in cages with different combinations of oviposition dishes. (E) Proportion of offspring of wasp-exposed parents that eclose. For (D) to (E), letters indicate significance groups at P < 0.01. For (B) to (E), error bars represent 95% confidence intervals (n = 4 experimental replicates).

To determine whether the fly oviposition switch is adaptive, we measured offspring eclosion success in various oviposition setups. In the absence of wasps, the offspring of flies in cages with only 0% ethanol dishes had significantly higher eclosion success than offspring from flies given 6% alcohol food, demonstrating that there is normally a fitness detriment to ovipositing in food with such high alcohol levels (Fig. 1D). When female wasps were present, however, the offspring of flies given an opportunity to oviposit on alcohol-laden food had significantly higher eclosion success than offspring of flies given no such opportunity (Fig. 1E). This prophylaxis probably arises from both decreased offspring infection and increased offspring success at curing infections (5). Such an induced fly behavioral immune response may serve as an alternative to the energetically costly cellular encapsulation response that flies mount against wasp eggs.

Mutant strains were used to determine whether flies require olfactory or visual cues to sense wasps. Or83b mutants fail to respond to most olfactory stimuli (11) but retained an oviposition preference for ethanol food in the presence of wasps (Fig. 2A, fig. S1A, and table S3), suggesting that this general olfactory receptor is not required for wasp detection or alcohol sensing (12). GMR-Hid flies express an apoptotic activator in the developing retina leading to dramatically reduced eyes (13), and ninaBP315 mutants fail to synthesize rhodopsin, eliminating vision while leaving the basic morphology of the eye intact (14). Neither vision mutant showed an oviposition preference for ethanol food in the presence of wasps (Fig. 2A, fig. S1A, and table S3), indicating that flies rely on sight to sense wasps in their environment and initiate the oviposition preference switch.

Fig. 2

Sight and NPF signaling control fly ability to sense and respond to wasps. (A to C) Proportion of eggs laid on ethanol dishes by (A) smell and sight mutants, (B) NPF and NPFR1 overexpression mutants, and (C) NPF and NPFR1 knockdown mutants in the presence and absence of wasps. For (A) to (C), the y axis is the same; error bars represent 95% confidence intervals (*P < 0.05, ***P < 0.001, n = 4). (D) NPF immunostain of an unexposed fly brain. *, NPF-expressing neurons; FSB, fan-shaped body; Lat, lateral regions; SEG, subesophageal ganglion; OL, optic lobes. (E to H) NPF-immunostained fan-shaped bodies from control and sight mutant flies unexposed or exposed to wasps.

Reduced expression of neuropeptide F (NPF) and its receptor (NPFR1) in fly brains increases ethanol tolerance and preference, similarly to reducing NPY in mammals (15, 16). Using the yeast Gal4-UAS transcription factor–promoter system, we found that NPF-Gal4–driven ectopic expression of NPF eliminated the wasp-induced ethanol oviposition preference (Fig. 2B; fig. S1, B, and D to G; and table S3). Flies with elav-Gal4–driven pan-neuronal ectopic expression of NPFR1 showed a weak but significant increase in alcohol preference in the presence of wasps, but there was a dramatic reduction in oviposition preference for alcohol food as compared to wild-type flies (Fig. 1B). RNA interference–mediated knockdown of NPF and NPFR1 levels in the brain, using NPF-Gal4 and elav-Gal4 drivers, respectively, led to increased ethanol oviposition preference regardless of wasp presence (Fig. 2C; fig. S1, C to E and H to I; and table S3).

These results suggest that the visual perception of wasps by flies might cause decreased NPF levels in fly brains. We immunostained fly brains with NPF antiserum after exposure to wasps (Fig. 2, D to H) and found a marginally significant decrease in whole-brain fluorescence levels in wild-type flies exposed to wasps for 24 hours but no such change in sight-impaired ninaBP315 flies (fig. S1J). NPF-expressing neurons send projections to the fan-shaped body, subesophageal ganglion, and lateral regions of the lower central brain (Fig. 2D) (16, 17). There was a significant loss of NPF immunofluorescence in the fan-shaped body of wild-type flies exposed to wasps, whereas the other NPF-positive regions remained unchanged (Fig. 2, E to F, and fig. S1, K to O). Despite constitutive differences between wild-type and ninaBP315 flies, there was no difference in the staining of the fan-shaped body or other brain regions between unexposed and wasp-exposed ninaBP315 flies (Fig. 2, G to H, and fig. S1, K to O). The fan-shaped body is part of the central complex of the fly brain that regulates both visual pattern recognition and ethanol-stimulated locomotion (18, 19).

To determine how long fly oviposition preferences are altered after sensing wasps (20), we "pre-exposed" flies to wasps before assaying oviposition preferences in cages devoid of wasps. Flies pre-exposed to wasps showed a strong preference for the ethanol oviposition dishes across a 4-day choice assay (Fig. 3A and table S4). The oviposition switch occurred even when flies were pre-exposed to wasps in food bottles containing alcohol or completely different media, demonstrating that flies do not learn to associate wasp presence with food type (Fig. 3A: P > 0.120 for preference comparisons between pre-exposure food types at each time point, table S4). Thus, the oviposition preference switch that flies maintain after seeing wasps is a natural example of nonassociative memory (21).

Fig. 3

Flies form long-term memories of seeing wasps. (A to C) Proportion of eggs laid on ethanol dishes (A) after wasp pre-exposure on different food types, (B) in the presence of wasps by the long-term memory mutant Adf1nal, and (C) after wasp pre-exposure (on molasses food) in Adf1nal mutants. For (A) to (C), all y axes are the same; error bars represent 95% confidence intervals (*P < 0.05, ***P < 0.001, n = 4).

The D. melanogaster transcription factor Adf1 is required for long-term memory formation and for regulating Alcohol dehydrogenase expression (21, 22). Adf1nal, a mutant that has normal early memory but lacks long-term memory, switched its oviposition preference to alcohol food in the presence of wasps like wild-type flies (Fig. 3B and table S4). When Adf1nal mutants were pre-exposed to wasps and then put into cages without wasps, however, the flies failed to retain the wasp exposure memory, showing no oviposition preference for alcohol food (Fig. 3C and table S4). This result is not explained by any reduced ethanol tolerance of Adf1nal flies (fig. S2, A and B). Furthermore, both vision and NPF signaling were required for initiating memory formation (fig. S2, C to F, and table S4). These data suggest that a single protein (Adf1) may simultaneously be responsible for memory of wasp presence and regulation of a gene that controls tolerance to the alcohol-laden food flies become permanently attracted to, and they also outline a simple model of wasp-mediated alcohol seeking in flies (fig. S3).

Innate and learned search images are important for numerous organismal interactions (23, 24). To further delimit the innate wasp search images D. melanogaster maintains, we assayed fly oviposition behavior during exposure to two more Figitid wasps, L. boulardi and L. clavipes; two Braconids, Aphaereta sp. and Asobara tabida; and a Diapriid pupal endoparasitoid, Trichopria sp. (25). Flies preferred ethanol-laden oviposition dishes in the presence of females of each endoparasitoid species that infects fly larvae, but the preference was weaker upon exposure to the wasp that infects fly pupae, reaching only marginal significance at 0 to 24 hours (Fig. 4A and table S1). This might make adaptive sense given that Drosophila larvae often move off their food source before pupating. Thus, D. melanogaster has evolved search images specific enough to distinguish female from male L. heterotoma and a pupal endoparasitoid from larval endoparasitoids, but broad enough to recognize two families of larval endoparasitoids (see fig. S4 for wasp images).

Fig. 4

Breadth of fly search images and the evolution of medication behavior. (A) Proportion of eggs laid on ethanol dishes in response to different wasp species. Error bars represent 95% confidence intervals (*P < 0.05, ***P < 0.001, n = 4). (B) Correlation between phylogenetically independent contrasts for ethanol tolerance (LD50) and ethanol oviposition preference index (EOPI) across seven Drosophila species.

To understand the evolution of this behavioral immune mechanism, we tested oviposition preferences in six more Drosophila species and found that three species (D. simulans, D. hydei, and D. virilis) showed a significant increase in oviposition preference for alcohol dishes in the presence of female L. heterotoma wasps, whereas the other species actively avoided ethanol dishes regardless of wasp presence (fig. S5, A to F, and table S5). Ethanol tolerance median lethal dose (LD50) values for each fly species (fig. S5, G to L) were positively correlated with the ethanol oviposition preference index, a measure of how strongly the preference for ovipositing in ethanol food increases in response to wasp exposure (fig. S5M). This relation was even stronger when the phylogenetic relationships between the Drosophila species were taken into account, using phylogenetically independent contrasts (26) (Fig. 4B). Thus, alcohol tolerance and the alcohol oviposition preference switch have coevolved across the genus Drosophila.

Supplementary Materials

Materials and Methods

Figs. S1 to S5

Tables S1 to S5

References and Notes

  1. Acknowledgments: We thank P. Shen for NPF antiserum; P. Shen, K. Moberg, and S. Sanyal for Drosophila strains; and J. van Alphen and B. Wertheim for wasp strains. This work was supported by NIH grant AI081879 to T.A.S. and the Integrated Cellular Imaging Microscopy Core of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077. Data are deposited in the Dryad Repository:
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