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. 2024 Oct 21;121(44):e2410874121. doi: 10.1073/pnas.2410874121

Tolerance and efficient metabolization of extremely high ethanol concentrations by a social wasp

Sofia Bouchebti a,1,2, Yael Gershon a, Alexander Gordin b, Dorothée Huchon a,c, Eran Levin a,2
PMCID: PMC11536130  PMID: 39432778

Significance

Many animals naturally consume low ethanol concentrations despite its inherent toxicity. Still, even species well adapted to ethanol consumption face detrimental effects when exposed to concentrations above 4%. Here, we show that an animal species, the Oriental hornet, can consume extremely high amounts of high concentrations of alcohol, without experiencing any detrimental effects on lifespan or behavior. This remarkable ethanol tolerance results from their high rates of ethanol metabolism, most likely enabled by their multiple copies of the alcohol dehydrogenase (NADP+) gene. Thus, social wasps can offer valuable insights into the physiological and behavioral aspects of ethanol tolerance and could help in understanding the underlying mechanisms and potential treatments for alcohol use disorder.

Keywords: Vespa orientalis, ethanol tolerance, stable carbon isotope

Abstract

Ethanol, a natural by-product of sugar fermentation, can be found in various fruits and nectar. Although many animals routinely consume ethanol in low concentrations as part of their natural diets, its inherent toxicity can cause severe damage. Even species particularly well adapted to ethanol consumption face detrimental effects when exposed to concentrations above 4%. Here, we investigated the metabolism of ethanol and its impact on survival and behavior in the Oriental hornet (Vespa orientalis), a social wasp that naturally consumes ethanol. We show that chronic ethanol consumption, even at concentrations as high as 80%, had no impact on hornet mortality, construction behavior, or agonistic behavior. Using 13C1 labeled ethanol, we show that hornets efficiently metabolized ingested ethanol and at a much higher rate than honey bees. The presence of multiple copies of the alcohol dehydrogenase (NADP+) gene in the Vespa genera suggests a potential mechanism for ethanol tolerance. These findings support the hypothesis that the mutualistic relationship between ethanol-producing organisms and vespid hosts may be at the origin of their remarkable capacity to utilize and metabolize ethanol.

Ethanol is a natural by-product of sugar fermentation by yeasts and can be found in many fruits and floral nectars (1). However, despite ethanol’s nearly double caloric value compared to sugar (7 vs. 4 calories per gram), its inherent toxicity prevents animals from safely utilizing it as a routine source of energy (2). Ethanol consumption increases mortality, usually as a function of ethanol concentration (3–8). In addition, ethanol ingestion affects various behaviors as well as motor and cognitive performances (1). Yet, many animals regularly consume ethanol in their natural diets (1). Among vertebrates, tree shrews exhibit the highest level of adaptation to ethanol consumption, as they periodically ingest food with concentrations of up to 3.8% ethanol in their natural habitat without apparent consequences (9). However, in laboratory conditions, chronic consumption of 10% ethanol induced severe liver damage within only 14 d (10). In invertebrates, fruit flies are considered highly adapted to ethanol consumption, as it is an integral part of their natural diet throughout their life stages (11, 12). Similarly to tree shrews, ethanol consumption under 4% does not lead to any negative effects in fruit flies, but concentrations above that threshold significantly increased mortality (11, 13–15).

In humans, alcohol use disorder, or the abuse of ethanol consumption, represents a substantial global problem with significant medical and social consequences (16). Recently, the World Health Organization issued a warning stating that even moderate ethanol consumption can be harmful, and emphasized that there is no safe level of ethanol intake that does not have adverse effects on health (17). Consequently, animal models for alcoholism are of great interest (12, 18).

Social wasps naturally consume ethanol in addition to insect prey and carrion (19), as their diet includes nectar (20) and ripe fruits (21, 22). While they have been observed to be strongly attracted to manufactured alcoholic beverages (23, 24), the phenomenon of “oenophilia” among these insects remains largely unexplored. Previous research by Stefanini et al. (25) has suggested that hornets and wasps may play a critical role in the evolution and ecological maintenance of the brewer’s yeast Saccharomyces cerevisiae. During winter, queens of social wasps harbor S. cerevisiae and transfer it to their workers in spring, who then pass it to the fruits upon consumption (25). In addition to yeasts, foragers of social wasps transfer various other ethanol-producing microorganisms, such as fungi and bacteria, from ripe fruits to nectar by passively carrying them on their bodies (26). Here, we hypothesized that the relationship between ethanol-producing organisms and their vespids hosts might be mutualistic and could impact their ability to utilize ethanol as an energy-rich resource.

Results

Ethanol Consumption Does Not Affect Hornet Mortality and Behavior.

To test this hypothesis, we used the Oriental hornet (Vespa orientalis) as a vespid model. We fed these hornets a sucrose solution that contained ethanol as their sole energy source for 7 d, with ethanol concentrations ranging from 0 to 80%. We found no significant effect of ethanol consumption on hornet survival (Fig. 1A, worker hornets, cox proportional hazards regression model, z = 0.97, P = 0.33; Fig. 2B, male hornets, cox proportional hazards regression model: z = 0.65, P = 0.52), construction behavior (Fig. 2A, linear mixed-effects model: F6,23.11 = 2.09, P = 0.093), or agonistic behavior (Fig. 2B, contact latency to an intruder, linear mixed-effects model: F6,57.00 = 1.09, P = 0.38; Fig. 2C, collective response to an intruder, linear mixed-effects model: F6,16.01 = 1.28, P = 0.32). When testing for the effect of chronic ethanol consumption on lifespan, we found that even the highest concentration of ethanol (80%) did not reduce the lifespan of hornets either (Fig. 1C, cox proportional hazards regression model, z = −1.3, P = 0.19). In order to control precisely the amount of ethanol ingested, we harnessed hornet and honey bee workers and fed them manually twice a day (10:00 to 18:00) a sucrose solution containing 0 or 80% ethanol for 7 d (5 µL for honey bee and 10 µL for hornet). We found no difference in survival between hornets fed with ethanol compared to hornets fed with sucrose solution only, but honey bees did not survive ethanol consumption beyond 24 h (Fig. 1D, log-rank test: χ2 = 44.00, df = 3, P = 2 × 10−9; pairwise comparisons: Hornets 0% vs. Hornets 80%, P = 0.76, Honey bees 0% vs. Honey bees 80%, P = 2.8 × 10−5, Hornets 0% vs. Honey bees 0%, P = 0.74, Hornets 0% vs. Honey bees 80%, P = 2.6 × 10−5, Hornets 80% vs. Honey bees 0%, P = 0.74, Hornets 80% vs. Honey bees 80%, P = 1.2 × 10−5).

Fig. 1.

Fig. 1.

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Ethanol consumption does not affect hornet survival or lifespan. (A) Effect of ethanol consumption at different concentrations (0, 1, 10, 20, 40, 60, and 80%) on worker hornet survival during 7 d (n = 50 individuals per concentration group). (B) Effect of ethanol consumption at different concentrations (0, 1, 10, 20, 40, 60, and 80%) on male hornet survival during 7 d (n = 50 individuals per concentration group). (C) Effect of ethanol consumption at different concentrations (0 and 80%) on worker hornet lifespan (n = 50 individuals per concentration group). (D) Effect of ethanol consumption at different concentrations (0 and 80%) on harnessed worker hornets and honey bees (n = 10 individuals per concentration group and species).

Fig. 2.

Fig. 2.

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Ethanol consumption does not affect hornet behaviors. (A) Effect of ethanol consumption at different concentrations (0, 1, 10, 20, 40, 60, and 80%) on hornet nest construction process at the seventh day of experiment (n = 10 boxes per group). (B) Effect of ethanol consumption at different concentrations (0, 1, 10, 20, 40, 60, and 80%) on hornet contact latency to an intruder (n = 10 boxes per group). (C) Effect of ethanol consumption at different concentrations (0, 1, 10, 20, 40, 60, and 80%) on hornet collective response to an intruder (n = 10 boxes per group). The boxes represent the first and third quartiles and the median. The whiskers represent the maximum and minimum values. The circles represent the outliers.

Hornets readily consumed large amounts of ethanol and did not seem to modulate their ethanol ingestion (Fig. 3A, worker hornets, linear mixed-effects model: Solution, F1,111.82 = 32.08, P = 1.16 × 10−7; Group, F6,40.31 = 3.41, P = 0.008, Solution × Group, F6,112.14 = 1.07, P = 0.38, but see SI Appendix, Table S1 for pairwise comparisons; and Fig. 3B, males hornets, linear mixed-effects model: Solution, F1,120.00 = 0.12, P = 0.73; Group, F6,13.32 = 1.14, P = 0.39, Solution × Group, F6,120.00 = 4.24, P = 0.0006, see SI Appendix, Table S2 for pairwise comparisons). Even when given a choice between a sucrose solution and an ethanol solution, hornets did not show any preference for either solution, regardless of the ethanol concentration offered (Fig. 3C, linear mixed-effects model: Solution, F2,151.00 = 10.70, P = 4.49 × 10−5; Group, F5,151.00 = 1.43, P = 0.21, Solution × Group, F5,151.00 = 0.66, P = 0.76; see SI Appendix, Table S3 for pairwise comparisons). In honey bees, a hymenopteran model extensively used in ethanol-related studies (27–30), even small doses of ethanol are enough to increase mortality, with the effect being dependent on the concentration of ethanol (31). Acute consumption of 1 to 5% ethanol solution impairs bees’ locomotion (31, 32) and cognitive abilities (29, 33, 34) and affects their aggressive (27) and foraging behavior (30, 35, 36).

Fig. 3.

Fig. 3.

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Hornets do not regulate their ethanol consumption. (A) Ethanol and water consumption per worker hornet (g) during 7 d (N = 50 individuals per concentration group). (B) Ethanol and water consumption per male hornet (g) during 7 d (N = 50 individuals per concentration group). (C) Ethanol, sugar solution, and water consumption per worker hornet (g) during 7 d (N = 50 individuals per concentration group). The boxes represent the first and third quartiles and the median. The whiskers represent the maximum and minimum values. The circles represent the outliers.

Hornets Metabolize Ethanol at High Rates.

Most animals metabolize ethanol via three enzymatic steps into Acetyl-CoA, which can be used for fatty acid synthesis or oxidized by the citric acid cycle (37). In humans, derivatives of ethanol and metabolites, such as acetaldehyde, primarily contribute to its toxic effects (38, 39), and ethanol tolerance is directly linked to highly efficient metabolization (40). As insects are thought to possess a similar ethanol-metabolizing pathway to humans (41, 42), we hypothesized that the unique ethanol tolerance exhibited by hornets could result from highly efficient ethanol metabolization. We fed hornets with 13C1-labeled ethanol and assessed the rate of ethanol metabolism in vivo across their different castes (adult workers, gynes, and males) and life stages (larvae) and compared it to worker honey bees. We found that both hornets and honey bees can metabolize ingested ethanol. However, hornets, regardless of caste or life stage, metabolize ethanol at a significantly faster and higher rate than honey bees (Fig. 4A, linear mixed-effects model: Treatment, F1,2464.01 = 34.11, P = 5.88 × 10−9; Type, F4,2464.01 = 31.85, P < 2.20 × 10−16, Time, F1,9.49 = 91.28, P = 3.55 × 10−6; Treatment × Type, F4,2464.01 = 21.98, P < 2.20 × 10−16, Treatment × Time, F1,2464.07 = 733.11, P < 2.20 × 10−16, Type × Time, F4,2464.07 = 45.70, P < 2.20 × 10−16, Treatment × Type × Time, F4,2464.07 = 30.04, P < 2.20 × 10−16; see SI Appendix, Table S4 for pairwise comparisons). Among hornets, workers exhibit the highest rate of ethanol metabolism (Fig. 4A). To track the incorporation of ethanol’s carbons in the hornet’s body, we fed ad libitum worker and male hornets with the same 13C1-labeled ethanol solution for 7 d and we assessed the δ13C ratio in their tissues. Our results indicate that 13C from ethanol is integrated into all tested tissues (brain, muscles, and fat body) in both workers and males, but significantly more so in the fat body of worker hornets, which functions as an analog to the mammalian liver (43), including for ethanol metabolism (Fig. 4B, linear mixed-effects model: treatment, F1,98.20 = 1350.47, P < 2.20 × 10−16; Type, F1,2.06 = 206.80, P = 0.004, Tissue, F2,97.10 = 25.99, P = 9.09 × 10−10; Treatment × Type, F1,98.20 = 64.84, P = 1.94 × 10−12, Treatment × Tissue, F2,97.10 = 111.65, P < 2.20 × 10−16, Type × Tissue, F2,97.10 = 45.64, P = 1.06 × 10−14, Treatment × Type × Tissue, F2,97.10 = 0.85, P = 0.43; see SI Appendix, Table S5 for pairwise comparisons).

Fig. 4.

Fig. 4.

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Hornets metabolize ethanol at high rates. (A) δ13C values (‰) in the CO2 from exhaled breath of hornets (gynes, larvae, males, and workers) and honey bees (workers) fed with labeled ethanol (n = 10 for each type of individuals) and unlabeled ethanol (control, n = 10 for each type of individuals). Data are mean ± SEM. (B) δ13C values (‰) of body tissues of worker and male hornets fed with labeled ethanol (n = 10 for each type of individuals) and unlabeled ethanol (control, n = 10 for each type of individuals). The boxes represent the first and third quartiles and the median. The whiskers represent the maximum and minimum values. The circles represent the outliers. A higher delta indicates higher 13C levels.

Hornets Possess Duplications of the Alcohol Dehydrogenase Gene.

We hypothesized that the molecular mechanism responsible for the alcohol resistance in vespid wasps may involve duplications of the alcohol dehydrogenase gene or other genes involved in ethanol metabolism. This hypothesis was supported by previous work showing that duplication of genes involved in the ethanol pathway may have enhanced ethanol resistance in unrelated lineages. For example, duplication of the aldehyde dehydrogenase 1 A1 in the beaver genome may be responsible for the higher resistance of beaver cells to ethanol (44). Similarly, it has been suggested that duplication of the alcohol dehydrogenase genes may have allowed tephritid flies to infect ripening fruits that are richer in ethanol, while species living in inflorescences have a single copy of the gene (45).

The genome of V. orientalis has not been fully sequenced, and thus, we mined the genomes of other members of the genus Vespa, searching for evidence for such gene duplication events. Specifically, we searched for duplications of the genes encoding of the following enzymes: alcohol dehydrogenase (NAD+), alcohol dehydrogenase (NADP+), aldehyde dehydrogenases, aldehyde dehydrogenase (NADP+), and acetyl-coenzyme A synthetase. Surprisingly, two copies of the alcohol dehydrogenase (NADP+) genes were found in members of the genus Dolichovespula and two to four copies were found in members of the genus Vespa (Fig. 5A). In each species, all paralogs are encoded on the same chromosome, suggesting that they originated from tandem duplication events. A phylogenetic analysis of gene copies among hymenopteran species suggests that these duplications have occurred in the lineage leading to these two genera (SI Appendix, Fig. S1).

Fig. 5.

Fig. 5.

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Hornets have multiple copies of the alcohol dehydrogenase gene (A) Sequence alignment and syntenic map of the region encoding the alcohol dehydrogenase (NADP+) gene across 14 species: 13 vespidae and the honey bee used as an outgroup. The exons of the E3 ubiquitin-protein ligase synoviolin, the DNA replication licensing factor Mcm5 isoform X1, and the alcohol dehydrogenase (NADP+) are indicated in pink, red, and blue, respectively. A putative and unannotated copy of the alcohol dehydrogenase gene found in Vespa velutina is indicated in yellow. All other coding sequences are indicated in green. The thin black lines indicate gaps, while thicker black bars indicate nucleotides. (B) Maximum likelihood gene tree of the alcoholdehydrogenase NADP+ in Vespinae. Bootstrap support values are indicated near their corresponding branch. Each species is indicated by a different color.

To confirm that this gene duplication is also present in V. orientalis, we assembled the transcriptome of this species from publicly available sequence reads. Two transcripts of the alcohol dehydrogenase (NADP+) gene were found. We reconstructed a maximum-likelihood phylogenetic tree of all alcohol dehydrogenase (NADP+) gene copies (Fig. 5B). The obtained phylogeny agrees with previous studies (46, 47). In the reconstructed tree, the sequences of each genus are grouped together (Fig. 5B). Moreover, three of the four paralogs of V. velutina and Vespa crabro are also grouped together. This suggests that the copies of the alcohol dehydrogenase (NADP+) gene evolve under some level of concerted evolution. The alternative explanation of multiple independent duplications in each lineage is less likely. An additional support for concerted evolution is the observation that the three coding sequences of V. crabro are 100% identical.

Interestingly, previous taxonomic studies based on total evidence have suggested that V. orientalis is closely related to V. crabro (48). This suggests that similar to V. crabro, four copies of the alcohol dehydrogenase (NADP+) gene may exist in V. orientalis, of which, we could only identify two in the transcriptomic data we analyzed.

Discussion

In this study, we showed that, like no other known organisms, chronic ethanol consumption in hornets, even at extremely high concentrations (80%), did not affect mortality and behavior. The drunken monkey theory predicts that nectarivore and frugivore animals should be adapted to natural, i.e., low, dietary ethanol concentrations but be negatively affected by high concentrations (1). Genetic variations in the ability to metabolize ethanol, especially different allelic forms of alcohol dehydrogenase and aldehyde dehydrogenase, are supposed to correlate with natural levels of environmental ethanol exposure, shaped by natural selection (11). In nature, ethanol concentration can reach up to 3.8% (9) in nectar and up to 8% in ripe fruits (49). Cultivated fruits such as wine grapes, also consumed by hornets, have ethanol concentrations ranging from 0 to 12% (50). Natural fermentation limits ethanol concentration to 20%, as yeasts fermenting sugars, including the yeast S. cerevisiae used for brewing and winemaking, do not survive in higher concentrations (51). Ethanol concentrations higher than 20% can only be achieved by human techniques developed a few centuries ago, such as distillation (52). So why are hornets so well adapted to consumption of extremely high ethanol concentrations? The remarkable ethanol tolerance exhibited by hornets could be attributed to their extensive coevolutionary history with yeasts (25), which has led to their unique adaptation to ethanol use. As social wasps are involved in ethanol production through their food sources—either by harboring yeasts in their digestive systems (25) or by transporting bacteria and fungi on their bodies (26)—it stands to reason that they have developed a natural adaptation to thrive in high ethanol concentrations. This adaptation allows hornets to use ethanol as a source of energy, highlighting the mutualistic nature of their relationship with ethanol-producing organisms.

Additionally, ethanol’s antimicrobial properties may be particularly advantageous to hornet workers, who frequently collect carrion to nourish developing larvae (19).

Our results suggest that the gene duplications of alcohol dehydrogenases NADP+ may be a molecular mechanism contributing to alcohol resistance in Vespa. This genomic adaptation shares similarities with instances of insecticide resistance mechanisms observed in many insects, where increased activity through gene amplification leads to multiinsecticide resistance (53, 54).

In conclusion, this study represents the report of an animal species capable of effectively and safely utilizing high ethanol concentrations under chronic consumption, thereby challenging the conventional notion that ethanol at high concentrations or under chronic consumption is toxic with no adaptive significance. Our study establishes social wasps as a unique animal model for alcohol-related research with potential applications across multiple disciplines, including medicine. Additionally, our work employs stable isotope labeling methods to assess alcohol metabolism in living organisms. This robust, noninvasive technique could become a powerful tool in future research on alcohol use disorders, including studies involving human subjects.

Materials and Methods

General Methods.

Colonies of Oriental hornets were collected from areas around the university and transferred to wooden boxes (14 L) with a glass front wall. Hornet colonies were provided with ad libitum water and sugar solution (60% inverted sugar) and fed three times a week with raw chicken and dead adult bumble bees (Bombus terrestris). The water and solutions provided to the colonies during the experiments were supplied in 15-mL plastic tubes clogged with cotton wool. The colonies were maintained, and the experiments were conducted in a climate-controlled room (28 °C, 75 ± 10% RH). Hornet individuals (larvae, gynes, males, and workers) were haphazardly sampled with forceps after placing the boxes containing the colonies in a −20 °C chamber for 30 to 45 min to reduce hornets’ activity. According to the experiments, hornets were placed individually in a 50-mL plastic tube or placed by a group of five individuals (from the same colony) in small wooden boxes (14.4 × 12 × 10 cm) with a metal mesh ceiling allowing ventilation. Honey bee workers (Apis mellifera) used in this study were collected using a sweeping net from flower patches on the campus university, placed individually in 50-mL plastic tubes, and tested within 30 min. Foragers honey bees were chosen for their higher ethanol tolerance compared to other castes (55).

Survival Experiments.

Hornet males and workers were placed separately in groups of five in boxes for 7 d with ad libitum access to water and a sucrose solution (50%) containing one of the following ethanol percentages: 0, 1, 10, 20, 40, 60, or 80 (SI Appendix, Table S6).

To assess the ethanol and water consumption of the hornets, tubes containing the solution and water were weighed at the experiment’s beginning and end. Control tubes containing the experimental solutions and water were weighed similarly and placed in boxes without hornets to assess the evaporation rate. Dead hornets were counted and removed daily. Ten replicates (i.e., box of five individuals) were carried out per group. Males and workers were sampled from five and nine different colonies, respectively.

Longevity Experiment.

Hornet workers were placed in groups of five in boxes with ad libitum access to water and a sucrose solution (50%) containing 0% or 80% of ethanol. Dead hornets were counted and removed daily until the last one died. Ten replicates (i.e., box of five individuals) were carried out per group, and six colonies were used for this experiment.

Survival Experiment with Controlled Feeding.

Ten hornet and ten honey bee workers were placed in a −20 °C freezer for 5 min and harnessed individually in Eppendorf tubes according to standard harnessing procedures (56). The individuals were then placed in an incubator (Panasonic, MIR-254) at 28 °C and 70 ± 10% RH for 7 d. Twice a day, individuals were manually fed 10 µL (for hornets) or 5 µL (for honey bees) of a sucrose solution (50%) containing either 0% or 80% ethanol. Fresh solutions were prepared every day and stored in the refrigerator at 4 °C for the second feeding of the day. Dead individuals were counted and removed daily.

Choice Experiment.

Hornet workers were placed in groups of five in wooden boxes with ad libitum access to water, sucrose solution (50%), and a sucrose solution (50%) containing one of the following ethanol percentages: 1, 10, 20, 40, 60, or 80, for 7 d. Tubes containing the solutions and water were weighed at the experiment’s beginning and end. Ten replicates (i.e., box of five individuals) were carried out per group, and eight colonies were used for this experiment.

Behavior Experiments.

Hornet workers were placed in groups of five in wooden boxes with ad libitum access to water and a sucrose solution (50%) containing one of the following ethanol percentages: 1, 10, 20, 40, 60, or 80, for 7 d.

To assess the construction behavior, building materials (soil and paper) were provided, and a piece of cardboard was glued to the ceiling of each box to facilitate construction (57). At the end of the experiment, a construction stage was attributed to each box according to the advancement of the nest built by the workers as follows: stage 0 corresponded to no construction; stage 1 to the initiation of construction when stains derived from the building materials started to appear on the ceiling of the boxes; stage 2 to the construction of a stem; stage 3 to the construction of the first cell; stage 4 to the construction of two cells; stage 5 to the construction of three cells; stage 6 corresponded to the construction of four cells; and stage 7 corresponded to the construction of five cells.

We assessed the agonistic behavior at the end of the experiment by introducing a dead hornet worker from a different colony into the box (58). The latency of the first antennal contact made with the dead hornet and the proportion of collective response, i.e., hornets interacting (antennal contact, bite, or sting) simultaneously with the dead hornet, were recorded for 5 min.

Ten replicates (i.e., box of five individuals) were carried out per group and experiment (i.e., construction behavior experiment and agonistic behavior experiment). Workers were sampled from nine different colonies for each experiment.

δ13C Analysis in Respiration.

Hornets were placed individually in tubes and were manually fed with a calibrated pipette containing 2 µL of a sucrose solution (50%) containing 1% of 13C-labeled ethanol (Cambridge Isotope Laboratories, Tewksbury, MA). Individual honey bees were placed in a −20 °C freezer for 5 min and harnessed according to standard harnessing procedures (56). After a full recovery from chilling (2 to 5 min), honey bees were fed 2 µL of a sucrose solution (50%) containing 1% of 13C-labeled ethanol and released from their harness. Control individuals (hornets and honey bees) were fed 2 µL of a sucrose solution (50%) containing 1% of nonlabeled ethanol (Sigma-Aldreich). Immediately after the feeding, individuals (hornets and honey bees) were placed individually in a 40-mL sealed metabolic chamber. Pull-mode respirometry was used as follows: Room air was pulled at a constant rate (30 mL min−1 STP) through an ascarite® column (CO2 absorbent) into the chamber and then directly into a G2121-i cavity ring-down spectroscopy (CRDS) stable carbon isotope analyzer (Picarro, Santa Clara, CA) (59). The analyzer was calibrated before the measurements using commercially certificated standards. The 13CO2 and 12CO2 contents in the exhaled breath of the hornets and honey bees were measured for 25 min. Ten individuals per group were tested. Hornet larvae were sampled from two different colonies, gynes and males from three different colonies, and hornet workers from four different colonies.

δ13C Analysis in Body Tissues.

Hornet males and workers were placed separately in groups of five in boxes with ad libitum access to water and a sucrose solution (50%) containing 1% of 13C-labeled ethanol by volume. Control groups were provided with a sucrose solution (50%) containing 1% of nonlabeled ethanol. After 7 d, individuals were frozen (−80 °C), and the brain, fat body, and flight muscles were dissected and dried at 60 °C for 3 d. Dried samples of each tissue (1 mg) were then loaded into tin capsules. The δ13C (‰) values in the samples were measured and calculated using a Picarro G2121-i Cavity Ring-Down Spectroscopy δ13C stable isotope analyzer with an A0502 ambient CO2 interface, an A0201 Combustion Module, and an A0301 gas interface (CM-CRDS) (58). To confirm the analyzer’s calibration and accuracy, we ran a secondary standard of verified δ13C value (C3 and C4 plant origin sucrose) every ten samples. All 13C concentrations are expressed in δ13CVPDB. Two replicates (i.e., box of five individuals) were carried out per group, and males and workers were sampled from two different colonies, respectively.

Ethanol Analysis in Experimental Solutions.

To verify the presence of ethanol in the experimental solutions provided to hornets after 7 d in the experimental chamber, control tubes containing sucrose solution with 80% ethanol were placed in a box without hornets. Samples were collected daily for analysis. The ethanol content in the samples was analyzed by GC-FID using headspace sampling. Ethanol peak identification was confirmed by GC–MS.

Extra dry ethanol, 50% sucrose in water solution, isopropanol, used as internal standard (PN 001626022100), and deionized water (Milli-Q IQ7003, Merck-Millipore) were used for standards preparation. Stock standard solutions were prepared in 2 mL polypropylene vials (SI Appendix, Table S7).

Working standard solutions were prepared from the above solutions by mixing of 0.10 mL of the corresponding stock standard solution with 0.40 mL of deionized water and 0.50 mL of 2% v/v isopropanol in water solution. Six sample solutions, one solution for each experiment day, were diluted the same way: 0.10 mL of the sample was mixed with 0.40 mL of water and 0.50 mL of 2% isopropanol solution.

GC-FID instrument (6890N, Agilent) equipped with HP-5 column (30 m × 0.320 μm × 0.25 μm film thickness, 19091 J-413, Agilent) and 7863B autosampler with 10 µL gastight syringe (5181-3354, Agilent) was used for quantitative ethanol measurements. Confirmation of ethanol peak was done on GC–MS (7890N GC with 5977 Mass Selective Detector 5977A, Agilent) equipped with DB-5 ms UI (30 m × 0.250 μm × 0.25 μm film thickness, 122-5532UI, Agilent) and 7693A autosampler with a 10-μL gastight syringe (5181-3354, Agilent).

A sample of 0.10 mL of each diluted standard and sample solution was placed into 2 mL autosampler vials, closed with caps with septa, and allowed to stand for 5 h at room temperature to reach the liquid–gas phase equilibrium. Results are presented SI Appendix, Table S8.

Identification of Gene Duplication.

Protein sequences of enzymes involved in ethanol metabolism of the honey bee (A. mellifera) were downloaded from the KEGG database (last access January 2024). Specifically, we downloaded eight sequences: the alcohol dehydrogenase (NAD+) (EC 1.1.1.1) XP_393266; the alcohol dehydrogenases (NADP+) (EC 1.1.1.2) XP_394676 and XP_006567931; the aldehyde dehydrogenases (NAD+) (EC 1.2.1.3) XP_392104, XP_394614, and XP_623084 aldehyde dehydrogenase (NAD+) (EC 1.2.1.5) XP_026298645 and the acetyl-coenzyme A synthetase (EC 6.2.1.1) XP_026297767.

These sequences were used to conduct tblastn searches against the genome assemblies of Vespidae species present in the Reference Sequence Database (RefSeq) of the National Center for Biotechnology Information (NCBI). The following assemblies and versions were considered: Ancistrocerus nigricornis (GCA_916049575.1) (60); Vespa velutina (GCF_912470025.1) (61); Vespula germanica (GCA_905340365.1) (62, 63); V. crabro (GCF_910589235.1) (61); Vespula vulgaris (GCF_905475345.1) (63); Dolichovespula media (GCA_911387685.1) (64); Dolichovespula sylvestris (GCA_918808275.2) (65); Dolichovespula saxonica (GCA_911387935.1) (66); Vespula pensylvanica (GCF_014466175.1) (63); Polistes dominula (GCF_001465965.1) (67); Vespa mandarinia (GCF_014083535.2) (68); Polistes canadensis (GCF_001313835.1) (69); and Polistes fuscatus (GCF_010416935.1) (70).

Reciprocal blasts were then performed using the first five queries against the honey bee genome (GCF_003254395.2) to confirm gene orthology. This approach allowed us to detect gene duplications of the alcohol dehydrogenases (NADP+). Surprisingly, this region was found to be missing in the genome assembly of D. media.

Examination of the region flanking the alcohol dehydrogenase (NADP+) genes showed that the genes were flanked by E3 ubiquitin-protein ligase synoviolin and the DNA replication licensing factor Mcm5 isoform X1 (these genes were similarly annotated in most species). We thus extracted and aligned this region in all genomes. The sequence alignment was performed using MAFFT v7.490 as implemented in Geneious Prime 2023.2.1 under the E-ins-i algorithm. Alignment refinements were performed by realignment of specific regions of the alignment.

Assembly of the Transcriptome of V. orientalis.

Paired RNA reads of V. orientalis deposited under accession SRX20756118 (71) were downloaded from NCBI on August 2023. The reads were filtered against the Kraken database using GenomeFLTR (72) using default setting. The resulting analysis shows that 294,521 reads were likely Homo sapiens contamination while 33,918 reads were similar to Choristoneura fumiferana granulovirus sequences. These sequences were filtered out from the raw data. The remaining reads were assembled using the IDBA-tran assembler (version 1.1.3) (73) under default settings. We used tBlastn searches to identify the alcohol dehydrogenase (NADP+) transcripts.

Phylogenetic Reconstruction of the Alcohol Dehydrogenase (NADP+) Gene Tree in Vespidae.

Phylogenetic reconstructions were performed under the maximum likelihood criterion using IQ-Tree 2.2.2.6 (74). We used Model Finder as implemented in IQ-Tree to determine the best model of evolution. The best-fit model was a model with two partitions. The first partition consisted of codon positions 1 and 2 while the second partition consisted of codon position 3. According to the Bayesian information criterion, the best-fit model for both partitions was the TN+F+G4. The tree was reconstructed using an edge-linked-proportional partition model with separate model parameters between partitions. Nonparametric bootstrap supports were computed based on 1,000 replications.

Statistics.

Statistical analyses were performed in R version 4.2.1. Survival analyses were performed using a Cox proportional hazards regression model considering censured data with the colony as a random factor or a log-rank test, followed by pairwise comparisons with the BH P-value adjustment method for nonparametric data. In datasets used for linear mixed-effects models, normality was assessed by examining Q–Q plots of model residuals, and log10 transformation was applied when needed. Linear mixed-effects models were followed by post hoc pairwise comparisons (Tukey adjusted).

The model used for δ13C analysis in the respiration included the treatment (labeled or control), the type of individual (worker honey bee, gyne hornet, larva hornet, male hornet, or worker hornet), the time (minute), and their interactions as a fixed effect, with the time accounted for repeated measure. The model used for δ13C analysis in the body tissues included the treatment (labeled or control), the type of individual (male hornet or worker hornet), the tissue (brain, muscles, or fat body), and their interactions with the colony as a random factor. Models used for consumption analyses included the group (i.e., the ethanol concentration offered), the solution (ethanol solution, water, or sucrose solution in the choice experiment), and their interactions with the colony as a random factor. Models used for behavioral experiments included the group as a fixed effect and the colony as a random factor.

Supplementary Material

Appendix 01 (PDF)

pnas.2410874121.sapp.pdf (1.1MB, pdf)

Acknowledgments

We thank Yuval Saar, Danielle Nesher, and Roy Ben Bezalel for their help during the experiments; Levona Bodner, Meray Kadee, and Ehud Fonio for their help in collecting the colonies; and Marshall D. Mccue, Shai Meiri, Tal Pupko, and Inon Scharf for constructive comments on the manuscript.

Author contributions

S.B. and E.L. designed research; S.B., Y.G., and A.G. performed research; S.B. and D.H. analyzed data; and S.B., D.H., and E.L. wrote the paper.

Competing interests

The authors declare no competing interest.

Footnotes

This article is a PNAS Direct Submission.

Contributor Information

Sofia Bouchebti, Email: sofia.bouchebti@gmail.com.

Eran Levin, Email: levineran1@gmail.com.

Data, Materials, and Software Availability

Raw data, sequence alignments, and phylogenetic trees can be accessed in ref. 75 in an open repository at Zenodo with the following link: https://doi.org/10.5281/zenodo.13347489.

Supporting Information

References

  • 1.Dudley R., The Drunken Monkey: Why We Drink and Abuse Alcohol (University of California Press, 2014). [Google Scholar]
  • 2.Room R., Babor T., Rehm J., Alcohol and public health. Lancet 365, 519–530 (2005). [DOI] [PubMed] [Google Scholar]
  • 3.Davis J. R., Li Y., Rankin C. H., Effects of developmental exposure to ethanol on Caenorhabditis elegans. Alcohol Clin. Exp. Res. 32, 853–867 (2008). [DOI] [PubMed] [Google Scholar]
  • 4.Bilotta J., Barnett J. A., Hancock L., Saszik S., Ethanol exposure alters zebrafish development: A novel model of fetal alcohol syndrome. Neurotoxicol. Teratol. 26, 737–743 (2004). [DOI] [PubMed] [Google Scholar]
  • 5.Mustard J. A., Oquita R., Garza P., Stoker A., Honey bees (Apis mellifera) show a preference for the consumption of ethanol. Alcohol Clin. Exp. Res. 43, 26–35 (2019). [DOI] [PubMed] [Google Scholar]
  • 6.Fry J. D., Mechanisms of naturally evolved ethanol resistance in Drosophila melanogaster. J. Exp. Biol. 217, 3996–4003 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Liu S.-X., Du Y.-C., Zeng T., A mini-review of the rodent models for alcoholic liver disease: Shortcomings, application, and future prospects. Toxicol. Res. 10, 523–530 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Poikolainen K., Alcohol and mortality: A review. J. Clin. Epidemiol. 48, 455–465 (1995). [DOI] [PubMed] [Google Scholar]
  • 9.Wiens F., et al. , Chronic intake of fermented floral nectar by wild treeshrews. Proc. Natl. Acad. Sci. U.S.A. 105, 10426–10431 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Xing H., et al. , Establishment of the tree shrew as an alcohol-induced fatty liver model for the study of alcoholic liver diseases. PLoS ONE 10, e0128253 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Guarnieri D. J., Heberlein U., “Drosophila melanogaster, a genetic model system for alcohol research” in International Review of Neurobiology, Bradley R. J., Harris R. A., Jenner P., Eds. (Academic Press, Cambridge, Massachusetts, 2003), pp. 199–228. [DOI] [PubMed] [Google Scholar]
  • 12.Devineni A. V., Heberlein U., The evolution of Drosophila melanogaster as a model for alcohol research. Annu. Rev. Neurosci. 36, 121–138 (2013). [DOI] [PubMed] [Google Scholar]
  • 13.McKenzie J. A., Parsons P. A., Alcohol tolerance: An ecological parameter in the relative success of Drosophila melanogaster and Drosophila simulans. Oecologia 10, 373–388 (1972). [DOI] [PubMed] [Google Scholar]
  • 14.Chawla S. S., Perron J.-M., Radouco-Thomas C., Effects of ingested ethanol on adult Drosophila melanogaster (Diptera: Drosophilidae). Can. Entomol. 113, 315–323 (1981). [Google Scholar]
  • 15.McKechnie S. W., Geer B. W., Regulation of alcohol dehydrogenase in Drosophila melanogaster by dietary alcohol and carbohydrate. Insect Biochem. 14, 231–242 (1984). [Google Scholar]
  • 16.Carvalho A. F., Heilig M., Perez A., Probst C., Rehm J., Alcohol use disorders. Lancet 394, 781–792 (2019). [DOI] [PubMed] [Google Scholar]
  • 17.No level of alcohol consumption is safe for our health (2023). World Health Organization. https://www.who.int/europe/news/item/04-01-2023-no-level-of-alcohol-consumption-is-safe-for-our-health. Accessed 21 May 2023.
  • 18.Tabakoff B., Hoffman P. L., Animal models in alcohol research. Alcohol Res. Health 24, 77–84 (2000). [PMC free article] [PubMed] [Google Scholar]
  • 19.Hunt J. H., The Evolution of Social Wasps (Oxford University Press, 2007). [Google Scholar]
  • 20.Bouchebti S., Bodner L., Levin E., Continuous exchange of nectar nutrients in an Oriental hornet colony. Commun. Biol. 5, 1–6 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Halperin J., Forest-entomological research in Israel. Z. Angew. Entomol. 64, 325–339 (1969). [Google Scholar]
  • 22.Richter M. R., Social wasp (Hymenoptera: Vespidae) foraging behavior. Annu. Rev. Entomol. 45, 121–150 (2000). [DOI] [PubMed] [Google Scholar]
  • 23.Dvoøák L., Social wasps (Hymenoptera: Vespidae) trapped with beer in European forest ecosystems. Acta Mus. Morav., Sci. Biol. (Brno) 93, 105–130 (2008). [Google Scholar]
  • 24.Landolt P. J., et al. , Polistes spp. (Hymenoptera: Vespidae) orientation to wine and vinegar. Fla. Entomol. 97, 1620–1630 (2014). [Google Scholar]
  • 25.Stefanini I., et al. , Role of social wasps in Saccharomyces cerevisiae ecology and evolution. Proc. Natl. Acad. Sci. U.S.A. 109, 13398–13403 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ehlers B. K., Olesen J. M., The fruit-wasp route to toxic nectar in Epipactis orchids? Flora 192, 223–229 (1997). [Google Scholar]
  • 27.Abramson C. I., Place A. J., Aquino I. S., Fernandez A., Development of an ethanol model using social insects: IV. Influence of ethanol on the aggression of Africanized Honey Bees (Apis mellifera L.). Psychol. Rep. 94, 1107–1115 (2004). [DOI] [PubMed] [Google Scholar]
  • 28.Abramson C. I., et al. , Development of an ethanol model using social insects: III. Preferences for ethanol solutions. Psychol. Rep. 94, 227–239 (2004). [DOI] [PubMed] [Google Scholar]
  • 29.Abramson C. I., et al. , The development of an ethanol model using social insects. I: Behavior studies of the honey bee (Apis mellifera L.). Alcohol. Clin. Exp. Res. 24, 1153–1166 (2000). [PubMed] [Google Scholar]
  • 30.Abramson C. I., Sanderson C., Painter J., Barnett S., Wells H., Development of an ethanol model using social insects: V. Honeybee foraging decisions under the influence of alcohol. Alcohol 36, 187–193 (2005). [DOI] [PubMed] [Google Scholar]
  • 31.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). J. Insect Physiol. 52, 1243–1253 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wright G. A., Lillvis J. L., Bray H. J., Mustard J. A., Physiological state influences the social interactions of two honeybee nest mates. PLoS ONE 7, e32677 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Mustard J. A., et al. , Acute ethanol ingestion impairs appetitive olfactory learning and odor discrimination in the honey bee. Neurobiol. Learn. Mem. 90, 633–643 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Giannoni-Guzmán M. A., et al. , Ethanol-induced effects on sting extension response and punishment learning in the Western Honey Bee (Apis mellifera). PLoS ONE 9, e100894 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Mixson T. A., Abramson C. I., Božič J., The behavior and social communication of honey bees (Apis mellifera carnica Poll.) under the influence of alcohol. Psychol. Rep. 106, 701–717 (2010). [DOI] [PubMed] [Google Scholar]
  • 36.Bozic J., Abramson C. I., Bedencic M., Reduced ability of ethanol drinkers for social communication in honeybees (Apis mellifera carnica Poll.). Alcohol 38, 179–183 (2006). [DOI] [PubMed] [Google Scholar]
  • 37.Wilson D. F., Matschinsky F. M., Ethanol metabolism: The good, the bad, and the ugly. Med. Hypotheses 140, 109638 (2020). [DOI] [PubMed] [Google Scholar]
  • 38.Birková A., Hubková B., Čižmárová B., Bolerázska B., Current view on the mechanisms of alcohol-mediated toxicity. Int. J. Mol. Sci. 22, 9686 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Holbrook O. T., Molligoda B., Bushell K. N., Gobrogge K. L., Behavioral consequences of the downstream products of ethanol metabolism involved in alcohol use disorder. Neurosci. Biobehav. Rev. 133, 104501 (2022). [DOI] [PubMed] [Google Scholar]
  • 40.Le Daré B., Lagente V., Gicquel T., Ethanol and its metabolites: Update on toxicity, benefits, and focus on immunomodulatory effects. Drug. Metab. Rev. 51, 545–561 (2019). [DOI] [PubMed] [Google Scholar]
  • 41.Santos K. S., et al. , Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.). Insect Biochem. Mol. Biol. 35, 85–91 (2005). [DOI] [PubMed] [Google Scholar]
  • 42.Geer B. W., Langevin M. L., McKechnie S. W., Dietary ethanol and lipid synthesis in Drosophila melanogaster. Biochem. Genet. 23, 607–622 (1985). [DOI] [PubMed] [Google Scholar]
  • 43.Arrese E. L., Soulages J. L., Insect fat body: Energy, metabolism, and regulation. Annu. Rev. Entomol. 55, 207–225 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Zhang Q., et al. , Genomic expansion of Aldh1a1 protects beavers against high metabolic aldehydes from lipid oxidation. Cell Rep. 37, 109965 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Goulielmos G. N., Loukas M., Bondinas G., Zouros E., Exploring the evolutionary history of the alcohol dehydrogenase gene (Adh) duplication in species of the family Tephritidae. J. Mol. Evol. 57, 170–180 (2003). [DOI] [PubMed] [Google Scholar]
  • 46.Lopez-Osorio F., Pickett K. M., Carpenter J. M., Ballif B. A., Agnarsson I., Phylogenetic relationships of yellowjackets inferred from nine loci (Hymenoptera: Vespidae, Vespinae, Vespula and Dolichovespula). Mol. Phylogenet. Evol. 73, 190–201 (2014). [DOI] [PubMed] [Google Scholar]
  • 47.Lopez-Osorio F., Pickett K. M., Carpenter J. M., Ballif B. A., Agnarsson I., Phylogenomic analysis of yellowjackets and hornets (Hymenoptera: Vespidae, Vespinae). Mol. Phylogenet. Evol. 107, 10–15 (2017). [DOI] [PubMed] [Google Scholar]
  • 48.Carpenter J., Kojima J., Villemant C., Phylogeny of hornets: A total evidence approach (Hymenoptera, Vespidae, Vespinae, Vespa). J. Hymenoptera Res. 32, 1–15 (2013). [Google Scholar]
  • 49.Dudley R., Ethanol, fruit ripening, and the historical origins of human alcoholism in primate frugivory. Integr. Comp. Biol. 44, 315–323 (2004). [DOI] [PubMed] [Google Scholar]
  • 50.Dudley R., Fermenting fruit and the historical ecology of ethanol ingestion: Is alcoholism in modern humans an evolutionary hangover? Addiction 97, 381–388 (2002). [DOI] [PubMed] [Google Scholar]
  • 51.Casey G. P., Ingledew W. M. M., Ethanol tolerance in yeasts. CRC Crit. Rev. Microbiol. 13, 219–280 (1986). [DOI] [PubMed] [Google Scholar]
  • 52.Dietler M., Alcohol: Anthropological/archaeological perspectives. Ann. Rev. Anthropol. 35, 229–249 (2006). [Google Scholar]
  • 53.Schmidt J. M., et al. , Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus. PLoS Genet. 6, e1000998 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Hemingway J., The molecular basis of two contrasting metabolic mechanisms of insecticide resistance. Insect Biochem. Mol. Biol. 30, 1009–1015 (2000). [DOI] [PubMed] [Google Scholar]
  • 55.Miler K., et al. , Alcohol intoxication resistance and alcohol dehydrogenase levels differ between the honeybee castes. Apidologie 52, 230–241 (2021). [Google Scholar]
  • 56.Gong Z., Tan K., Nieh J. C., Hornets possess long-lasting olfactory memories. J. Exp. Biol. 222, jeb200881 (2019). [DOI] [PubMed] [Google Scholar]
  • 57.Bouchebti S., Bodner L., Bergman M., Magory Cohen T., Levin E., The effects of dietary proline, β-alanine, and γ-aminobutyric acid (GABA) on the nest construction behavior in the Oriental hornet (Vespa orientalis). Sci. Rep. 12, 7449 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Berthelot K., Portugal F. R., Jeanson R., Onset of fights and mutual assessment in ant founding queens. J. Exp. Biol. 220, 750–753 (2017). [DOI] [PubMed] [Google Scholar]
  • 59.Bodner L., Bouchebti S., Levin E., Allocation and metabolism of naturally occurring dietary amino acids in the Oriental hornet. Insect Biochem. Mol. Biol. 139, 103675 (2021). [DOI] [PubMed] [Google Scholar]
  • 60.Crowley L. M., Falk S., The genome sequence of the Early Mason-wasp, Ancistrocerus nigricornis (Curtis, 1826). Wellcome Open Research. https://wellcomeopenresearch.org/articles/8-461. Accessed 4 March 2024. [Google Scholar]
  • 61.Favreau E., et al. , Putting hornets on the genomic map. Sci. Rep. 13, 6232 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Crowley L. M., The genome sequence of the German wasp, Vespula germanica (Fabricius, 1793). Wellcome Open Res. 7, 60 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Harrop T. W. R., et al. , High-quality assemblies for three invasive social wasps from the Vespula genus. G3 10, 3479–3488 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Crowley L. M., The genome sequence of the Median Wasp, Dolichovespula media (Retzius, 1783). Wellcome Open Research. https://wellcomeopenresearch.org/articles/8-492. Accessed 4 March 2024. [Google Scholar]
  • 65.Falk S., Broad G. R., The genome sequence of the tree wasp, Dolichovespula sylvestris Scopoli, 1763. Wellcome Open Res 7, 113 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Crowley L. M., The genome sequence of the Saxon Wasp, Dolichovespula saxonica (Fabricius, 1793). Wellcome Open Res. 8, 498 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Standage D. S., et al. , Genome, transcriptome and methylome sequencing of a primitively eusocial wasp reveal a greatly reduced DNA methylation system in a social insect. Mol. Ecol. 25, 1769–1784 (2016). [DOI] [PubMed] [Google Scholar]
  • 68.Childers A., Geib S. M., Smith T. P. L., Foster L. J., Korlach J., Asian giant hornet, Vespa mandarinia, genome assembly. Ag Data Commons. 10.15482/USDA.ADC/1519179. Deposited 6 August 2020. [DOI] [Google Scholar]
  • 69.Patalano S., et al. , Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies. Proc. Natl. Acad. Sci. U.S.A. 112, 13970–13975 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Miller S. E., et al. , Evolutionary dynamics of recent selection on cognitive abilities. Proc. Natl. Acad. Sci. U.S.A. 117, 3045–3052 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Aljassani A. A., Al-Esawy M., Augul R. S., Alisawi O., Phylogenetic relationship of Iraqi Vespa orientalis Linnaeus 1771 wasps using mitochondrial CO1. IOP Conf. Ser.: Earth Environ. Sci. 1262, 032016 (2023). [Google Scholar]
  • 72.Dotan E., Alburquerque M., Wygoda E., Huchon D., Pupko T., GenomeFLTR: Filtering reads made easy. Nucleic Acids Res. 51, W232–W236 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Peng Y., et al. , IDBA-tran: A more robust de novo de Bruijn graph assembler for transcriptomes with uneven expression levels. Bioinformatics 29, i326–i334 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Minh B. Q., et al. , IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Bouchebti S.: Dataset manuscript. Zenodo. 10.5281/zenodo.13347489. Deposited 20 August 2024. [DOI]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix 01 (PDF)

pnas.2410874121.sapp.pdf (1.1MB, pdf)

Data Availability Statement

Raw data, sequence alignments, and phylogenetic trees can be accessed in ref. 75 in an open repository at Zenodo with the following link: https://doi.org/10.5281/zenodo.13347489.

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