Here, we test the hypothesis that wood ants apply self‐produced acids on tree‐collected resin to produce a more potent antimicrobial agent. Specifically, we examined whether (i) ants enhance the antifungal activity of resin, (ii) ants add endogenous acids to resin, and (iii) these acids increase the antifungal activity of resin.

Wood ants are also chemical factories. They produce large quantities of formic acid in their venom gland, which they spray at prey and enemies (Blum, 1992 ; Morgan, 2008 ). In other ant species, formic acid is also present in the trophallactic fluid following oral uptake from the venom gland (Tragust et al., 2013 ), and other acids have been found in the metapleural glands (Vieira, Morgan, Drijfhout, & Camargo‐Mathias, 2012 ). Formic acid has well‐known antimicrobial properties. It is widely used by humans, as cleaning agent and as preservative additive in livestock food (Iba & Berchieri, 2007 ). Moreover, formic acid is effective against Metarhizium , a common fungal pathogen of ants (Graystock & Hughes, 2011 ), and is used by Lasiu s neglectus ants to disinfect their brood (Tragust et al., 2013 ). This suggests that wood ants may combine endogenous acids with tree resin.

Wood ants and honeybees incorporate tree resin with antimicrobial properties into their nest (Christe, Oppliger, Bancala, Castella, & Chapuisat, 2003 ; Simone‐Finstrom & Spivak, 2010 ). In the wood ant Formica paralugubris , workers actively collect large amounts of resin from coniferous trees, which they bring back to their nest and place near their brood (Brütsch & Chapuisat, 2014 ; Castella, Christe, & Chapuisat, 2008 ). Coniferous resin is rich in secondary metabolites with antimicrobial properties (Phillips & Croteau, 1999 ). The presence of resin decreases the overall microbial load in wood ant nests and protects the ants against bacterial and fungal pathogens (Chapuisat, Oppliger, Magliano, & Christe, 2007 ; Christe et al., 2003 ).

An original way to fight enemies is to exploit the defensive chemicals produced by other organisms (de Roode, Lefèvre, & Hunter, 2013 ). Humans use a myriad of chemicals from multiple sources, alone or in synergistic combinations, to medicate themselves, clean their environment, or control pests (Mason & Singer, 2015 ). Animals also harness chemicals produced by other species for their own defense (de Roode et al., 2013 ; Mason & Singer, 2015 ). For example, many insect herbivores sequester plant secondary metabolites to gain protection against predators or parasites (Lefèvre, Oliver, Hunter, & de Roode, 2010 ; Nishida, 2002 ). It has been proposed that animals may combine multiple acquired chemicals to benefit from synergistic effects (Mason & Singer, 2015 ). However, evidence for the use of such “defensive cocktails” by animals remains scant (Mason & Singer, 2015 ).

Animals living in large social groups are exposed to a high risk of epidemics. In response to this threat, social animals have evolved sophisticated individual and collective means to control disease, which combine immunological, behavioral, and organizational defenses (Cremer, Armitage, & Schmid‐Hempel, 2007 ; Naug & Smith, 2007 ; Wilson‐Rich, Spivak, Fefferman, & Starks, 2009 ). Collective defenses include ways to keep the environment hygienic, for example, by removing or neutralizing infectious particles (Morelos‐Juárez, Walker, Lopes, & Hughes, 2010 ; Tragust et al., 2013 ).

2 Materials and Methods

2.1 Effect of wood ants on the antifungal activity of resin In a first experiment, we tested whether spruce tree resin that had been in contact with wood ants had increased inhibitory activity against the generalist entomopathogenic fungus Metarhizium brunneum, compared to resin that had not been contacted by ants. As controls, we used twigs and small stones. Twigs are major constituents of wood ant nests, and small stones are commonly found in some of the nests (Castella et al., 2008). We established experimental groups of workers from field colonies of Formica paralugubris (Chapuisat, Goudet, & Keller, 1997; Christe et al., 2003). We collected pieces of fresh resin from spruce trees, as well as twigs and stones of similar size, in areas away from ant colonies. The pieces of resin, twigs, and stones were disinfected under UV light (30 mn under a UV lamp radiating at 254 nm in a Biosafety Cabinet BSC—700II, HMC Europe). Each tested material (pieces of resin, twigs, and stones) was kept with and without ant workers for 2 weeks. In ant‐exposed treatments, four pieces of the tested material were kept with 40 workers in a small plastic box (13.5 × 15 × 5 cm; n = 25 replicates for each material). In ant‐free controls, four pieces of the tested material were kept in a box without workers (n = 25 replicates for each material). The edges of the boxes were treated with Fluon to prevent ant escape. The workers were free to interact with the pieces of resin, twigs, and stones. They had ad libitum access to water and jelly food consisting of chicken eggs, honey, water, and agar (Reber & Chapuisat, 2012a). After this 2‐week period of exposure to ants or ant‐free control conditions, we measured the inhibitory activity of resin, twigs, and stones against the fungus M. brunneum. We used a strain that had been isolated from Valais, Switzerland, and showed high virulence against Formica selysi (Reber & Chapuisat, 2012b). M. brunneum was described in 2009 and was previously known as M. anisopliae anisopliae (Bischoff, Rehner, & Humber, 2009). A strain of the latter species complex caused high mortality to F. paralugubris (Chapuisat et al., 2007). M. brunneum is used here as a model fungal pathogen, while other pathogens might be important in the field. Indeed, the resin affects a broad spectrum of fungi and bacteria that are potential pathogens of F. paralugubris (Chapuisat et al., 2007; Christe et al., 2003). Inhibitory activity was measured on Malt extract agar nutritive medium in 8.5‐cm‐diameter petri dishes, inoculated by plating 100 μl of 0.05% Tween 20 solution containing 7 × 105 asexual spores (=conidia) of M. brunneum. The four pieces of each material (resin, twigs, or stone) coming from the same experimental box were placed together in a petri dish. The petri dishes (n = 25 per material and treatment) were incubated at 25°C for 6 days. We then photographed each petri dish and measured the spore‐free areas around the tested material with the ImageJ software (Schneider, Rasband, & Eliceiri, 2012). Spore‐free areas either were void of both spores and mycelium or consisted of white and mostly sterile mycelium known as sectors (Ryan, Bridge, Smith, & Jeffries, 2002). For the statistical analysis, we used one estimate of inhibitory activity per experimental box (=replicate). We therefore measured the overall spore‐free area in each petri dish and divided it by four. This is a conservative estimate of the average inhibition halo around each item, because large halos were overlapping. We constructed a model with the spore‐free area as response variable, and the material (resin, twigs or stone) and previous contact with workers (presence or absence of workers in the box) as explanatory variables. The response variable was square‐root‐transformed to achieve homogeneity of variances and normal distribution of residuals, as required for an ANOVA. We carried out post hoc comparisons with Tukey's HSD tests.

2.2 Transfer of endogenous acids to resin and other types of nest material In a second experiment, we examined whether ants applied endogenous acids to pieces of resin, twigs, or stones. We placed four pieces of the tested material (resin, twigs, or stone) in boxes with and without ants as described above, except that there were 50 workers per box in the treatment with ants (n = 10 replicates for each material and treatment type, with or without ants). As organic acids are very soluble in water, we sampled the acids from each material (resin, twigs, or stone) by immersing the four items from the same experimental box in 1 ml of MilliQ water for 30 s. The samples were stored at −20°C until HPLC analysis (see below). We also tested whether the retention and subsequent detection of formic acid varied with the type of material (resin, twigs, and stone). For this, 1 μl of 60% synthetic formic acid (CAS number 64‐18‐6) was deposited on each type of material (10 replicates per material and treatment). After 24 hr, each item was immersed in 500 μl of MilliQ water for 30 s. The samples were stored at −20°C until HPLC analysis. To identify the origin of the acids detected on nest materials, we extracted the content of the venom gland, trophallaxis fluid, and metapleural glands from ten workers anesthetized with CO 2 . For venom and trophallaxis fluid, we gently pressed their gaster and collected the liquid with a microcapillary. For the metapleural glands, we introduced the tip of a microcapillary in the gland opening and extracted the liquid by capillarity. We diluted these extracts in 500 μl of MilliQ water. The samples were stored at −20°C until HPLC analysis. To measure the organic acids in the samples, we analyzed them by high‐performance liquid chromatography (HPLC), using an Agilent HP1100 HPLC system equipped with a diode array detector (DAD), with UV detection wavelength set at 210 ± 2 nm. To remove small particles, the samples were centrifuged (3 min at 182 g) and the supernatant was transferred to 2‐ml glass vials (Interchim, Swiss Labs, Mulhouse, F). We injected 40 μl of the samples onto a 300 mm × 7.8 mm BP‐100 H carbohydrate column (Benson Polymeric, USA). The temperature of the column was maintained at 40°C, and MilliQ water was used as a solvent with 20 mmol/L of sulfuric acid (analysis grade 95–97%, Honeywell, Germany) at a flow rate of 0.4 ml/min. Succinic (CAS number 150‐90‐3, Acros organics, USA) and formic (CAS number 141‐53‐7, Sigma Aldrich, USA) acids were quantified in the samples by external calibration. The linearity of the method was established using six standard solutions at concentration levels from 5 μg/mL to 1.3 mg/mL. We constructed a model with acid quantity as response variable and the material (resin, twigs, or stone) and previous contact with workers (presence or absence of workers in the box) as explanatory variables. We analyzed the data of each acid separately.