If ants have an antibiotic gland, how can they spread hospital infections?

If ants have an antibiotic gland, how can they spread hospital infections?

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Wikipedia describes how antibiotics are produced in ants:

"Metapleural glands… are responsible for the production of an antibiotic fluid that then collects in a reservoir… also referred to as the bulla… From the bulla, ants can groom the secretion onto the surface of their exoskeleton. This helps to prevent the growth of bacteria and fungal spores on the ants and inside their nest."

So how are ants often cited as an important problem in hospitals, spreading infections from garbage and through the patients? Shouldn't their natural antibiotics prevent that?

I have worked in hospitals (US) most of my life, treating both community-acquired, and more pertinently to this question, nosocomial (hospital acquired) infections, and have read many articles on the subject.

I have never, ever seen ants mentioned anywhere.

People, flies, cockroaches and rats, yes.

Ants, no.

However, ants are vectors in a few foreign studies. The key word in these studies is potential.

As you stated,

From the bulla, ants can groom the secretion onto the surface of their exoskeleton. This helps to prevent the growth of bacteria and fungal spores on the ants and inside their nest.

No antibiotic covers all organisms. Also, the secretions are to protect the ants against infection, not against the carrier state.

Pathogenic bacteria is cultured from ants themselves. The ant can be thought of as similar to a human: humans make antibodies to pathogens but are vectors of many diseases. Ants protect themselves via secretions, but are hosts to potential pathogens.

Part I: Review of Scientific Data Regarding Transmission of Infectious Agents in Healthcare Settings
Hospital hygiene and infection control
Ants associated with pathogenic microorganisms in brazilian hospitals: attention to a silent vector
Ants in a hospital environment and their potential as mechanical bacterial vectors Insect/Bacteria Association and Nosocomial Infection

Ants can and do carry loads that are several times their own weight. I grew up in an area with a lot of ants, and a common scene was a long trail of ants acting as a food supply line. Once a morsel is located, they create a long feremone trail to that morsel. A large clump of ants is always working to break the food into smaller pieces, and several ants are usually on their way back to the colony carrying large crumbs. Often these crumbs are the first thing you notice, rather than the actual ants, because they are often so much larger than the ants themselves.

In a hospital, most of the food that ants would scavenge would have fallen from the plate of a human being. Many of those human beings would be carriers of disease. I would expect that the trail that these ants walk is littered with plenty of crumbs-of-crumbs that fall off of their load along the way. If these were large enough for us to see, they would get the attention of the ants and they would carry them back to their colony. But a particle of the microscopic crumb dust they do leave behind might carry several specimens of the germ that sickened some messy patient, even if the ant that dropped it was relatively sterile.

Other than the fact that ants carry bacteria on their food particles, there could also be alternative reasons that they can spread infections.

Just like antibiotics derived from bacteria and fungi have caused resistant pathogen strains to evolve (MRSA for example), it could also be the case that the bacteria naturally living around the ants have evolved some degree of resistance to the antibiotics, perhaps by effluxing sufficient amounts of them by overexpressing pump proteins to the levels that the antibiotics are only mildly bacteriostatic instead of bacteriocidal. These bacteria can then fall off the ants while in the hospital and lead to disease.

It is reasonable to assume that this could evolve, since the ants frequently communicate socially with one another, and any resistant bacteria or fungi can then quickly colonise a previously empty niche, thus creating significant evolutionary pressure for this.

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

1. Introduction

Larger social groups tend to have higher burdens of disease, requiring compensatory measures for prophylaxis and control [1–4]. Human social evolution has seen major increases in disease burden since the dawn of our evolutionary history. Diseases were particularly harmful during the Neolithic agricultural revolution [5] and subsequent urbanization [6], and have only been effectively countered by cultural evolution during the last two centuries [7,8]. The fungus-growing ants (Attini) live in agrarian societies. Like other social animals [5,9], they are under threat from various disease agents (e.g. [10]), but they have also had millions of years of evolutionary time to adapt via natural selection. Across genera and species, attine colony sizes vary from several dozen to millions of individuals and the extent of worker polymorphism increases with colony size [10–12], providing interesting opportunities to understand how disease management strategies covary with social complexity [13–15].

The fungus-growing ants have two obligate symbionts, the basidiomycete cultivar lineages on which they depend for nutrition, and the ascomycete cultivar pathogen, Escovopsis neither symbiont has been found free-living, excepting cultivars from some basal attines [10–12]. To generally control infections, attines have evolved diverse prophylactic and public health tactics, which involve individual behaviours [13–17], collective behaviours [18] and an array of antimicrobial compounds [14,19,20]. Primary sources of these compounds are mutualistic microorganims, including Pseudonocardia and other actinomycete bacteria [19–23], and exocrine gland secretions, particularly from the metapleural glands (MGs) [14,15,24–26].

The paired MGs have clusters of secretory cells connected to a storage reservoir with a narrow opening [12,25–27]. Attine taxa differ in their reliance on bacteria-produced or glandular antimicrobials: species (or genera) with visible cuticular actinomycetes appear to apply MG secretions primarily to protect brood, while MG secretions are used more generally against fungus garden, brood and adult infections in ants that lack these bacteria [14,15]. The significance of MG-secretion use in disease control has largely been inferred from behavioural observations of MG grooming, combined with correlative data on pathogen growth inhibition ([14,15], but see [27–29]). The transfer of MG secretions to infected tissues has not been confirmed by target-specific chemical assays, and few studies have tested the efficacy of particular compounds as antibiotics [30]. In this study, we explicitly test the hypothesis that a single abundant component of MG secretion plays a key role in disease prophylaxis and infection control in Atta leaf-cutting ants.

In vivo, MG secretions of attine ants are known to suppress germination rates of conidia [14,30] and to increase survival of infected workers [31]. In vitro, conidia and mycelia of Escovopsis and other microorganisms show different sensitivity to an array of MG compounds of Acromyrmex leaf-cutting ants [30], but this cannot be expected to apply in the same way in Atta where workers have lost cuticular actinomycete cultures. In Atta cephalotes and Atta sexdens, phenylacetic acid (PAA) is known to be the primary component of MG secretions, accounting for more than 80% and 57.3% of the total mixture, respectively [32,33], whereas the MGs of Acromyrmex workers are not known to produce PAA [33], and MG secretions are rarely used for grooming during Escovopsis infections [14].

We hypothesized that fungal pathogens are sensitive to PAA secreted by Atta leaf-cutting ants, and that this form of chemical defence evolved to replace ancestral biological control via antibiotics derived from cuticular actinomycete cultures [14,20,28]. For PAA to have evolved as a targeted disease management adaptation against specialized garden mycopathogens, including Escovopsis, we expected ants to have a series of correlated behaviours to ensure that the use of PAA is specific and precise, so that the probability that resistance evolves remains as low as possible. We present in vivo and in vitro assays on the pharmacology of PAA in the MG secretion of A. cephalotes to test the extent to which this compound is instrumental in inhibiting Escovopsis, using other generalist pathogens as controls. We discuss the results of our study in relation to complementary information on disease control in fungus-growing ants in general, and Acromyrmex and other Atta leaf-cutting ants in particular. Finally, we address some of the evolutionary factors that may prevent resistance problems in chemical disease control by fungus-farming ants.

2. Material and methods

(a) Collections and hygienic response behaviours

For all experiments, we used subcolonies of A. cephalotes and 15 other attine ant species created from field-collected colonies taken from Soberania National Park or near Gamboa in central Panama between 2004 and 2010 (see the electronic supplementary material). Voucher specimens of the ants are deposited in the Museo de los Invertebrados, Universidad de Panamá. Subcolonies were used to monitor four specific disease defence behaviours: (i) fungal grooming, which involves ants ‘licking’ the garden surface to remove particles that are presumably contaminated [17] (ii) MG grooming, which occurs when a worker extends her legs to raise the body from the substrate, and flexes a foreleg along the femoro-tibial joint to bring the posterior surface of the metatarsus into contact with the opening of one of the MGs (iii) cultivar planting, which involves a worker cutting a piece of healthy fungus garden and transplanting it to an infected garden area to swamp pathogen growth with compensatory growth of the fungal mutualist and (iv) weeding, which occurs when an ant removes a piece of garden and places it in a garbage dump [15,17]. Some prophylactic behaviours result in the accumulation of waste particles in the infrabuccal pockets of worker ants, which are then discarded in the dump [15,16].

Five colonies of A. cephalotes were collected to quantify prophylactic behaviour. From each colony, we established five subcolonies with 1.0 g of fungus garden and 20 medium-sized workers (headwidth (HW) = 1.2–1.6 mm). These subcolonies were subjected to one of four experimental treatments or were left unmanipulated as controls (cf. [28]). The treatments involved single inoculations with dry conidia from the insect pathogens Beauveria bassiana or Metarhizium brunneum, or from one of two strains of Escovopsis isolated from fungus gardens of A. cephalotes. Each experimental subcolony was inoculated with ca 1.5 × 10 6 dry conidia [14,15], and controls were sham-inoculated by rubbing a sterile piece of paper on the fungus garden. Subcolonies were observed for 60 min after inoculation, and the frequency of each prophylactic behaviour recorded. For fungal and MG grooming behaviours, which were relatively frequent, behavioural frequencies were then converted to rates (behaviours per minute) and analysed using a linear mixed model, with experimental treatment as a fixed main effect and colony as a random effect.

(b) Identification of phenylacetic acid in metapleural gland secretion and its distribution in gardens

We selected five small (HW ≈ 1.0 mm) and five large workers (HW > 1.8 mm) from each of five colonies. Workers with full MG reservoirs were selected by the visible presence of milky liquid on the MG bulla, the externally visible cover of the gland reservoir. A fine capillary tube was inserted through the meatus of the bulla, which allowed us to collect ca 0.5–1.0 µl of accumulated secretion from both MGs of each ant [33]. MG secretions of the five workers in each size class were pooled for each nest in a 0.5 ml vial, and dissolved in 20 µl of pentane. We injected 2 µl of each sample into a gas chromatograph–mass spectrometer (GC–MS) to confirm the presence and relative abundance of PAA in MG secretions. We identified PAA by comparison of the mass spectra, gas chromatographic retention indices and retention times with those of a pure reference sample (Sigma-Aldrich). Relative abundance was estimated from the peak area of PAA relative to the sum of all MG components. Full GC–MS protocols are given in the electronic supplementary material.

We confirmed that PAA is not naturally present in the ants’ basidiomycete fungal symbiont or in M. brunneum and B. bassiana cultures. We sampled fungal symbionts from pure cultivars isolated from three colonies of A. cephalotes, using nine samples of symbiont per colony, and 10 samples from pure cultivars of each pathogen. We collected ca 0.05 g of fungus from Petri dishes and placed these fragments directly in a vial with HPLC-grade pentane for GC–MS analyses. To determine whether PAA was present in the fungus gardens after MG grooming, we used three colonies of A. cephalotes, constructing 20 subcolonies with 1 g of fungus garden, 20 medium workers and six pupae from each colony. We inoculated the fungus gardens of 10 of these with ca 1.5 × 10 6 dry conidia of M. brunneum before adding the workers, and in the remaining 10 a sterile piece of paper was rubbed on the fungus garden as a control treatment before adding workers [14]. Three hours later, we froze the Petri dishes at −20°C for 20 min, and then placed 0.05 g of the fungus garden in a vial with solvent for GC–MS analyses.

(c) Phenylacetic acid transfer during metapleural gland grooming

To determine whether PAA is transferred from the ants' MG to infection targets in the garden, we compared extracts from the surface of the tarsi of workers from infected and uninfected A. cephalotes subcolonies using GC–MS. Twenty subcolonies from each of three A. cephalotes colonies were established with 15 medium workers and 1.0 g of fungus garden each (total subcolonies, N = 60). Subsequently, 30 subcolonies were used as unmanipulated controls and 30 other subcolonies were each inoculated with ca 2.5 × 10 6 dry conidia of M. brunneum. We increased the concentrations of conidia in this experiment to ensure that almost all ants groomed their MG after the infection [28]. Sixty minutes after infection, the subcolonies were transferred to a −20°C freezer for approximately 20 min to kill all ants. For each infected and control subcolony, we randomly selected six workers, removed their fore-, middle- and hind-legs and placed these in separate vials with pentane. After allowing to stand for 5 min, 2 µl of the supernatant was injected into the GC–MS to determine the presence of PAA. Presence–absence data were analysed using a contingency table.

To assess the quantitative transfer of PAA to pathogens, we created nine subcolonies from each of four A. cephalotes colonies, each one containing 1.0 g of fungus garden, three pupae and 30 randomly selected media workers. Three subcolonies were each infected with ca 1.5 × 10 6 dry conidia of M. brunneum, B. bassiana or Escovopsis, as described above. The Escovopsis used was derived from a separate colony of A. cephalotes collected from the same field site as the experimental colonies. After inoculation with fungal conidia, workers groomed the fungus garden so that detritus was accumulated in the infrabuccal pockets and subsequently discarded as infrabuccal pellets [15]. Three hours after inoculation, the subcolonies were frozen for 20 min after which 30 intact pellets were collected and pooled in a single vial that was stored at −20°C prior to chemical analysis. The chemical composition of pellets was assayed by placing the 30 pellets per treatment in 25 µl pentane, containing 0.001 µg µl −1 pentadecane as an internal standard, and allowed to stand for 5 min. We then injected 2 µl of this extract into the GC–MS and estimated absolute PAA concentration by comparison to the internal standard. Concentrations of PAA were compared using a linear mixed model with pathogen type (M. brunneum, B. bassiana or Escovopsis) as a fixed main effect, and colony, and colony by pathogen interaction as random effects. PAA concentrations were log-transformed to homogenize within-group variance.

(d) Inhibitory effects on pathogen spore germination in Atta

Eight colonies of A. cephalotes were used to quantify the inhibitory effect of MG secretions on germination rates of conidia that ant workers had accumulated in their infrabuccal pellets after infection. Four colonies were exposed to M. brunneum and four to Escovopsis isolated from a separate A. cephalotes colony from the same collection site. To assess whether ants reduced the viability of pathogenic conidia, we recorded the germination of infrabuccal pellets following infection for four HW size classes of ants: minims (HW less than 0.8 mm), minors (HW 1.0–1.2 mm), media (HW 1.2–1.6 mm) and majors (HW more than 1.8 mm). From each colony, we created 12 subcolonies (three replicates for each HW size class), each having 1 g of fungus garden, 20 workers, three larvae and three pupae, and exposed them to ca 2.5 × 10 6 dried conidia of M. brunneum or Escovopsis, a high enough dose for the ants to rapidly generate and discard infrabuccal pellets with fungal pathogen conidia.

Three hours after infection, all infrabuccal pellets deposited by ants were collected from the subcolonies with a sterile needle and plated on Petri dishes with potato dextrose agar (PDA, 19.5 g per 500 ml distilled water) [28]. Seventy-two hours later, we counted the number of pellets showing fungal germination consistent with the morphology of the pathogen used. Germination rates were compared using a generalized linear mixed model using binomial errors. Main effects were pathogen type (M. brunneum or Escovopsis), worker size class and their interaction. Colony was included as a random variable nested within pathogen type, because different colonies were treated with different pathogens.

(e) Comparative analyses of phenylacetic acid production and inhibitory efficiency across attine ants

To determine the distribution of PAA in MG secretions across the attine ants, we assayed 16 representative species: Mycocepurus smithii, Apterostigma collare, Ap. goniodes, Myrmicocrypta ednaella, Cyphomyrmex longiscapus, Trachymyrmex sp. 10, Trachymyrmex sp. 3, T. cornetzi, T. zeteki, Sericomyrmex amabilis, amabilis, Acromyrmex echinatior, Ac. octospinosus, A. cephalotes, A. sexdens and A. colombica. Colonies were maintained in the laboratory for at least two weeks prior to experiments (see the electronic supplementary material). We sampled five nests for each species and created one subcolony from each of them with 0.5 g of fungus garden, three pupae and 20 workers. These subcolonies were infected with ca 1.5 × 10 6 dry conidia of M. brunneum and 4–7 h later infrabuccal pellets deposited by the workers were collected (N = 25 pellets per subcolony for leaf-cutting ants and N = 50 pellets from non-leaf-cutting species, to obtain a similar amount of pellet material). The pellets were placed in a vial and frozen at −20°C until GC–MS analyses, when we added 20 µl of pentane to each vial, and gently shaken until the pellet dissolved. For each sample, 2 µl of this solution was injected into the GC–MS. We used data from an earlier study [34] to compare the frequency of MG grooming with the presence of PAA in the infrabuccal pellets.

We measured the inhibitory effect of PAA on Escovopsis strains isolated from the gardens of 10 attine species, representing eight genera (Myc. smithii, Ap. pilosum, Myr. ednaella, C. costatus, S. amabilis, T. zeteki, Ac. echinatior, Ac. octospinosus, A. cephalotes and A. colombica). Macromorphology, conidia colour and growth rates on plates were used to distinguish Escovopsis morphotypes [35–38], which confirmed that we worked with a good representation of the overall phylogenetic diversity (electronic supplementary material, figure S1). In addition to Escovopsis, we also tested the sensitivity towards PAA of two generalist entomopathogens, M. brunneum, B. bassiana and two weedy Trichoderma contaminants collected from Atta fungus gardens (see the electronic supplementary material for mycological methods).

Using PDA as a growth medium in Petri dishes (15 × 60 mm), we added different concentrations (100, 200, 400, 500 and 800 µg ml −1 ) of analytical standard PAA (Sigma-Aldrich), using the polar aprotic solvent dimethyl sulfoxide (DMSO) as a carrier. Three days later, we inoculated an 8-mm diameter plug of pure culture from each fungus on three replicate dishes for each PAA concentration and added three controls (PDA only, PDA + DMSO and 100 µg ml −1 of the fungicide cycloheximide as a positive control). Ten days after treatment, we used a Nikon Coolpix L110 camera to photograph each dish to determine the extent of fungal growth, after which we calculated growth areas (cm 2 ) using the I mage J v. 1.37v software (following [39]) (electronic supplementary material, figure S1). The effect of PAA concentration on fungal growth was modelled by fitting a generalized linear model with binomial errors for each Escovopsis strain, based on the proportional cover out of the total available medium area (21 cm 2 ). The PAA concentration at which the fungal growth was restricted to 50% of the dish area (ID50) was estimated by inverse prediction from the fitted models.

3. Results

(a) Phenylacetic acid as defensive compound to control infection with fungal pathogens

The frequency of fungal grooming and MG grooming by A. cephalotes workers was significantly different between the infection treatment groups and controls (one-way ANOVA, F4,16 = 8.71, p < 0.001 and F4,16 = 4.44, p = 0.013, respectively electronic supplementary material, table S1), but these frequencies did not differ across the four types of pathogen challenges (Tukey post hoc tests p > 0.05). No differences across treatments were observed in either cultivar planting or weeding behaviours shortly after infection (both p > 0.5 electronic supplementary material, table S1).

PAA was present in all samples of A. cephalotes MG secretions collected (figure 1), but the collection procedure meant that many samples were contaminated with compounds typically found in haemolymph. The four samples without such contamination confirmed that PAA was the dominant compound in the MG secretion in both large (relative abundance: 54 and 88%) and small (78 and 81%) workers. PAA was also detected in all pellets produced by the four worker size classes. No PAA was detected in pure cultures of the fungal symbiont or any of the pathogens, but PAA was detected in a total of five out of 30 fungus gardens of subcolonies following infection with M. brunneum (electronic supplementary material, table S2). No PAA was detected on fore-, mid- or hind-legs prior to infection, and after infection PAA was found only on the forelegs (electronic supplementary material, table S2 N = 171, χ 2 = 20.03, d.f. = 2, p < 0.0001) implying that worker ants transferred PAA to point sources of infection by grooming the MG opening with their forelegs and then contacting the target.

Figure 1. Typical gas chromatogram profile of the secretion of the metapleural gland of an A. cephalotes worker. The large peak at a retention time of 10.21 min was identified as PAA (structure shown to the right of the peak) based on its mass spectrum (inset).

Colonies did not differ significantly in their inhibitory effect against pathogens (random effect Z = 1.55, p = 0.122) but there were clear differences among worker size classes (figure 2 F3,88 = 42.2, p < 0.001). The smallest workers more strongly inhibited conidia germination of pellets of both M. brunneum and Escovopsis sp. than medium and larger workers (figure 2). All four worker-size classes inhibited growth from Escovopsis pellets significantly more than growth from M. brunneum pellets (figure 2 F1,7 = 49.6, p < 0.001), with differences in inhibition efficiency across size classes also being highly significant (size class × fungus type interaction: F3,88 = 6.87, p < 0.001).

Figure 2. Use of PAA from metapleural glands of different worker size classes of A. cephalotes, showing the mean (+s.e.) proportion of infrabuccal pellets that did not germinate. Minim workers (HW = 0.6–0.8 mm) and minor workers (HW = 1.0–1.2 mm) more strongly inhibited germination of M. brunneum spores (black bars) than Escovopsis sp. (isolated from an A. cephalotes nest) (grey bars) relative to media (HW = 1.4–1.6 mm) and major workers (HW = 1.8–2.2 mm).

Medium-sized workers of A. cephalotes did not differ in the quantity of PAA transferred to pellets following infections with different pathogens (figure 3 F2,6 = 0.375, p = 0.702), and there was no effect of colony of origin (treated as a random effect) in this analysis (F3,6 = 0.576, p = 0.652), suggesting that colonies use PAA for a wide range of fungal infections. There was, however, a significant pathogen by colony interaction (F6,24 = 4.15, p = 0.005, figure 3), suggesting that colonies may respond differently to different fungal infections.

Figure 3. Mean quantity of PAA + s.e. extracted from infrabuccal pellets produced by A. cephalotes workers when challenged with three different fungal pathogens across four test colonies.

(b) Phenylacetic acid as an evolutionarily derived substance for chemical disease control in Atta

PAA was found in all fungal pellets deposited by workers of A. colombica, A. sexdens and A. cephalotes, but was never detected across a representative selection of sympatric attine species from seven other genera, including species such as Trachymyrmex sp. 10 and S. amabilis that are known to have high rates of MG grooming [14,15,34] (electronic supplementary material, table S3) and few if any actinomycete bacteria as alternative defence. PAA was also absent in two species of the sister leafcutter ant genus, Ac. echinatior and Ac. octospinosus, that have cuticular actinomycetes and intermediate rates of MG grooming when their nests are infected with M. brunneum (electronic supplementary material, table S3).

Bioassays showed that PAA inhibited the growth of Escovopsis morphotypes to a different extent. Morphotypes obtained from lower attine species were more sensitive (ID50 i.e. the concentration required for 50% inhibition: mean ± s.e. = 156.8 ± 32.8 μg ml −1 ), while morphotypes isolated from higher attines (including leaf-cutting ants) required higher concentrations for inhibition (figure 4: ID50 = 373.6 ± 15.1 μg ml −1 t8 = 6.76, p < 0.001). Within the higher attines, however, there was no significant difference between the sensitivity of morphotypes from leaf-cutting and non-leaf-cutting ants (t4 = −0.235, p = 0.826). No Escovopsis morphotype grew at PAA concentrations of 800 μg ml −1 , but the entomopathogen M. brunneum and the two strains of Trichoderma showed less than 50% inhibition at this high concentration. Beauveria bassiana showed generally low growth in the Petri dishes, but was rather insensitive to PAA concentration (range of inhibition always 50–85% across the 0–800 μg ml −1 range).

Figure 4. Inhibitory effects of PAA on Escovopsis strains taken from the fungus gardens of different species of attine ants and on other more general fungal pathogens. Inhibition was measured as the proportion of the Petri dish area with PDA medium that remained free of Escovopsis growth after 10 days. Circles mark the estimated ID50 (concentration of PAA that restricted growth to 50% of the plate area) for each fungal strain, with error bars showing the 95% confidence limits of the estimate. Estimated ID50 values for non-Escovopsis fungi are either less than 0 or more than 800 µg ml −1 . The attine ants from which Escovopsis strains were isolated are grouped according to the type of fungal agriculture they practice [10].

4. Discussion

This study documents the use of an antimicrobial agent by Atta leaf-cutting ants for controlling both specialized and generalized mycoparasites. Atta fungus-growing ants have evolved truly large-scale farming and ‘organismal’ colonies, characterized by extreme caste differentiation and almost complete absence of the typical reproductive conflicts that characterize many eusocial Hymenoptera [40,41]. Our results suggest that they have also evolved unique innovations in disease management. Advances in disease management have also characterized the cultural evolution of large-scale human societies [5], so it is of interest to explore the extent of analogy across these two domains of social evolution. We address how our study complements earlier work on leaf-cutting ants and their close relatives, what makes Atta disease management unique and how even the most sophisticated insect societies continue to face threats from rapidly evolving diseases.

(a) Unravelling the details of prophylactic and acute disease control in leaf-cutting ants

Results confirmed the hypothesis that PAA has antimicrobial activity [42], and that it is the most abundant MG compound in A. cephalotes [33]. PAA has previously been shown to be the most abundant MG compound in A. sexdens [24,33], and second most abundant in A. laevigata [25]. While it has previously been inferred that the less abundant organic acid Myrmicacin (3-hydroxydecanoic acid) is used to control fungus garden ‘weeds’ by Atta [32], we did not detect this compound in the secretions of A. cephalotes. We estimated that a medium-sized worker can produce 0.45 µg of PAA during an infection, which is considerably less than the 1.4 µg of PAA that was estimated to be produced by an ‘average sized’ A. sexdens worker [12]. Heuristically, using our lower value, a mature colony of A. cephalotes with 1 million medium workers could thus produce roughly 0.5 g of PAA. Future studies are needed to determine the effective in vivo dosage of PAA to inhibit the spread of Escovopsis and other pathogens within the garden, and whether this massive production helps explain why there is little evidence that Escovopsis regularly kills large colonies of any Atta species, even though chronic infections are widely prevalent [43]. Earlier studies have reported that PAA is only found in Atta, but not in other genera of attine ants [24,25,32,33,44], a result that we confirmed across a substantial selection of Panamanian attine ants. This study complements our earlier findings that Atta has adopted synthesized chemical disease control, in contrast to its sister taxon of leaf-cutting ants, Acromyrmex, which has maintained ancestral cuticular cultures of Pseudonocardia actinomycetes to control Escovopsis infections [14,45]. However, a shift from biological to chemical control of a specialized pathogen is not necessarily more sustainable [14,28], unless enhanced care and flexibility of use outweigh possible higher risks of resistance evolution over time.

The results of our study confirm that the cocktail of behavioural and chemical tools that Atta workers employ largely lives up to common-sense sustainability criteria [14,15]. The main active control component PAA appears to be dynamically transferred from the MGs to the forelegs of workers to infection targets of both insect and fungus garden pathogens, after which a series of other grooming behaviours ensures that infective particles accumulate in the infrabuccal pockets of (particularly small and medium size) workers, where they are killed prior to permanent removal from the nest. Our study thus provides experimental evidence that has so far been lacking [27,28] in showing that Atta workers can control conidia germination rates of specialized (Escovopsis strains) and generalized (B. bassiana and Metarhizium anisopliae) fungal pathogens, but that they are especially efficient in controlling different morphotypes of Escovopsis, the only known pathogen that specializes on attine fungus gardens. Due to this specialization, it is therefore most likely to show coevolutionary responses to increased control efficiency.

The in vivo experimental results confirmed that A. cephalotes workers increased their frequency of MG grooming when exposed to a challenge by fungal conidia, but that this increase was independent of the fungal species used, as observed in Acromyrmex when workers were inoculated with conidia of Metarhizium and Escovopsis [29], indicating that PAA is active against both specialized and generalist pathogens. Our finding that the amount of PAA transferred may be different for different colonies exposed to different pathogens suggests that Atta workers can adjust both the quantity and quality of their MG secretion in response to the type of disease challenge, as was recently shown for sympatric Acromyrmex [29]. Two studies have now shown that MG compounds and secretions from Acromyrmex species can reduce mycelial growth or inhibit germination of Escovopsis conidia [29,30], but Acromyrmex workers only occasionally target Escovopsis infections behaviourally with their MG secretions [14], consistent with their actinomycetes being the main control strategy [14,45]. Our in vitro assays further showed that the PAA concentrations required to control Escovopsis were lower than those needed to control other pathogens, consistent with the hypothesis that these MG secretions, and PAA in particular, are alternative functional defences that have replaced actinomycete-bacteria-produced antibiotics used by Acromyrmex and most sympatric Trachymyrmex to control Escovopsis [14,15,28].

(b) Can chemical pest control in large-scale ant farms meet resistance problems?

Atta and Acromyrmex both process live vegetation for fungus farming, and their alternative systems of chemical and biological pest control have likely been evolutionarily elaborated since the genera split about 10 Mya [10]. The hypothesis that Atta would not have been able to overtake Acromyrmex in terms of ecological footprint (and pest-ant status) unless it had evolved ways to prevent or severely limit the evolution of resistance against chemical pest control seems reasonable. Understanding how this was achieved is important, because of the analogies with human large-scale farming that is plagued by problems of resistance to pesticides after only a few decades of use [46]. It is interesting that higher concentrations of PAA are required to inhibit other pathogens (figure 4) with the exception of B. bassiana, which suggests that PAA synthesis could have evolved because Escovopsis is particularly vulnerable to this compound. It is also noteworthy that Escovopsis strains from lower attine species (i.e. more basal attine ant genera) were more susceptible to PAA (ID50 around 160 µg ml −1 ) than Escovopsis isolated from the gardens of higher attines (ID50 around 370 µg ml −1 ). This result agrees with previous studies of Escovopsis isolates from Atta showing that these strains are generally different from those found in non-leaf-cutting higher attine ants [36] and even more different from those normally found in lower attines [37,38]. Only Escovopsis strains from the basal attine genus Apterostigma are highly variable, with some strains being similar to those found in Atta [36–38].

Differential patterns of susceptibility and resistance are often inferred to be the result of an evolutionary arms race in which natural antagonists have achieved a dynamic tug-of-war equilibrium. As Atta and Acromyrmex are likely to share the same strains of Escovopsis [35] and rear very similar strains of the symbiotic cultivar [47,48], it is not surprising that Escovopsis strains isolated from Atta and Acromyrmex were equally sensitive to PAA. However, horizontal transmission of fungus garden strains between sympatric colonies of Atta and Acromyrmex does not appear to occur at our Panamanian study site [48], so that any horizontally transmitted Escovopsis strain parasitizing both Atta and Acromyrmex will face the challenge of having to cope with both actinomycete bacterial antibiotics and ant PAA across generations. This may make it harder for Escovopsis to evolve higher virulence and raises the question of why Escovopsis lineages do not seem to have evolved specificity for either Atta or Acromyrmex [35].

The PAA-based chemical control practices of Atta have many characteristics that may help to maintain sustainability even if there would be no sympatric Acromyrmex hosts to co-infect. First, the results of our present study show that PAA is never used prophylactically (electronic supplementary material, table S1), i.e. it is not detected in gardens or on the legs of ants unless there is an infection. Second, PAA application is extremely precisely targeted only to sources of infection and reinforced by elaborate complementary behaviours such as active weeding, manual grooming and concentrated treatment in the infrabuccal cavity (i.e. away from the garden) to reduce germination success of spores of pathogenic fungi. Remarkably, the infrabuccal pellet treatment was considerably less effective against Metarhizium, a generalist insect pathogen that will never evolve resistance [49], than against the specialist garden pathogen Escovopsis (figure 2). Although Escovopsis spores in deposited pellets can still partly germinate in vitro (figure 2), this would hardly be a selection factor for resistance unless these hyphae can find their way back to a fungus garden to sporulate, which seems highly unlikely as they will be unattractive for foraging workers. Further research in the dynamic interactions between Atta, Acromyrmex and their cultivar and Escovopsis symbionts will remain highly rewarding for understanding the general emergence or avoidance of resistance problems in farming symbioses.

(c) The comparative perspective of disease management in fungus-growing ants

Despite substantial progress, we lack sufficient comparative data to fully understand the parallel transitions in public health systems that co-occurred with evolutionary transitions in attine fungiculture and social organization [10,11,14,15,17]. We know that dramatic changes in the sources of antimicrobial compounds have occurred across the attine phylogenetic tree, such that actinomycete-bacteria-produced antimicrobials appear to be key for controlling Escovopsis in many species of Trachymyrmex and Acromyrmex, and in several genera of basal attine ants, while MG secretions fulfil this role in Atta, Sericomyrmex [14] and possibly in some Trachymyrmex species [15] that lack visible actinomycetes. However, the interactions among different antimicrobials are incompletely understood. For example, we do not know if other MG components, other exocrine products or bacterial metabolites may enhance the inhibitory effect of PAA or constrain responses due to trade-offs with other vital functions. Furthermore, information about the antimicrobial compounds produced by the cuticular actinomycetes associated with Acromyrmex and Trachymyrmex species is very scarce [21–23], limiting our understanding of the biocontrol system that ancestral Atta species abandoned when evolving PAA-based chemical control.

Finding that the smallest Atta workers have the greatest inhibitory effect on pellet germination is of interest. These tiny workers are actively recruited by larger workers to sites of infection [50], which suggests they have a special role in disease management, consistent with their MGs being disproportionately large compared with those of larger workers [51]. Comparative studies between attine and non-attine ants, and between leaf-cutting versus non-leaf-cutting ants, have shown that Atta and Acromyrmex both have significantly enlarged MGs across all worker castes [52,53]. Relative MG-sizes also vary between patrilines in the same colony of two Panamanian Acromyrmex species [54]. MG size is thus heritable in the sterile castes but not in queens, posing intriguing questions about trade-offs with other costly performance traits, both in general and for disease management in particular [54].

Relative abundance of visible actinomycetes on ants' exoskeletons [14] and MG-grooming are key traits, but we still know rather little about the pathogen pressure on leaf-cutting ant nests in the field ([20,28], but see [55]), seasonal variation in disease pressure and defence [56], and the possible roles of myrmecophilous arthropods in disease transmission or control [12]. Detection and cognition are also likely to be of key significance, as the smallest Atta worker castes invest more biomass in their brains relative to larger nest-mates [57,58], but it remains unknown whether they have a heightened sensitivity to cues indicating infection. Large workers of Ac. echinatior are better at discriminating between fungal symbiont strains than medium and small workers, consistent with division of labour in cognitive tasks across worker castes [59] and with disease removal tasks that require significant time and metabolic efforts being delegated to the small worker castes.

Finally, it is intriguing that PAA has evolved repeatedly as an antimycotic compound in microorganisms (e.g. [42,60]) and that some actinomycete bacteria of the genus Streptomyces also produce PAA [42] while other strains of this actinomycete genus have been isolated from the cuticle of Acromyrmex ants producing other antibiotics [22,23]. However, nothing is known about in vivo effective dosages of PAA when used as an antimycotic by attine ants, and little is known about the quantitative production of PAA by bacteria (e.g. [42,60]). In humans, PAA is a very potent compound that controls pathogens at concentrations of 2 µg ml −1 [60], further adding to the unresolved question why this compound is not more common among attines if it is so potent for fungal disease control.

Regulation and specificity of antifungal metapleural gland secretion in leaf-cutting ants

Ants have paired metapleural glands (MGs) to produce secretions for prophylactic hygiene. These exocrine glands are particularly well developed in leaf-cutting ants, but whether the ants can actively regulate MG secretion is unknown. In a set of controlled experiments using conidia of five fungi, we show that the ants adjust the amount of MG secretion to the virulence of the fungus with which they are infected. We further applied fixed volumes of MG secretion of ants challenged with constant conidia doses to agar mats of the same fungal species. This showed that inhibition halos were significantly larger for ants challenged with virulent and mild pathogens/weeds than for controls and Escovopsis-challenged ants. We conclude that the MG defence system of leaf-cutting ants has characteristics reminiscent of an additional cuticular immune system, with specific and non-specific components, of which some are constitutive and others induced.

1. Introduction

The metapleural glands (MGs), paired structures at the posterolateral margin of the mesosoma, are found only in ants and are one of the defining apomorphies of the family Formicidae [1,2]. The secretions of MGs may have many functions, but the production of anti-microbial compounds for general nest sanitation is the most widespread and general [3–6]. The evolution of glands that produce secretions with generalized prophylactic functions seems a logical adaptation for ants, because colonies are often densely packed and individuals interact continuously with the nest substrate (usually soil) and food (often cadavers), where micro-organisms abound [7,8]. Despite their likely importance for the evolution and diversification of the ants, few studies have investigated the details of MG function. The taxonomic coverage of these studies is scattered, and most have primarily focused on ants with exceptionally derived MG functions [9,10].

The leaf-cutting ants (Attini) are a partial exception to this dearth of information, as MG reservoir size can be measured relatively easily, and these sizes show interesting allometries across worker castes [11–13]. The anti-microbial role of MG secretions in leaf-cutting ants is potentially of particular significance, because these ants have to protect both themselves against entomopathogens, and their mutualistic fungus gardens against parasites and competitors [14], which appears to have led to significantly enlarged MGs in Atta and Acromyrmex leaf-cutting ants [13], where the secondary evolution of queen multiple-mating has also allowed considerable genetic variation for MG size to be maintained [15,16].

Studies on MG secretions in attine ants have shown that the production of these secretions is metabolically costly [17], that they contain both a diversity of carboxylic acids of various chain lengths and proteinaceous compounds [18,19], and that their functioning may be subject to metabolic trade-offs [20,21]. However, specific tests of antimicrobial function have remained rare, mainly due to the technical difficulties of extracting the very small quantities of these glandular secretions [9,18,19]. Hence, the antimicrobial role of MG secretions has usually been inferred rather than measured, or has focused on single compound testing rather than condition-dependent variation in natural MG secretions [5].

MG secretions were believed to mainly spread passively over the ant cuticle [22], but a recent study by Fernández-Marín et al. [23] showed that active acquisition of small quantities of secretion with the front legs followed by focused grooming frequently occurs across the fungus-growing ants. Most bacterial, viral and protozoan disease propagules must be ingested by ants to become infective, but pathogenic fungi enter the ant hosts via the cuticle [8]. This has led to the expectation that MG secretions in leaf-cutting ants may function predominantly as an antifungal defence, a notion that was corroborated by Fernández-Marín et al. [21,23], showing that a number of attine species increase their active grooming rate with MG secretion after being exposed to fungal conidia (asexual spores). The ants also apply MG secretions to fungus garden infections, after which the compromised but treated mycelial debris is stored in the infrabuccal pocket (a cavity just behind the ant mouthparts), where any remaining conidia are likely to be killed by the mixture of MG and labial gland secretions before debris-pellets are discarded [23,24]. However, it has remained unknown whether any infection-induced increase in grooming with MG secretions is accompanied by an increase in quantity or adjustment in the specific potency of the secretions.

The present study examined whether the MG secretions of Acromyrmex octospinosus leaf-cutting ant workers can be adjusted according to the fungal conidia to which the ants are exposed, using five species of fungi representing three broad categories of threat (entomopathogen, generalist saprophyte and fungus garden pathogen).

2. Material and methods

(a) Fungal inoculation experiments

Four representative colonies of Acromyrmex octospinosus, collected in Gamboa, Panama, in 2005 (colony Ao273) and 2007 (colonies Ao404, Ao482 and Ao492), were used in the experiments. Colonies were picked because of their similarity in size, with approximately 1 litre of fungus garden and 50–100 large garden workers being easily available at each of the consecutive bouts of sampling. The colonies were kept under standardized conditions in a climate room at 25°C and 70 per cent relative humidity at the University of Copenhagen. To avoid age- or caste-specific variation in the composition of the MG secretions, we used only large garden workers of approximately the same intermediate age class [25].

Two entomopathogenic fungi (Beauveria bassiana and Metarhizium brunneum—previously called Metarhizium anisopliae, but now distinguished as a sibling species [26]), two saprophytic fungi with the potential of causing low pathogenicity (Aspergillus niger and Gliocladium virens) and a specialized parasite that attacks the fungal cultivar of the ants (Escovopsis weberi) were chosen as disease agents. Beauveria bassiana (KVL 03-90) and M. brunneum (KVL 04-57) were obtained from the stock collection of the Department of Agriculture and Ecology, University of Copenhagen, whereas A. niger (DSM 1957) and G. virens (DSM 1963) were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The E. weberi strain was collected from an A. octospinosus nest in Gamboa by Hermogenes Fernández-Marín in early 2010, and identified based on morphological characters [27].

We first tested the pathogenicity and virulence of these five fungal species on A. octospinosus workers by inoculating ants with fungal conidia and monitoring their survival. From each colony, we collected a random sample of 60 major workers, of which we inoculated half and used the other half as controls. Inoculations were conducted by gently grasping individuals with a pair of sterilized soft forceps, and pipetting 2 μl of fungal (approx. 10 7 conidia ml −1 ) suspension in 0.05 per cent Triton-X (to avoid conidia clumping) onto the propodeum. Inoculated individuals were placed separately into plastic pots (diameter 2.5 cm, height 4 cm), where they were maintained at 25°C with an ad libitum supply of 10 per cent sucrose water. The other half of the workers (control group) were treated with 2 μl 0.05 per cent Triton-X solution applied in the same way. Subsequently, ant mortality was assessed daily for 14 days (survival data in electroic supplementary material, table S1). Ants that died during this period were surface-sterilized by rinsing with 70 per cent ethanol followed by 96 per cent ethanol to avoid fungal growth from external contaminants. The surface-sterilized ants were placed in a plastic pot lined with damp filter paper. Cadavers were inspected after 10 days to score the presence or absence of hyphal growth on the cuticular surface, and to check whether these hyphae produced conidia of the fungus with which the ants were inoculated (sporulation data in electronic supplementary material, table S2). The fungal inoculations and controls were carried out simultaneously so that ant mortality assessment and cadaver inspections were completed within two weeks from the start of the experiments.

The workload of the experiments was such that the treatments could only be handled in parallel by same person (the first author) for two colonies at a time. The experiments with colonies Ao273 and Ao404 were therefore carried out six months before the experiments with colonies Ao482 and Ao492. This reduced error variance as the variance across colonies could be partialled out in subsequent statistical analyses (where it turned out to be insignificant).

Table 1. Repeated-measures MANOVA, testing the inhibitory efficiency of metapleural gland secretions of major workers of A. octospinosus (dependent variable) across five fungal infections and controls, after inoculation with the same fungal species (treatment) and with four different colonies as independent factors. For within-bioassays, we give approximate F statistics based on Wilks's lambda for between bioassays, we report exact F statistics.

(b) Extraction and quantification of metapleural glands secretions

To examine the quantitative responses of MG secretion in inoculated ants, we set up a second experiment structured as two-way repeated measures factorial design, to measure the amount of MG secretions from inoculated and control ants for each of the five different fungal species. To inoculate ants with each of the five fungal species and obtain sufficient amounts of the glandular secretions after these treatments, we took out 30 major workers from each colony at a time and divided these randomly into six groups of five individuals to be inoculated with the five different kinds of conidia or to serve as controls.

The inoculation procedure was as mentioned before, but the ants were killed by freezing in liquid nitrogen 12 h after inoculation, and gland extractions were carried out immediately after killing. Dipping in liquid nitrogen serves two purposes in glandular extractions: (i) to prevent passive flow or active grooming of MG secretions during the extraction process, and (ii) to halt the chemical reactions of MG secretions so that all secretions collected in the reservoir (a sclerotized atrium where glandular secretions were stored [6]) represented the same state (after fungal inoculations or control solution application) at the time of extraction. The amount of secretion was measured by piercing the MG reservoir (externally visible as the bulla [28]) with a very fine insect-mounting needle, after which a graduated 10 μl Hamilton syringe was inserted to extract accumulated secretion. Secretions from both reservoirs of the paired glands were extracted in the same syringe (i.e. pooled), and their volume recorded (MG quantity data in electronic supplementary material, table S3). To ensure that all secretions present were extracted with our method, we dissected the reservoir from a sample of ants after glandular extractions with a fine razor blade and examined the reservoir under the microscope. These reservoirs were found to be empty and dry [17]. Similar dissections were carried out on a sub-sample of ants prior to glandular extractions, and these reservoirs were found to be either wet or with secretions ‘oozing’ from the punctured point. Extraction and quantification procedures were done for all five individuals of the same treatment group in quick succession (usually spanning 10–20 min). Secretions extracted from the same treatment for all five individuals were deposited as droplets on a sterilized microscope slide, after which 10 μl of the pooled sample of MG secretions was collected and immediately dissolved in 1 ml of pentane, capped to prevent evaporation, and used for testing the quality of these MG secretions.

(c) Testing quality of metapleural gland secretions after challenging with fungal conidia

The efficacy of MG secretions in inoculated and control ants was measured with in vitro inhibition assays measuring zone of inhibition. All five fungal species were grown on potato dextrose agar (PDA) plates. Conidia (asexual spores borne on hyphae) were harvested, cleaned and diluted to approximately 10 5 conidia ml −1 , a concentration low enough to prevent fungal overgrowth that could mask inhibition halos. Prior to using the fungal species for these inhibitive bioassays, assessments of conidia germination were carried out to ensure that the fungal conidia were viable. All fungal species had more than 90 per cent germination (M. brunneum 99% B. bassiana 96% A. niger 97.8% G. virens 93.6% E. weberi 91.5%).

To achieve an even fungal growth, 1 ml volumes of fungal conidia suspensions were pipetted onto the surface of PDA plates and evenly spread out using a Drigalski spatula. PDA plates were divided into six equal segments, and a disc of sterilized filter paper (5 mm diameter) was placed at the centre of each segment. About 10 μl of the pooled MG secretion extracted from ants after inoculation was dissolved in 1 ml pentane (see §2b) and 10 µl was then pipetted onto the paper discs. The sixth segment was used as a control and received 10 μl of pentane. To examine whether the inhibitive effects of MG secretions were due to enzymatic activities, we performed a sub-sample of inhibition assays (MG secretions from ants treated with M. brunneum and control solution) by incubating the MG secretions in a water bath at 100°C for 5 min before application to denature the proteinaceous components.

All agar plates were incubated at 25°C for 24 h, after which the halos in the fungal mats caused by the inhibitive action of the applied secretions were measured (see panel in figure 3). The zone-of-inhibition assay was replicated on ten PDA plates for each fungal species (inhibitive halo radius data in electronic supplementary material, table S4).

(d) Statistical analyses

The survival of inoculated ants over 14 days was analysed using a proportional-hazards model, with colonies, fungal treatments and their interaction as main effects. Surviving individuals were included as censored cases. Post hoc pairwise differences between colonies and fungal treatments were based on risk-ratio tests, with the significance level being adjusted with the Bonferroni procedure to correct for multiple comparisons. The proportion of sporulating ants was analysed using a generalized linear model with binomial error structure, with fungal treatments and colonies as main effects.

Differences in the amounts of MG secretion produced by ants inoculated with different fungal conidia were analysed using two-way ANOVA, testing for differences between both colonies and fungal treatments, with each ant providing a data point consisting of pooled secretion volumes of its two MGs. In the case of overall significance, post hoc multiple-comparison Tukey's tests were performed to examine which treatments made ants produce significantly more MG secretion. Approximately one-third of the worker MG reservoirs have previously been reported as being filled with secretion under unchallenged conditions [17], so we assumed that we would be able to measure any increases of secretion volume relative to such controls.

To test the differences in inhibitive efficiency between MG secretions from ants inoculated with different fungal species, we used a repeated-measures multivariate analysis of variance (MANOVA), with halo area of fungal bioassay species as the dependent variable set, and fungal species and colony as independent variables. This allowed us to evaluate whether the identity of inoculated fungal species had an effect on the inhibitive activity of MG secretions, and whether any such effect was specific to the fungus tested.

To test the differences in inhibitive efficiency between boiled MG secretions and non-heated MG secretions, we performed a Student's t-test for the available comparisons. Analyses were carried out using jmp software (v. 9.02, SAS institute).

3. Results

The survival of ants differed significantly between the fungal treatments (likelihood-ratio, LR: χ 2 = 654.18, d.f. = 5, p < 0.0001), with treatment explaining approximately 60 per cent of the variation in survivorship (R 2 LR = 0.597). Ants inoculated with the entomopathogenic fungi M. brunneum and B. bassiana consistently suffered greater mortality than those inoculated with the control solution. Ants inoculated with A. niger also consistently suffered greater mortality than those inoculated with the control solution, but significantly lower mortality compared with ants inoculated with both entomopathogenic fungi (figure 1). Mortalities of ants inoculated with G. virens and E. weberi did not differ significantly from mortalities of ants inoculated with the control solution (figure 1), confirming previous findings of E. weberi conidia not being harmful to ants [29]. The overall survival of ants differed significantly between the four experimental colonies (LR: χ 2 = 14.47, d.f. = 3, p = 0.0023), reflecting resistance variation between colonies. There was also a significant interaction between colony and fungal treatment (LR: χ 2 = 36.41, d.f. = 15, p = 0.0015), reflecting small but consistent differences in how each colony responded to each fungus (figure 1), although colony and the interaction between colony and treatment each explained less than 5 per cent of the variance in survival (R 2 LR = 0.020 and 0.049, respectively). The proportion of dead ants sporulating differed significantly between the fungal treatments (LR: χ 2 = 150, d.f. = 5, p < 0.0001), but not between experimental colonies (LR: χ 2 = 8.58, d.f. = 3, p = 1.0000). No sporulation was detected for G. virens, E. weberi and control treatments, except minor contamination from unknown saprophytic fungi that also occurred in the controls and were therefore ignored. However, all cadavers from ants exposed to B. bassiana and M. brunneum, and (depending on colony) between 33 and 63 per cent of the cadavers of ants exposed to A. niger, produced characteristic conidia.

Figure 1. Survival of ants from four Acromyrmex octospinosus colonies (ad) challenged with conidia of a range of fungal species or a control solution. Different symbols represent fungal conidia challenges or controls: filled circle, an entomopathogenic fungus Metarhizium brunneum open circle, an entomopathogenic fungus Beauveria bassiana filled triangle, a potentially mild insect pathogen Aspergillus niger open triangle, a likely garden weed Gliocladium virens filled diamond, an ant cultivar parasite Escovopsis weberi and open diamond, a control solution. Fungal challenges and controls marked with the same letter did not differ significantly in their survival (post hoc Tukey's test using Bonferroni correction for multiple treatments).

Workers from the four colonies did not differ significantly in the quantities of MG secretion that their large workers produced (F3,120 = 0.08, p = 0.97), nor in their respective responses to the different fungal treatments (F15,120 = 0.77, p = 0.70), so data were pooled for presentation in figure 2. There were, however, significant differences in the amount of MG secretion produced following the different inoculation treatments (F5,120 = 19.45, p < 0.0001), with B. bassiana and M. brunneum treatments eliciting about twice as much MG secretion compared with the controls. Treatments with A. niger, G. virens and E. weberi elicited significantly less secretion than M. brunneum and B. bassiana, and did not differ significantly from the controls (Tukey's tests figure 2).

Figure 2. Histograms showing the frequency distributions of the volume of metapleural gland secretion extracted from workers (n = 20) exposed to each of the five fungi, and control treatments. Because there was no effect of colony on volume of secretion, frequency distributions were drawn pooling individuals from all four colonies.

Across all five treatment and control groups, we extracted on average 3.6 ± 0.1 μl (mean ± s.e.) of secretion from 120 workers MG reservoirs. From the control treatments, we extracted 2.3 ± 0.1 μl of secretion from 20 worker MG reservoirs. Using the scale of MG reservoir content developed by Poulsen et al. [17], we estimated that these corresponded to approximately one-third-filled reservoirs on average, whereas the 40 M. brunneum- and B. bassiana-challenged ants had 4.8 ± 0.3 μl and 4.8 ± 0.3 μl of secretion in their respective reservoirs, indicating that they were approximately two-third-filled on average. The 40 ants inoculated with A. niger and G. virens had 3.7 ± 0.1 μl and 3.0 ± 0.2 μl of secretion in their respective reservoirs, which amounts to them being roughly half-filled. The 20 E. weberi inoculated ants had 2.8 ± 0.2 μl of secretion in their reservoirs on average, which was not significantly different from the control ants. In our total sample, four ants (3%) had an empty reservoir for unknown reasons: one each from the M. brunneum, B. bassiana, G. virens and E. weberi treatment group (figure 2). Using our volume approximation, four workers had their reservoir almost fully filled with secretion (approx. 6.5 μl)—three from the B. bassiana and one from the M. brunneum treatment group (figure 2)—indicating an induced response to infection with entomopathogenic fungi.

There were highly significant differences in the efficiency of antifungal activity of MG secretions from ants treated with different fungal species (table 1 and figure 3). Secretions taken from ants inoculated with B. bassiana, M. brunneum, A. niger and G. virens exhibited higher antifungal activity compared with MG secretions taken from E. weberi and control treatments. There were no significant between-colony differences in efficacy of MG secretions or interactions between colony and fungal treatment, indicating that the response was colony-independent. Across colonies, however, there were significant differences in the sensitivity of the different fungi to MG secretions (table 1), with G. virens being somewhat more sensitive and B. bassiana less sensitive to MG secretions than the other species (figure 3). There was also a significant interaction between the species of fungus to which worker ants were exposed and the sensitivity of the fungus to the MG secretions of those ants (table 1), which was primarily due to a relatively constant response of the bioassay fungi to MG secretions for controls and ants treated with E. weberi, and a different pattern of efficacy of MG secretions from ants inoculated with M. brunneum or B. bassiana (figure 3).

Figure 3. Antifungal activity of metapleural gland secretions extracted from worker ants exposed to six treatments and tested under five types of fungal bioassays, measured as the width of the halo in a zone-of-inhibition assay (mean ± 95% CI). Metapleural gland secretions from ants treated with entomopathogenic fungi (Metarhizium brunneum and Beauveria bassiana) and saprophytic/weedy/mildly pathogenic fungi (Aspergillus niger and Gliocladium virens) had significantly higher antifungal activities than those treated with the fungus garden parasite (Escovopsis weberi) and the controls. Within each bioassay, clusters of treatments marked with the same letter did not differ significantly in the size of halo (post hoc Tukey's test using Bonferroni correction for multiple bioassays). To the right, an example PDA bioassay is shown, with the halo radius for B. bassiana marked.

There were no significant differences in the inhibitive efficiency between boiled MG secretions and non-heated MG secretions (t = 0.94, d.f. = 1, p = 0.350 for boiled control versus control t = −0.77, d.f. = 1, p = 0.443 for boiled secretions treated with M. brunneum inoculations versus non-heated secretions treated with M. brunneum inoculations), indicating that the antifungal ingredients were not enzymatic. This result concurs with an earlier study of MG secretion antibiotic properties in Myrmecia gulosa ants [30].

4. Discussion

(a) Functional plasticity in metapleural gland responses

Infections with virulent M. brunneum and B. bassiana entomopathogens triggered both more MG secretion and more efficient inhibition per unit of MG secretion. These results are consistent with those of Bot et al. [5], who showed that both conidia and hyphae of these pathogens are inhibited similarly by seven different classes of chemical compounds from the MG secretions of A. octospinosus. The mild pathogen A. niger has an interesting intermediate position, as infections resulted in more potent MG secretions (figure 3) but without increasing the secretion volume (figure 2). This may be related to conidia germination, but not hyphal growth, of both A. niger and G. virens being less efficiently inhibited by several compounds in the MG secretion than conidia of the two virulent entomopathogens [5].

While previous authors [28,31] concluded that ant MGs seemed to be somewhat disappointingly simple, because no muscles were present to directly regulate the emergence of secretion to the cuticular surface, our present data suggest that the production of MG secretion is remarkably plastic and appears to be both quantitatively (figure 2) and qualitatively (figure 3) adjusted to specific fungal infection threats. Given these conditional responses, the question now seems almost the reverse: why have behaviourally controlled muscular release mechanisms for MG secretion not been favoured by selection?

Our results suggest that the MGs of leaf-cutting ants may function as analogues of a simple prophylactic immune system that operates on the cuticular surface of ant workers. This hypothesis is based on the evidence provided here that MG secretions have both general and specific functions, and that their production is partly constitutive and partly induced. We will discuss this hypothesis below, using a modified version of a framework diagram from Schmid-Hempel & Ebert [32] that maps immune defences along perpendicular specificity and inducibility axes (figure 4). We also include our current understanding of MG chemistry and function in Atta leaf-cutting ants, which are the sister genus of Acromyrmex, but different in several key life-history traits. We use this analogy with the immune system proper because it helps to interpret our findings in a coherent conceptual framework emphasizing evolutionary adaptation, not because we would claim that the mechanisms and metabolic pathways involved are similar or even related to those of the normal immune system of these insects.

Figure 4. Diagrammatic representation of the analogies between generally established immune system functions and metapleural gland defences in Acromyrmex and Atta leaf-cutting ants, with references to published studies where they were available. The upper-left quadrat refers to hypothetical inferences of specific chemicals having constitutive hygiene functions in leaf-cutting ants that remain to be tested for their specific efficacy.

(b) Constitutive, non-specific defence components of leaf-cutting ant metapleural glands

The bottom-left quadrat of figure 4 summarizes the most basal antimicrobial MG functions, which were the first to be discovered [3]. General antimicrobial activity probably stems from the high acidity (pH 2.5–4) of MG secretions [3,9,30]. This acidity and its pH-reducing effects in fungus gardens are well documented in a series of studies on leaf-cutting ants [5,33], and are probably due to the abundant presence of organic acids in MG secretions [19].

The second component in this quadrat is that at least some secretion is normally present in the MGs of all ants, although the amount in the reservoirs of unchallenged ants varies, probably as a function of nutrition and age [19], so that some ants (3% in our study) end up having reservoirs that are scored as empty [3,17]. There is thus almost always secretion present for the ants to work with, even under benign circumstances, and this constitutive defence amounts to about one-third of the maximal holding capacity of the MG reservoirs, on average [17] (figure 2).

The third component is the direct supply of MG secretion to the external environment via the rather large opening of the reservoir [6]. The lack of any autonomous or behaviourally controlled muscles to control release of the secretion [28] indicates a constitutive and non-specific defence component of the MGs.

(c) Induced, non-specific defence components

The bottom-right quadrat of figure 4 summarizes the two known mechanisms by which the application of MG secretion for non-specific defence can be induced. The first is active grooming. The location of the MG openings just above the hind-legs allows secretions to be picked up by leg movements as soon as they emerge. This form of dispersing MG secretion [22] is particularly employed when leaf-cutting ants are challenged with fungal conidia [21,23].

The frequency of MG grooming and the target of application is known to vary between Acromyrmex and Atta, with Atta substantially increasing their MG grooming rate and expanding their grooming targets to the garden, and Acromyrmex maintaining much lower MG grooming rates and primarily targeting the brood [21]. This is consistent with Atta workers relying more on MG grooming and less on antibiotic production by mutualistic actinomycete bacteria, whereas the opposite combination appears to apply in Acromyrmex [21] (see §4d for more details).

(d) Induced, specific defence components

The top-right quadrat of figure 4 summarizes two known mechanisms in Acromyrmex and Atta leaf-cutting ants by which MG secretion appears to be differentially induced depending on the type of challenge. The fact that A. octospinosus workers significantly increase the quantity of MG secretion only after being challenged with directly lethal insect pathogens such as B. bassiana and M. brunneum indicates that the ants are able to classify pathogens according to the degree of threat to themselves and their nest-mates. The same results (figure 2) suggest that this recognition process is a continuum, rather than an all-or-nothing response, as the amount of MG secretion was also elevated somewhat after challenges with G. virens and A. niger.

Interestingly, induction specificity also included increases in potency of the MG secretion, and here responses were generally similar for all fungal infections except Escovopsis, in spite of the variation in threat across these challenges (figure 3). This underlines that the MG secretion responses are analogous to immune system functioning, because challenges both increase overall investment in defence (cf. lymphocytes) and induce the production of specific defence agents (cf. antibodies) after the kind of challenge has been identified [34]. There was difference in how the fungal species responded to MG secretions that were induced by particular fungal conidia (table 1), which suggests that there is potential for changing MG secretions to target particular threats. However, we found no match between which fungal species had induced a particular MG secretion and the susceptibility of that species to the secretion (figure 3). From our heat-denaturing testing and an earlier study [30], we infer that proteinaceous/enzymatic compounds are unlikely to be important as antifungal ingredients. Hence, the identities of compounds that make MG secretion more potent after infections remain unknown, but will be interesting targets for future research.

The specific defence responses obtained in our present study are similar to those obtained by Fernández-Marín et al. [21,23], who showed that MG grooming rates in Atta colombica were elevated when inoculated with conidia of Metarhizium and Escovopsis but not by inert talcum powder controls. Whether this response is mediated by quantitative and/or qualitative change in MG secretion is unknown, but it seems reasonable to infer that an increase in grooming rate would only make sense if at least the amount of available MG secretion would also be proportionally increased. There is a significant difference between Atta and Acromyrmex in that MG grooming in the former also has a major function for infection control in the fungus garden, whereas this is not so in the latter [21]. This is consistent with Acromyrmex maintaining cultures of actinomycete bacteria on their exoskeleton to control Escovopsis infections, whereas Atta has abandoned this form of biological control (possessed by most basal attine ants [35]) in exchange for chemical control via MG grooming [21]. It is therefore not surprising that Atta has inducible specific MG defences against Escovopsis and Acromyrmex has not.

(e) Constitutive, specific defence components

The upper-left quadrat in figure 4 largely represents a ‘black box’, but there is enough evidence for this defence component to hypothesize that this aspect of the immune system analogy is also likely to be important in fungus-growing ants [5,19]. There are major compounds of MG secretions that are likely to qualify as specific constitutive defences, which are different in Atta and Acromyrmex in spite of other compounds being present in the MG secretions of both genera [6]. The prime candidate compound in Atta is phenylacetic acid, which makes up 72 to 80 per cent of the total MG secretion, but is absent in the MG secretion of Acromyrmex. However, Acromyrmex MG secretion has indoleacetic acid (IAA) as a major component (24–25% of total secretion), whereas this compound is found only in trace amounts in the MG secretion of Atta. Further work will be needed to unravel the specific targets of these major components. The role of IAA seems particularly intriguing, as this compound is a well-known plant growth hormone [36], but initial tests have shown that it does not inhibit fungal conidia or hyphae [5].

It has been repeatedly found that in addition to chemical compounds that make MG secretion acidic, there is a significant fraction of proteinaceous compounds of unknown identity in the MG secretions of leaf-cutting ants [3,18]. However, the results of our boiling assay indicate that any such proteins do not seem to have the specific functions found in other insects [37–39]. This underlines that secretions of the MGs of leaf-cutting ants might have a combination of adaptive traits that are specific for this clade of ants only.


Study sites and species

In Peru, we collected ants from several habitats at the Centro de Investigación y Capacitación Rio Los Amigos (“CICRA,” 12°34′S, 70°05′W elevation

270 m), which is a biological station located at the confluence of the Madre de Dios and Los Amigos rivers. Surrounding the station is the Los Amigos conservation concession, which comprises 146,000 ha of mostly primary tropical rainforest on a mixture of upland terraces and floodplains. Annual rainfall at Los Amigos is between 2700 and 3000 mm, with more than 80% of the precipitation falling during the October–April wet season (Pitman 2008 ). Mean monthly temperatures range from 21 to 26°C and humidity averages 87% (Pitman 2008 ). In Canada, we collected ants from the Koffler Scientific Reserve at Jokers Hill (“KSR,” 44°02′N, 79°32′W elevation

300 m) and elsewhere in the greater Toronto area. Annual precipitation in southern Ontario averages 790 mm and mean monthly temperatures range from −6°C in January to 21°C in July (Environment Canada, Canadian Climate Normals 1971–2000).

Excluding callows, we collected live workers (minors only in polymorphic species), gynes, and virgin males of nine ant species (Table 1) for use in survival assays. Ants were brought back to the laboratory and kept in plastic containers, fed standard artificial diet (Bhatkar and Whitcomb 1970 ), and given water via a damp piece of cotton. All ants were assayed within 24 h of collection. In Peru, we hand-collected arboreal ants from bamboo culms (Camponotus mirabilis and C. longipilis) and from the hollow stems of myrmecophytic Cordia nodosa trees (Allomerus octoarticulatus and an unidentified and possibly undescribed species of Azteca). We also collected colonies of Odontomachus bauri from decaying wood or soil. In Canada, we collected colonies of four species of ground-dwelling ants: Aphaenogaster cf. rudis and Lasius cf. nearcticus from decaying wood and Myrmica rubra and Brachymyrmex depilis from soil. We chose these ant species because they had multiple colonies producing alates at the time of our study they are not a random sample from across the ant phylogeny, but we have no reason to think they differ systematically from other ants with respect to immunity-related traits. All colonies contained workers and at least one of the alate sexes (sometimes both). Because split sex ratios occur in many ant species, some colonies may contain only gynes or males at a given time (Meunier et al. 2008 ).

Subfamily Species Nest type Colonies Ants in fungus, control treatments Average body length, mm (replicates)
Workers Gynes Males Workers Gynes Males
Myrmicinae Allomerus octoarticulatus Arboreal 6 36, 35 10, 10 22, 22 1.72 (12) 5.46 (7) 4.79 (6)
Dolichoderinae Azteca sp. Arboreal 2 11, 12 0, 0 9, 9 2.42 (15) 1.9 (16)
Formicinae Camponotus mirabilis Arboreal 6 27, 27 9, 7 23, 23 5.92 (12) 12.25 (8) 5.99 (4)
Formicinae Camponotus longipilis Arboreal 4 14, 13 8, 8 10, 10 7.42 (7) 10.54 (1) 6.76 (3)
Ponerinae Odontomachus bauri Soil 4 26, 26 13, 12 4, 4 5.53 (17) 6.37 (4) 4.16 (6)
Myrmicinae Aphaenogaster cf. rudis Soil 7 43, 39 37, 37 18, 16 3.29 (15) 5.17 (5) 3.54 (12)
Formicinae Brachymyrmex depilis Soil 2 12, 12 11, 9 6, 6 1.05 (5) 2.99 (5) 1.47 (5)
Formicinae Lasius cf. nearcticus Soil 2 12, 12 10, 10 12, 12 2.61 (12) 4.95 (5) 2.88 (21)
Myrmicinae Myrmica rubra a a Exotic species.
Soil 2 30, 30 0, 0 26, 27 3.36 (15) 3.52 (17)

Survival assays

We exposed ants to the generalist entomopathogenic fungus, Beauveria bassiana, which infects over 200 species of arthropods and has been used in other studies of ant immunity (Feng et al. 1994 Diehl and Junqueira 2001 Schmidt et al. 2011 ). Beauveria bassiana is not actively used as an insecticide at our field sites (CICRA: M. Frederickson, pers. obs. KSR: A. Weis, pers. comm.). We extracted conidia from the commercial insecticide Botanigard ES (strain GHA) by first growing a suspension on 6.5% sabouraud dextrose agar plates in a darkened environment. To avoid contamination by other chemicals in Botanigard ES, conidia from these initial plates were not used directly. Instead, we collected these conidia and grew them on new plates conidia arising from these secondary plates were used for survival assays. We suspended the conidia in a 0.05% solution of the surfactant Triton X-100 [Sigma-Aldrich, Oakville, Ontario, Canada]. We counted conidia densities using a haemocytometer and diluted the suspension to a concentration of 1 × 10 7 conidia/mL. This was procedure was performed daily to ensure a fresh supply of conidia. Conidia suspensions were checked to be viable by plating them on 6.5% sabouraud dextrose agar plates. In the fungal and control treatments, respectively, we placed 0.5 μL of the conidia suspension or the same amount of a 0.05% solution of the surfactant only on ant thoraces.

The number of workers and alates collected from each colony varied depending on the quantity available. We used approximately equal numbers of each caste (i.e. workers, males, gynes) for the fungal and control treatments (Table 1). For two species, Azteca sp. and M. rubra, we were unable to collect gynes (Table 1). All individuals within a colony were exposed to the same fungal suspension and the same suspension was used for colonies and species collected on the same date. We placed each fungus-treated or control ant in a 50-mL falcon tube and kept the tubes at ambient temperatures (in Peru,

18–33°C) or in environmental chambers (in Canada, L14:D10 light cycle, 15–25°C). Ants were fed a standard artificial diet (Bhatkar and Whitcomb 1970 ) and provided with water via a damp piece of cotton. We monitored ants every day for 14 days, recording the day of death if it occurred in this period. After an ant died, it was removed from its falcon tube and placed into a 2-mL microcentrifuge tube with a small piece of damp cotton to keep the environment moist. We then monitored the deceased ants for fungal growth daily for 7 days. Over 95% of ants that died in the fungal treatment and just 1% of ants that died in the control treatment had B. bassiana hyphae growing out of their corpses within 7 days. This suggests that the differences in survival between the treatments were due to B. bassiana exposure. In total, we monitored the survival of 445 fungus-treated and 434 control ants.

Body size

We measured ant body size on a different set of individuals from the ants used in the survival assays, but all were collected at the same time and from the same sites. Under a Leica M205 dissecting microscope with a digital micrometer, we measured the maximum length of the head, mesosoma, petiole, and gaster of each ant, and then summed these to get a measure of body length. The number of individuals per caste per species varied from 1 to 20 (Table 1). Although these measures are not from the individuals used in the survival assays, we have no reason to expect biases in body size among the ants used in the assays and the ants used for size measurements.

Statistical analysis

We assessed variation among workers, gynes, and males in susceptibility to B. bassiana only for species in which the fungal treatment significantly affected ant mortality. We tested whether the fungal treatment significantly affected the mortality of all nine species independently using a Cox proportional hazard model, with caste, treatment, and colony as main effects. We found that the fungal treatment had no significant effect on Azteca sp. and L. nearcticus mortality. We checked this by performing a likelihood ratio test between the full model and a model with just caste and colony (fungal treatment removed) for these two species. The full model did not provide a better fit for the data in Azteca sp. (P = 0.1564) or L. nearcticus (P = 0.4039). This lack of response is largely attributable to the high baseline mortality of males in both species (3.33 days in Azteca sp. and 4 days in L. nearcticus, respectively), which greatly constrains the potential effect size of the fungal treatment. Our study would be unable to adequately assess variation in susceptibility among castes for either species, and thus they were removed from further analyses.

For the remaining seven species (C. mirabilis, C. longipilis, A. octoarticulatus, O. bauri, M. rubra, A. rudis, and B. depilis), we used survival analysis to analyze our data, with the Cox proportional hazards model censored at 14 days. Treatment, caste, and species were included as main effects site of origin (Peru or Canada), colony of origin, and type of nest (arboreal or ground) were included as random factors but were not significant predictors of survival and were removed from the final model. The regression coefficients for all three-way interactions (treatment × caste × species) were nonsignificant in the full model, so we included only the two-way interactions in the final model (i.e., treatment × caste, treatment × species, and caste × species). A log-likelihood ratio test indicated that the full model, including three-way interactions, did not improve model fit, compared with a model with only two-way interaction terms (P = 0.122). We also created Kaplan–Meier curves for each caste with treatment as the main factor. Unlike our main statistical model, it pools all species together, but nonetheless provides a useful visualization of the results.

We calculated hazard ratios (HR) from the coefficients in the Cox regression. The hazard ratio represents the probability of one group dying relative to another group at any point in time. Values above one indicate an elevated risk of dying, while values below one indicate the opposite. We only use regression coefficients significant at the ≤0.05 level. By calculating the HR of gynes or males relative to workers in the control treatment, we can determine if baseline mortality differed among castes. However, we are most interested in whether mortality due to the fungal treatment differed among castes. To examine this, we calculated the treatment-HR (HRtrt), which is the HR of the fungus-treated group relative to the control group for each caste and species individually this takes into account any differences in baseline mortality between castes and species. We can then make comparison among HRtrt values to determine whether they differed among castes within a species (Altman and Bland 2003 ). Significant differences in HRtrt indicate that the fungus treatment had different effects on castes, suggesting differences in caste immunity.

Lastly, we investigated the relationship between body size and immunity. As mentioned previously, body size was measured from a different set of ants collected from the same colonies as those used for the survival assays and thus cannot be incorporated directly into the survival analysis. To investigate if body size is a predictor of immunity, we used Ln (HRtrt) as a proxy for immunocompetence. We fit an ANCOVA with average body size, caste, and species as main effects to determine whether these are significant predictors of Ln (HRtrt). Due to limitations in statistical power, we could not create a full model that accounts for all two-way and three-way interactions. Instead, we fit the data to two separate models. Model 1: main effects and interaction effects of body size and caste with species as a cofactor. Model 2: main effects and interaction effects of body size and species with caste as a cofactor. However, backwards elimination removed interaction effects from both Model 1 and Model 2. Log-likelihood ratio tests favored a reduced model without interaction effects in body size x caste (Model 1 P = 0.475) and in body size x species (Model 2 P = 0.826). We report results from this reduced model. We also report Bonferroni-corrected P-values to account for the use of HRtrt in multiple tests.

For all analyses, we utilized the downloadable packages, Survival and ggplot2, in the statistical software R (R Development Core Team RFFSC 2013 ).

Why do wounds become infected?

Wounds heal best when they are clean and free from germs. Normally, many germs live harmlessly on our skin and in the environment around us. Normally the germs live on our skin, or in areas such as our nose, without causing any problems.

Usually the skin acts as a barrier. If the skin is broken germs may spread into the more sensitive tissues underneath. This is how an infection can start. This makes the tissues sore and swollen, and less likely to heal. Open wounds are more likely than closed wounds to develop infections. This is because the break in the skin provides a route for the germs to travel from the outside to the inside.

Some circumstances make it more likely that wounds will become infected. These include:

  • If you have diabetes type 1 or type 2.
  • If the object which caused the wound was dirty and contained germs.
  • If the wound was caused by a human or animal bite.
  • If the wound still contains a 'foreign body', ie bits of whatever caused the injury. For example, bits of glass, splinters of wood, thorns, etc.
  • The size and depth of the wound. Larger or deeper wounds have a higher chance of becoming infected.
  • Jagged edges to the wound.
  • If proper precautions were not taken before an operation.
  • If you are an older person. Your skin heals less well as you get older.
  • If you are very overweight.
  • If your immune system does not work as well as normal. For example, if you are on medication such as steroids or chemotherapy, or if you have HIV/AIDS.

The medicinal uses of insects and other arthropods worldwide have been reviewed by Meyer-Rochow, [1] who provides examples of all major insect groups, spiders, worms and molluscs and discusses their potential as suppliers of bioactive components. Using insects (and spiders) to treat various maladies and injuries has a long tradition and, having stood the test of time, can be effective and provide results. However, sometimes folk-medicinal "logic" was based on the Doctrine of Signatures = "let likes be cured by likes"and had, if any at all, little more than a psychological effect. For example, to treat cases of constipation, dung beetles were prescribed to slim down stick insects were thought to help hairy tarantulas seemed the right treatment for hair loss and fat grubs resembling the swollen limb caused by the parasite Wuchereria bancrofti were expected to help the elephantiasis sufferer. An organism bearing parts that resemble human body parts, animals, or other objects, was thought to have useful relevance to those parts, animals or objects. So, for example, the femurs of grasshoppers, which were said to resemble the human liver, were used to treat liver ailments by the indigenous peoples of Mexico. [2] This doctrine is common throughout traditional and alternative medicine, but is most prominent where medical traditions are broadly accepted, as in traditional Chinese medicine and Ayurveda, and less by community and family based medicine, as is more common in parts of Africa.

Traditional Chinese medicine Edit

Traditional Chinese medicine includes the use of herbal medicine, acupuncture, massage, exercise, and dietary therapy. It is a typical component of modern medical care throughout East Asia and in some parts of Southeast Asia (such as Thailand). Insects are very commonly incorporated as part of the herbal medicine component of traditional Chinese medicine, and their medical properties and applications are broadly accepted and agreed upon. Some brief examples follow:

The Chinese Black Mountain Ant, Polyrhachis vicina, is supposed to act as a cure all and is widely used, especially by the elderly. It is said to prolong life, to have anti-aging properties, to replenish Qi, and to increase virility and fertility. Recent interest in the ants' medicinal qualities has led to British researchers to study the extract's potential to serve as a cancer-fighting agent. [3] [ medical citation needed ] Chinese Black Mountain Ant extract is typically consumed mixed with wine.

India and Ayurveda Edit

Ayurveda is ancient traditional Indian treatment almost universally incorporated alongside Western medicine as a typical component of medical treatment in India. Although Ayurvedic medicine is often effective, doses can be inconsistent, and may sometimes be contaminated with toxic heavy metals. [4] Some brief examples to follow:

Termite is said to cure a variety of diseases, both specific and vague. Typically the mound or a portion of the mound is dug up and the termites and the architectural components of the mound are together ground into a paste which is then applied topically to the affected areas or, more rarely, mixed with water and consumed. [5] This treatment was said to cure ulcers, rheumatic diseases, and anemia. [4] It was also suggested to be a general pain reliever and health improver. [4]

The Jatropha Leaf Miner, a lepidopteran which feeds preferentially on Jatropha, is an example of a major insect agricultural pest which is also a medicinal remedy. [5] The larvae, which are also the form of the insect with the greatest economic impact on agriculture, are harvested, boiled, and mashed into a paste which is administered topically and is said to induce lactation, reduce fever, and soothe gastrointestinal tracts. [5]

Africa Edit

Unlike China and India, the traditional insect medicine of Africa is extremely variable. It is largely regional, with few, if any, major agreements on which insects are useful as treatments for which ailments. [5] Most insect medicinal treatments are passed on through communities and families, rather than being taught in university settings, as Traditional Chinese Medicine and Ayurveda sometimes are furthermore, most traditional medicine practices necessitate a person in a "healer" role. [5] Some brief examples to follow:

Grasshopper is both commonly eaten as a delicacy and an excellent source of protein and is consumed for medicinal purposes. [5] These insects are typically collected, dried in the sun, and then ground into a powder. [5] The powder can then be turned into a paste when mixed with water and ash and applied to the forehead to alleviate the pain of violent headaches. [5] Additionally, the headaches themselves can be prevented by a "healer" inserting the paste under the skin at the nape of the afflicted person's neck. [5]

Termites are also used in parts of Africa much like they are in India. [4] Parts of the mound are dug up, boiled, and turned into a paste, which can then be applied to external wounds to prevent infection or consumed to treat internal hemorrhages. [5] termites are used not only as a form of medicine, but also as a medical device. If a "healer" wants to insert a medicine subcutaneously, they will often spread that medicine on the skin of the patient, and then agitate a termite and place the insect on the skin of the patient. [5] When the termite bites, its mandibles effectively serve as an injection device. [5]

Americas Edit

The Americas were more highly influenced by the Doctrine of Signatures than China, India, or Africa, most likely because of their colonial history with Europe. The majority of insect use in medicine is associated with Central America and parts of South America, rather than North America, and most of it is based on the medical techniques of indigenous peoples. [2] Currently, insect medicine is practiced much more rarely than in China, India, or Africa, though it is still relatively common in rural areas with large indigenous populations. [2] Some examples to follow:

Chapulines, or grasshoppers, are commonly consumed as a toasted regional dish in some parts of Mexico, but they are also used medicinally. [2] They are said to serve as diuretic to treat kidney diseases, to reduce swelling, and to relieve the pain of intestinal disorders when they are consumed. [2] However, there are some risks associated with consuming chapulines, as they are known to harbor nematodes which may be transmitted to humans upon consumption.

Much like the termites of Africa, ants were sometimes used as medicinal devices by the indigenous peoples of Central America. [2] The soldier cast of the Army ant would be collected and used as living sutures by Mayans. [2] This involved agitating an ant and holding its mandibles up to the wound edges when it bit down, the thorax and abdomen were removed, leaving the head holding the wound together. [2] The ant's salivary gland secretions were reputed to have antibiotic properties. [2] The venom of the Red harvester ant was used to treat rheumatism, arthritis, and poliomyelitis via the immunological reaction produced by its sting. This technique, in which ants are allowed to sting afflicted areas in a controlled manner, is still used in some arid rural areas of Mexico. [2]

The silkworm, Bombyx mori, was also commonly consumed both as a regional food and for medicinal purposes in Central America after it was brought to the New World by the Spanish and Portuguese. [2] Only the immatures are consumed. Boiled pupae were eaten to treat apoplexy, aphasy, bronchitis, pneumonia, convulsions, hemorrhages, and frequent urination. [2] The excrement produced by the larvae is also eaten to improve circulation and alleviate the symptoms of cholera (intense vomiting and diarrhea). [2]

Honey bee products Edit

Honey bee products are used medicinally across Asia, Europe, Africa, Australia, and the Americas, despite the fact that the honey bee was not introduced to the Americas until the colonization by Spain and Portugal. They are by far the most common medical insect product, both historically and currently. [5]

Honey is the most frequently referenced medical bee material. It can be applied to skin to treat excessive scar tissue, rashes, and burns, [6] and can be applied as a poultice to eyes to treat infection. [4] It is also consumed for digestive problems and as a general health restorative, and can be heated and consumed to treat head colds, cough, throat infections, laryngitis, tuberculosis, and lung diseases. [2]

Additionally, apitoxin, or honey bee venom, can be applied via direct stings to relieve arthritis, rheumatism, polyneuritis, and asthma. [2] Propolis, a resinous, waxy mixture collected by honeybees and used as a hive insulator and sealant, is often consumed by menopausal women because of its high hormone content, and it is said to have antibiotic, anesthetic, and anti-inflammatory properties. [2] Royal jelly is used to treat anemia, gastrointestinal ulcers, arteriosclerosis, hypo- and hypertension, and inhibition of sexual libido. [2] Finally Bee bread, or bee pollen, is eaten as a generally health restorative, and is said to help treat both internal and external infections. [2] All of these honey bee products are regularly produced and sold, especially online and in health food stores, though none are yet approved by the FDA.

Though insects were widely used throughout history for medical treatment on nearly every continent, relatively little medical entomological research has been conducted since the revolutionary advent of antibiotics. Heavy reliance on antibiotics, coupled with discomfort with insects in Western culture limited the field of insect pharmacology until the rise of antibiotic resistant infections sparked pharmaceutical research to explore new resources. Arthropods represent a rich and largely unexplored source of new medicinal compounds. [7]

Maggot therapy Edit

Maggot therapy is the intentional introduction of live, disinfected blow fly larvae (maggots) into soft tissue wounds to selectively clean out the necrotic tissue. This helps to prevent infection it also speeds healing of chronically infected wounds and ulcers. [8] Military surgeons since classical antiquity noticed that wounds which had been left untreated for several days, and which had become infested with maggots, healed better than wounds not so infested. [9] Maggots secrete several chemicals that kill microbes, including allantoin, urea, phenylacetic acid, phenylacetaldehyde, calcium carbonate, proteolytic enzymes, and many others. [10]

Maggots were used for wound healing by the Maya and by indigenous Australians. More recently, they were used in Renaissance Europe, in the Napoleonic Wars, the American Civil War, and in the First and Second World Wars. [11] [12] It continues to be used in military medicine. [13]

Apitherapy Edit

Apitherapy is the medical use of honeybee products such as honey, pollen, bee bread, propolis, royal jelly and bee venom. One of the major peptides in bee venom, called Melittin, has the potential to treat inflammation in sufferers of Rheumatoid arthritis and Multiple sclerosis. Melittin blocks the expression of inflammatory genes, thus reducing swelling and pain. It is administered by direct insect sting, or intramuscular injections. Bee products demonstrate a wide array of antimicrobial factors and in laboratory studies and have been shown to kill antibiotic resistant bacteria, pancreatic cancer cells, and many other infectious microbes. [14]

Blister beetle and Spanish fly Edit

Spanish fly is an emerald-green beetle, Lytta vesicatoria, in the blister beetle family (Meloidae). It and other such species were used in preparations offered by traditional apothecaries. The insect is the source of the terpenoid cantharidin, a toxic blistering agent once used as an aphrodisiac. [15] [16]

Blood-feeding insects Edit

Many blood-feeding insects like ticks, horseflies, and mosquitoes inject multiple bioactive compounds into their prey. These insects have been used by practitioners of Eastern Medicine for hundreds of years to prevent blood clot formation or thrombosis. [17] However, modern medical research has only recently begun to investigate the drug development potential of blood-feeding insect saliva. These compounds in the saliva of blood feeding insects are capable of increasing the ease of blood feeding by preventing coagulation of platelets around the wound and provide protection against the host's immune response. Currently, over 1280 different protein families have been associated with the saliva of blood feeding organisms. [18] This diverse range of compounds may include: [14] [19]

  • inhibitors of platelet aggregation, ADP, arachidonic acid, thrombin, and PAF.
  • anticoagulants
  • vasodilators
  • vasoconstrictors
  • antihistamines
  • sodium channel blockers
  • complement inhibitors
  • pore formers
  • inhibitors of angiogenesis
  • anaesthetics
  • AMPs and microbial pattern recognition molecules.
  • Parasite enhancers/activators

Currently, some preliminary progress has been made with investigation of the therapeutic properties of tick anticoagulant peptide (TAP) and Ixolaris a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis. [20] Additionally, Ixolaris, a tissue factor inhibitor has been shown to block primary tumor growth and angiogenesis in a glioblastoma model. [21] Despite the strong potential of these compounds for use as anticoagulants or immunomodulating drugs no modern medicines, developed from the saliva of blood-sucking insects, are currently on the market. [14]

Like plants and insects, arachnids have also been used for thousands of years in traditional medical practices. Recent scientific research in natural bioactive factors has increased, leading to a renewed interest in venom components in many animals. In 1993 Margatoxin was synthesized from the venom of the Centruroides margaritatus the Central American bark scorpion. It is a peptide that selectively inhibits voltage-dependent potassium channels. Patented by Merck, it has the potential to prevent neointimal hyperplasia, a common cause of bypass graft failure. [22]

In addition to medical uses of arachnid defense compounds, a great amount of research has recently been directed toward the synthesis and use of spider silk as a scaffolding for ligament generation. Spider silk is an ideal material for the synthesis of medical skin grafts or ligament implants because it is one of the strongest known natural fibers and triggers little immune response in animals. Spider silk may also be used to make fine sutures for stitching nerves or eyes to heal with little scarring. Medical uses of spider silk is not a new idea. Spider silks have been used for thousands of years to fight infection and heal wounds. Efforts to produce industrial quantities and qualities of spider silk in transgenic goat milk are underway. [23] [24]

Psychoactive scorpions Edit

Recent news reports [25] claim that use of scorpions for psychoactive purposes is gaining in popularity in Asia. Heroin addicts in Afghanistan are purported to smoke dried scorpions or use scorpion stings to get high when heroin is not available. The use of scorpions as a psychoactive drug reportedly gives an instant high as strong or stronger than heroin. However, there is little information on the long-term effects of using scorpion toxins. [26] The 'scorpion sting craze' has also increased in India with a decreasing availability of other drugs and alcohol available to youth. [27] Young people are reportedly flocking to highway sides where they can purchase scorpion stings that after several minutes of intense pain, supposedly produce a six- to eight-hour feeling of wellbeing. [28]

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Chapter Summary

Fungi are eukaryotic organisms that appeared on land more than 450 million years ago, but clearly have an evolutionary history far greater. They are heterotrophs and contain neither photosynthetic pigments such as chlorophyll, nor organelles such as chloroplasts. Fungi that feed on decaying and dead matter are termed saprobes. Fungi are important decomposers that release essential elements into the environment. External enzymes called exoenzymes digest nutrients that are absorbed by the body of the fungus, which is called a thallus. A thick cell wall made of chitin surrounds the cell. Fungi can be unicellular as yeasts, or develop a network of filaments called a mycelium, which is often described as mold. Most species multiply by asexual and sexual reproductive cycles. In one group of fungi, no sexual cycle has been identified. Sexual reproduction involves plasmogamy (the fusion of the cytoplasm), followed by karyogamy (the fusion of nuclei). Following these processes, meiosis generates haploid spores.

24.2 Classifications of Fungi

Chytridiomycota (chytrids) are considered the most ancestral group of fungi. They are mostly aquatic, and their gametes are the only fungal cells known to have flagella. They reproduce both sexually and asexually the asexual spores are called zoospores. Zygomycota (conjugated fungi) produce non-septate hyphae with many nuclei. Their hyphae fuse during sexual reproduction to produce a zygospore in a zygosporangium. Ascomycota (sac fungi) form spores in sacs called asci during sexual reproduction. Asexual reproduction is their most common form of reproduction. In the Basidiomycota (club fungi), the sexual phase predominates, producing showy fruiting bodies that contain club-shaped basidia, within which spores form. Most familiar mushrooms belong to this division. Fungi that have no known sexual cycle were originally classified in the “form phylum” Deuteromycota, but many have been classified by comparative molecular analysis with the Ascomycota and Basidiomycota. Glomeromycota form tight associations (called mycorrhizae) with the roots of plants.

24.3 Ecology of Fungi

Fungi have colonized nearly all environments on Earth, but are frequently found in cool, dark, moist places with a supply of decaying material. Fungi are saprobes that decompose organic matter. Many successful mutualistic relationships involve a fungus and another organism. Many fungi establish complex mycorrhizal associations with the roots of plants. Some ants farm fungi as a supply of food. Lichens are a symbiotic relationship between a fungus and a photosynthetic organism, usually an alga or cyanobacterium. The photosynthetic organism provides energy from stored carbohydrates, while the fungus supplies minerals and protection. Some animals that consume fungi help disseminate spores over long distances.

24.4 Fungal Parasites and Pathogens

Fungi establish parasitic relationships with plants and animals. Fungal diseases can decimate crops and spoil food during storage. Compounds produced by fungi can be toxic to humans and other animals. Mycoses are infections caused by fungi. Superficial mycoses affect the skin, whereas systemic mycoses spread through the body. Fungal infections are difficult to cure, since fungi, like their hosts, are eukaryotic, and cladistically related closely to Kingdom Animalia.

24.5 Importance of Fungi in Human Life

Fungi are important to everyday human life. Fungi are important decomposers in most ecosystems. Mycorrhizal fungi are essential for the growth of most plants. Fungi, as food, play a role in human nutrition in the form of mushrooms, and also as agents of fermentation in the production of bread, cheeses, alcoholic beverages, and numerous other food preparations. Secondary metabolites of fungi are used as medicines, such as antibiotics and anticoagulants. Fungi are model organisms for the study of eukaryotic genetics and metabolism.

Contact Diseases

Contact Diseases are transmitted when an infected person has direct bodily contact with an uninfected person and the microbe is passed from one to the other. Contact diseases can also be spread by indirect contact with an infected person&rsquos environment or personal items. The presence of wound drainage or other discharges from the body suggest an increased potential for risk of transmission and environmental contamination. Precautions that create a barrier and procedures that decrease or eliminate the microbe in the environment or on personal belongings, form the basis of interrupting transmission of direct contact diseases.

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