Two summaries for two ecology papers- for phyllis young

rspb.royalsocietypublishing.org Research Cite this article:Mathis KA, Tsutsui ND. 2016 Dead ant walking: a myrmecophilous beetle predator uses parasitoid host location cues to selectively prey on parasitized ants.

Proc. R. Soc. B 283: 20161281.

http://dx.doi.org/10.1098/rspb.2016.1281 Received: 8 June 2016 Accepted: 20 July 2016 Subject Areas:

behaviour, ecology Keywords:

myrmecophily, phorid fly parasitism, Azteca sericeasur , Staphylinidae, predation strategy, complex interactions Author for correspondence:

Kaitlyn A. Mathis e-mail: [email protected] †Present address: Department of Ecology and Evolutionary Biology, University of Arizona, 1041 East Lowell Street, Tucson, AZ 85721, USA.

Electronic supplementary material is available at http://dx.doi.org/10.1098/rspb.2016.1281 or via http://rspb.royalsocietypublishing.org. Dead ant walking: a myrmecophilous beetle predator uses parasitoid host location cues to selectively prey on parasitized ants Kaitlyn A. Mathis †and Neil D. Tsutsui Department of Environmental Science, Policy, and Management, University of California, 130 Mulford Hall, Berkeley, CA 94702-3114, USA KAM, 0000-0002-3809-9128; NDT, 0000-0002-1868-3941 Myrmecophiles (i.e. organisms that associate with ants) use a variety of ecological niches and employ different strategies to survive encounters with ants. Because ants are typically excellent defenders, myrmecophiles may choose moments of weakness to take advantage of their ant associates. This hypothesis was studied in the rove beetle, Myrmedonota xipe, which associates with Azteca sericeasur ants in the presence of parasitoid flies. A combination of laboratory and field experiments show that M. xipebeetles selectively locate and prey upon parasitized ants. These parasitized ants are less aggressive towards beetles than healthy ants, allowing beetles to eat the parasitized ants alive without interruption. Moreover, behavioural assays and chemical analysis reveal that M. xipeare attracted to the ant’s alarm pheromone, the same secretion used by the phorid fly parasitoids in host location. This strategy allows beetles access to an abundant but otherwise inaccessible resource, as A. sericeasur ants are typically highly aggressive. These results are the first, to our knowledge, to demonstrate a predator sharing cues with a parasitoid to gain access to an otherwise unavailable prey item. Furthermore, this work highlights the importance of studying ant – myrmecophile interactions beyond just their pairwise context. 1. Introduction Ant societies attract a suite of symbiotic organisms that take advantage of a col- ony’s abundant resources. Beetles in the family Staphylinidae are common ant associates, yet relatively little is known about the role of these beetles in their host colonies. Owing to the formidable chemical and behavioural defences of ants, beetles often possess complex strategies to safely interact with their ant symbionts [1,2]. Many beetles act as scavengers that hide in refuse piles, or as ant-mimicking social parasites within a host colony, draining them of resources.

Others are predators of the ants themselves, locating their prey by ‘eavesdrop- ping’ on the ants’ communication system [3]. While commensal, parasitic and predatory beetles are common within ant societies, little is known about how bee- tles might benefit their ant associates [4]. Furthermore, although most biological communities function as complex networks of context-dependent interactions, ant – beetle associations are rarely observed outside of their pairwise context. While often studied in isolation, predator – prey and host – parasitoid inter- actions have a wide array of effects within food webs and are thus increasingly approached from a community perspective [5]. Natural enemies can directly influence hosts or prey by reducing population size or inducing changes in phenotype (e.g. behaviour, morphology). These interactions often have cascading effects on other species within the community [6 – 8]. Cascading (or indirect) effects can be mediated by both changes in host/prey density (density-mediated indirect effects) and changes in the traits of host/prey species (trait-mediated indirect effects) (reviewed in [9]).

& 2016 The Author(s) Published by the Royal Society. All rights reserved. on August 10, 2016 http://rspb.royalsocietypublishing.org/ Downloaded from Trait-mediated interactions may be particularly relevant to host – parasitoid systems because host behaviour and/or physi- ology is frequently modified as a result of parasitism, with subsequent consequences for str ucturing biological communities [10]. Trait modifications can oc curbeforeorafterparasitismand are either adaptive or non-adaptive to the parasitoid and/or host [11 – 13]. For example, in the presence of parasitoids, hosts will often suspend normal activity in order to implement chemical or behavioural defensive strategies [14 – 16]. After parasitism, immature parasitoids within the host may alter the host’s physi- ology to encourage behaviours that optimize conditions for parasitoid development [17 – 19]. Alternatively, host immune response or even self-sacrifice behaviour may increase to prevent parasitoids from developing [20,21]. Here, we experimentally demonstrate a context-dependent predation strategy involving: (i) phorid fly parasitoids, where parasitism reduces ant aggression; and (ii) predatory beetles, where beetles selectively prey upon parasitized ants, thus potentially reducing phorid fly populations without impacting ant populations. We also demonstrate that this interaction is mediated by the alarm pheromone of the ant, which both natural enemies use in host/prey location. Azteca sericeasur (referred to asAzteca instabilis in prior pub- lications, but recently identified as A. sericeasur; J. Longino 2014, personal communication) is a highly aggressive and territorial arboreal ant species that lives in large polydomous carton nests [8]. This species frequently nests in the shade trees of coffee plantations and forages in the coffee below, preying on and removing herbivores [22]. Ants in the genus Aztecaare known for their pungent alarm pheromone that they disperse liberally from large pygidial gland sacs when disturbed.

Asuiteof Pseudacteon phorid fly species uses this alarm phero- mone to locate and parasitize A. sericeasur[23].Pseudacteon lasciniosus is the largest of the three species and the most abun- dant at our field sites and Pseudacteon planidorsalisis a smaller species and the second most abundant [24]. The presence of phorid fly parasitoids not only reduces the ants’ ability to forage by as much as 50% [16,25], but also indirectly affects inter- actions between the ants and a wide range of competitors and mutualistic partners [8]. The newly described species of rove beetle, Myrmedonota xipe (Staphylinidae), has been observed in association with A. sericeasurants.Myrmedonota xipe beetles are found near disturbed A. sericeasur, often mating or preying on A. sericeasur ants after the arrival of phorid flies [26]. These interactions suggest two questions. First, because A. sericeasur workers are notoriously aggressive, how are the beetles able to prey upon the ants? Second, are the beetles using the same alarm pheromone as the phorid flies to locate the ants? We test the hypothesis that parasitism by phorid flies reduces ant aggression, allowing beetles to gain access to the ants as a prey item. Furthermore, we hypothesize that this context-dependent interaction is induced by the ant’s alarm pheromone, which is released during phorid attack. 2. Material and methods (a) Study site We conducted all fieldwork on a shaded coffee plantation, Finca Irlanda, in the Soconusco region of Chiapas, Mexico (15 811 0N, 928 20 0W) between July 2012 and March 2013. Finca Irlanda is approximately 280 ha in size, located between 950 and 1150 m elevation, and receives approximately 4500 mm of precipitation per year.

Azteca sericeasur is the most abundant ant of the approximately 60 species of arboreal ants on the farm [25].

Azteca sericeasur builds carton nests on the trunks of shade trees within the coffee plantation, where their colonies tend to be distributed in patches [27]. Ants, beetles and phorid flies were all collected for laboratory experiments at five different A. sericeasur nests within the field site using an aspirator. (b) Arena experiment To determine whether M. xipeselectively attack parasitized ants, and whether parasitized and unparasitized ants respond to beetle attacks differently, we placed parasitized and unparasitized ants in an arena with the beetles, filmed their interactions, and ana- lysed the resulting footage (electronic supplementary material, S1).

We placed A. sericeasur ants (n¼ 5) in small plastic container with an individual phorid fly until the ants were parasitized (approx. 1 h). We then chilled the containers in the 220 8C freezer for 2 min until the fly and ants were anaesthetized. While the ants were anaesthetized, we confirmed that ants were successfully parasitized by inspecting for oviposition wounds from the phorid fly. If the ant was successfully parasitized, we added a single dot of paint (green, white or blue chosen at random for each observation) on the head of each ant and identified the anaes- thetized phorid flies to species under a microscope (electronic supplementary material, S2). New phorid flies were used for each parasitism event. We also anaesthetized and painted five unparasitized ants using the same method. We confirmed the ants to be unparasitized by inspecting them for a lack of ovipos- ition wounds. We allowed the ants a 1 h recovery period, which was enough time for ants to resume normal activities. Parasitized ants were used within 4 h of the initial parasitism event. We placed the five unparasitized ants and five parasitized ants (by either P. lasciniosus orP. planidorsalis ) with twoM. xipebeetles in a plastic container (arena) coated with fluon (Northern Products, Rhode Island, USA) to prevent the insects from climbing the walls of the arena. We sealed the arena with a transparent glass lid. We then filmed the arena for 15 min. A total of 82 arena experiments were conducted (50 P. lasciniosus-parasitized versus healthy and 32 P. planidorsalis -parasitized versus healthy treatments). We later analysed video footage of the arena experiments using O BSERVER XT software (v. 11, Noldus Information Technology, Wageningen, The Netherlands), by recording the duration of each behaviour. The behaviours we included were: (i) attack (one animal bites another animal; and (ii) mandible flare (an ant opens and closes her mandibles repeatedly in the direction of another animal) [28]. We recorded behaviours with the O BSERVER Software, blind to the treatment type. We calculated the total duration of each behaviour by organism type ( parasitized ant, unparasitized ant or M. xipe) and the corresponding target organism type for each observation.

(c) Beetle trap experiment Previous studies have shown that dying ants will leave their nests or are restricted entry to their nests [29]. To determine whether M. xipe beetles are able to selectively locate individual parasitized ants within the coffee plantation, we constructed beetle traps from small plastic cups with plaster of Paris on the bottom to retain moisture and lids with holes large enough for the beetles to enter the traps (electronic supplementary material, S3). Each cup contained an A. sericeasurworker randomly assigned to one of four treatment types. The four treatments included: (i) A. sericeasur parasitized by P. lasciniosus, (ii)A. sericeasur parasitized by P. planidorsalis , (iii)A. sericeasur manually injured by puncturing their mesothorax with a Minuten pin (0.20 mm diameter, Bioquip Products, Rancho Dominguez, CA, USA) to simulate a phorid attack wound, and (iv) A. sericeasuruntreated as a control. Ants in parasitism treatments were parasitized according to the same rspb.royalsocietypublishing.org Proc. R. Soc. B283: 20161281 2 on August 10, 2016 http://rspb.royalsocietypublishing.org/ Downloaded from methods in the arena experiment. We placed a total of 624 traps, divided evenly by treatment. We chose 39 sites in both high shade (10 sites with focal trees containingA. sericeasurnests and 10 sites without A. sericeasur nests ) and low shade (10 sites with focal trees containing A. sericeasurnests and nine sites without A. sericeasur nests). We placed 16 traps at each site: one cup of each treatment type 0 m and 5 m from each sites’ focal tree, both on the ground and suspended 1 m in the nearest coffee plant.

We placed the cups for each site in the field on the same day, and retrieved them 2 days later (electronic supplementary material, S4). We identified M. xipebeetles in each cup, and recorded whether the ant was living or completely consumed.

(d) Extraction and analysis of Aztecaalarm pheromone To determine the primary components of A. sericeasuralarm phero- mone, we collected A. sericeasurvolatile alarm pheromone compounds by first placing an A. sericeasurworker in a 50 ml glass beaker. Then we disturbed the worker by pinching her tibia and covered the beaker with aluminium foil. We then inserted a solid phase microextraction (SPME) fibre into the beaker through the aluminium foil for 10 min to adsorb the headspace volatiles.

We conducted five ant volatile collections. SPME fibres were immediately inserted into a Finnigan Trace MS þgas chromato- graph/mass spectrometer equipped with a DB-5 capillary column (30 m 0.32 mm 0.25 mm, Agilent Technologies, CA, USA).

Extracts were analysed in splitless mode, with a temperature programme that started at 100 8C for 1 min, which then increased by 20 8Cmin 2 1 until it reached 150 8C, and then increased by 5 8Cmin 2 1 until it reached 325 8C where it stayed for 5 min.

Injector and transfer line temperatures were kept at 325 8Cand 280 8C, respectively. (e) Alarm pheromone bioassays To determine whether M. xipeare attracted by the alarm pheromone of A. sericeasur, we obtained commercially availa- ble synthetic 2-heptanone and 2-pentanone (Sigma-Aldrich, St Louis, USA), the two primary compounds released by disturbed ants. We prepared four treatment solutions: (i) 1 ml of pesticide- grade hexanes as a negative solvent control, (ii) 20 crushed A. sericeasur pygidial glands in 1 ml of hexane, (iii) 5 ml2-heptanone in 1 ml of hexane, and (iv) 5 ml of 2-pentanone in 1 ml of hexane. We then placed treatment solutions in 2 dram glass vials, open, with a filter paper wick at 20 field sites [23]. All field sites were at least 25 m apart, at the base of trees within the coffee farm that contain an A. sericeasur nest. At each site, we placed the treatment solution vial on the ground with leaf litter removed from the surrounding area. Once we opened the vial with treatment solution, we observed a 10 cm 2area surrounding the vial for 15 min, and col- lected and identified beetles attracted to the area using an aspirator. We quantified the total number of beetles from each species collected at each site with each treatment type. ( f ) Data analysis All statistical analyses were performed in R (v. 3.1.2) [30]. We tested for significant differences in prey choice ( parasitized or healthy ants) and ant response (attack and mandible flare beha- viours) in the arena experiments ( n¼ 83 observations) using separate two-way analysis of variance (ANOVA) followed by Tukey HSD means comparison tests. To examine whether ant removal via beetle predation differed with treatment or habitat types in the beetle trap experiments, we compared ant removal by beetles with generalized linear mixed models (GLMMs) in the ‘lme4’ package. For all models we used the binomial error dis- tribution with the probit link to improve fit. We compared three models. In the first model, we included treatments ( parasitized by P. lasciniosus , parasitized by P. planidorsalis, manually injured, or healthy control), sites (1 – 39), distance from focal tree (0 m or 5 m), the presence or absence of an A. sericeasurnest at focal tree, trap height placement (0 m or 1 m) and shade cover (high or low) as fixed effects. In the second model, we added interactions between treatment and height, and between treatment and shade. In the third model, we added all interactions between treat- ment and each habitat variable. To select the best model, we used the Akaike’s information criterion (AIC) provided by the ‘mass’ package and after we fitted the models we checked homoscedasti- city of the residuals [31]. The model that best predicted ant removal by beetle predators was the first model, thus including interactions between habitat variable and treatments did not improve fit of the model. For the alarm pheromone bioassays, we tested for significant differences between treatments (2-heptanone, 2-pentanone, pygidial gland extract positive control or hexane negative control) using a one-way ANOVA followed by Tukey HSD mean comparison tests. 3. Results (a) Do beetles prefer to prey on parasitized ants?

In the arena experiment, beetles attacked ants that were parasitized by P. lasciniosusmore often than they attacked unparasitized ants ( figure 1 a;ANOVA; F 1,48 ¼24.59, p , 0.001). Ants parasitized by P. planidorsaliswere not attacked more than unparasitized ants ( figure 1 b;‘%of observation spent attacking’: 11.21 +5.67% spent attacking parasitized ants, 8.97 +6.23% spent attacking unparasitized ants; ANOVA; F 1,31 ¼0.81, p¼ 0.37). In the beetle trap exper- iment, beetles were found consuming only ants parasitized by either P. lasciniosus (14.7% of ants) or P. planidorsalis(2.5%) and did not consume control or injured (sham-parasitized) ants. Consumption of P. lasciniosus-parasitized ants was significantly higher than consumption of control or injured ants (GLMM, x2 3¼ 50.33, p, 0.0001; figure 2), and consump- tion of P. planidorsalis -parasitized ants was not significantly higher than control or injured ants (GLMM; x2 3¼ 50.33, p ¼ 0.20). (b) Do parasitized and unparasitized ants display different levels of aggression towards predatory beetles?

In the arena experiment, parasitized ants were less aggressive than healthy ants. During the observations, parasitized ants attacked beetles significantly less often than did unparasitized ants, regardless of which phorid species parasitized them ( figure 1 c,d;P. lasiniosus- parasitized ants unparasitized ants: F 1,34 ¼4.124, p, 0.05; P. planidorsalis- parasitized ants unparasitized ants: F 1,26 ¼4.495, p, 0.05). However, ants parasitized by the two species of phorid fly differed in their mandible flare performance when compared with unparasi- tized ants. Ants parasitized by P. lasciniosusflared their mandibles less often than did unparasitized ants ( figure 1 e; F 1,40 ¼ 11.38, p, 0.002), but ants parasitized by P. planidorsalis did not ( figure 1 f; F 1,28 ¼ 0,p¼ 0.99). (c) Are beetles able to successfully locate parasitized ants in different habitat types?

In the beetle trap experiment, ants were completely consumed in all traps where M. xipewere collected after 2 days. Beetles rspb.royalsocietypublishing.org Proc. R. Soc. B283: 20161281 3 on August 10, 2016 http://rspb.royalsocietypublishing.org/ Downloaded from successfully located and consumed ants parasitized by P. lasciniosusin both habitat types. Attacks on ants in high shade were significantly more frequent than attacks in low shade (GLMM; figure 3 a, P. lasciniosus shade, x2 1¼15.41, p , 0.0001) and significantly more frequent on the ground than above ground (GLMM; figure 3 b; x2 1¼ 9.21, p, 0.003).

Furthermore, there were no differences based on distance from focal tree (GLMM; figure 3 d; x2 1¼ 2.65, p¼ 0.10), or with and without A. sericeasurpresence (GLMM; figure 3 c; x2 1¼ 0.06, p¼ 0.80). (d) How do beetles locate ants?

Analysis of SPME fibres containing volatiles from disturbed A.

sericeasur workers showed that workers have two primary components present within their alarm pheromone, 2-penta- none and 2-heptanone, in a roughly 3 : 8 ratio (electronic supplementary material, S5; 0.374 +0.02 2-pentanone : 2-hep- tanone ratio; n¼ 5). In alarm pheromone bioassays, M. xipe was attracted to both pygidial gland extracts and synthetic 2-heptanone (the most abundant compound in the pheromone blend) significantly more than the hexane control (ANOVA; F 3,76 ¼ 7.072, p, 0.03). Myrmedonota xipe beetles were not attracted to 2-pentanone (the secondary component of the blend) more than the hexane control (ANOVA; F 3,76 ¼7.072, p ¼ 1.0; figure 4). 4. Discussion Our results show that M. xipe selectively preys on parasitized ants, particularly those parasitized by P. lasciniosus.Thesepara- sitized ants display reduced aggression (including a reduced frequency of mandible flaring), which may allow beetles to more easily gain access to them as a prey item. Furthermore, in the field, beetles were able to consume up to 14.7% of ants parasitized by P. lasciniosus, which suggests that these beetles may have an important role in reducing P. lasciniosuspopu- lations. This preliminary estimate of beetle predation rates should be tested in further studies. However, it is probably con- servative, given that M. xipeare generally already present just after phorid parasitism and are frequently observed preying on ants near disturbed colonies shortly after parasitism takes place (KA Mathis 2014, personal observation). Myrmedonota ant mandible flare t ype ant attack type beetle attack t ype 100 percentage of observation spent percentage of observation spent 80 60 40 20 0 100 15 0 12 8 6 0 15 10 5 0 5 10 15 0 5 10 80 *** *** ** * 60 40 20 0 (a)( b)(c)( d) (e) (f) Figure 1. Box plots of arena experiment behaviours. ( a) Beetles attacking ants parasitized by P. lasciniosus(left column) and healthy ants (right column). ( b) Beetles attacking ants parasitized by P. planidorsalis(left column) and healthy ants (right column). ( c) Pseudacteon lasciniosus -parasitized ants (left column) and by healthy ants (right column) attacking beetles. ( d)Pseudacteon planidorsalis -parasitized ants (left column) and by healthy ants (right column) attacking beetles. ( e) Mandible flares by P. lasciniosus -parasitized ants (left column) and by healthy ants (right column). ( f) Mandible flares by P. planidorsalis-parasitized ants (left column) and by healthy ants (right column). The box plots indicate the median (large horizontal bars), the 25th and 75th percentiles (white rectangles), the minimum and maximum values (whiskers) and outliers (white circles). Asterisks represent significance level (* 0.05, ** 0.01, *** 0.001). (Online version in colour.) control 0.4 injureplanidorsalis lasciniosus *** 0 0.1 0.2 0.3 proportion of ants consumed by M. xipe Figure 2.

Proportion of ants consumed by M. xipebeetles in beetle trap experiments. Bars represent the four treatment types: controls are traps baited with healthy ants, injure treatments are traps baited with manually injured ants, P. planidorsalis treatments are traps baited with ants parasitized by P. planidorsalis andP. lasciniosus treatments are traps baited with ants parasitized by P. lasciniosus. Asterisks indicate significance level (*** 0.001). rspb.royalsocietypublishing.org Proc. R. Soc. B283: 20161281 4 on August 10, 2016 http://rspb.royalsocietypublishing.org/ Downloaded from xipepreyed on ants parasitized by P. planidorsalissignificantly less often than it preyed ants parasitized by P. lasciniosus, probably because ants parasitized by P. planidorsaliswere more aggressive than ants parasitized by P. lasciniosus.These experiments indicate that beetles probably have a stronger impact on ant – P. lasciniosusinteractions than ant – P. planidorsalis interactions. This study also demonstrates that M. xipeusesA. sericeasur alarm pheromone as a cue to locate prey. Although phorid flies also use the alarm pheromone to locate A. sericeasurhosts, they are attracted to 1-acetyl-2-methylcyclopentane, a less abundant compound within the alarm pheromone blend that is only found in Aztecaspecies ants [23,32,33]. The com- pound that attracts M. xipe, 2-heptanone, is relatively common in the alarm pheromone of dolichoderine ants. The use of 2-heptanone by M. xipesuggests that these beetles may be less selective and may prey on other dolichoderine ant species in addition to A. sericeasur. However, of the approximately 15 species at our study sites, A. sericeasuris over- whelmingly the most abundant, and are probably the ant that these beetles encounter most frequently.

Several other studies have shown intraguild predation of parasitoids, including examples where: (i) a predator preys on the adult parasitoid as well as the host, (ii) a predator preys on the unparasitized hosts as well as hosts with an ecto- parasitoid, (iii) a predator preys more on parasitized hosts than healthy hosts owing to host vulnerability after parasit- ism, and (iv) a predator preys more on unparasitized hosts owing to the more advantageous location of parasitized hosts in a nest (reviewed in [34]). These studies differ from ours in that M. xipeappears to almost exclusively prey on parasitized ants, as they are unable to access unparasitized ants as a resource.

The unique strategy of M. xipemay have adaptive impli- cations for both A. sericeasurand the phorid parasitoids. First, as beetles are only consuming individuals that harbour phorid fly eggs, beetle predation may, counter-intuitively, 0.30 no. ants consumed per treatment no. ants consumed per treatment 0.25 0.20 0.15 0.10 0.05 0 0.30 0.25 *** ** 0.20 0.15 0.10 0.05 low shade Azteca present Azteca absenthigh shade 0 m 1 m 0m 5m 0 0.30 0.25 0.20 0.15 0.10 0.05 0 0.30 0.25 0.20 0.15 0.10 0.05 0 (a)( b) (c)( d) Figure 3. The proportion of P. lasciniosus-parasitized ants consumed by M. xipebeetles in beetle trap experiments, broken down by habitat type. ( a) Average number of beetles found in traps with ants parasitized by P. lasciniosusin low shade (left bar) and high shade (right bar) habitats. ( b) Average number of beetles found in traps with ants parasitized by P. lasciniosuson the ground (left bar) and 1 m above the ground (right bar). ( c) Average number of beetles found in traps with ants parasitized by P. lasciniosus at sites withA. sericeasur nests (left bar) and without A. sericeasurnests (right bar). ( d) Average number of beetles found in traps with ants parasitized by P. lasciniosus at 0 m (left bar) and 5 m (right bar) from the focal tree. Asterisks represent significance level (* 0.05, ** 0.01, *** 0.001). 2-heptanoneMyrmedonota xipe attraction to Azteca sericeasur alarm pheromone no. beetles to arrive a 6 5 4 3 2 1 0 a bb 2-pentanone treatment hexane pygidial Figure 4.

Box plots of the average number of M. xipebeetles to arrive at alarm pheromone bioassays. Each box represents a treatment type, where 2-heptanone is synthetic 2-heptanone, 2-pentanone is synthetic 2-pentanone, hexane is a solvent control and pygidial is an extract from crushed pygidial glands of A. sericeasur. The box plots indicate the median (large horizontal bars), the 25th and 75th percentiles (white rectangles), the minimum and maximum values (whiskers) and outliers (white circles). Lowercase letters show significant differences between treatment types. rspb.royalsocietypublishing.org Proc. R. Soc. B283: 20161281 5 on August 10, 2016 http://rspb.royalsocietypublishing.org/ Downloaded from benefit its prey by reducing the phorid fly population and thus predation may be adaptive toA. sericeasur. Adaptive suicide in social insects is relatively common as a pre-emptive strategy (e.g. honeybees using their stings to defend the colony), although it is notably difficult to evaluate in the con- text of host – parasitoid interactions [29,35 – 39]. The host suicide hypothesis postulates that mature parasitoids emer- ging from hosts are more likely to infect host’s kin than non-kin. Therefore, when maturation of the parasitoid is pre- vented, the inclusive fitness of the host should be increased.

Even a very small increase in inclusive fitness will be enough to drive the system, and favourable situations for adaptive suicide include systems where the host is a social insect, when there is high host inbreeding, and when parasi- toids have small search ranges [20]. This system appears to be a good candidate to meet these criteria, as A. sericeasuris not only a social insect, but is also polygynous and polydomous, forming colonies that can have territories spanning several hectares [40]. Although the lifetime dispersal distances of these phorid flies are currently unknown, P. lasciniosusand P. planidorsalis are rarely found farther than two meters from any given A. sericeasurnest [41]. Therefore, it is unlikely that phorid flies are dispersing beyond the boundaries of a single A. sericeasur colony prior to oviposition, although future work on the precise dispersal distance of these phorid flies is needed. However, despite the potential benefits, selective predation on parasitized ants may still have costs for the ant colony, and therefore, not support the host suicide hypothesis. First, it is possible that some small percentage of phorid fly larvae do not successfully mature to adulthood. In these cases, ants that survive parasitism and go on to benefit the colony would be at a greater risk for predation, which would ulti- mately cost the colony as a whole. Second, if the parasitized workers are active colony members during the phorid’s devel- opment period, the benefit of eliminating the phorid fly larvae may be offset by the costs incurred from losing productive parasitized ant workers. Future work is needed to confirm true costs and benefits of beetle predation on parasitized ants. Additionally, it will be worthwhile to determine the phys- iological mechanism by which this behavioural switch in aggression occurs in A. sericeasur. Nonetheless, our study shows that the effects of the M. xipe association with A. sericeasuris dependent upon phorid fly presence and that these beetles may be indirectly beneficial to A. sericeasur , by reducing the number of developing P. l asc in os us and P. planidorsalis parasitoids by approximately 14.7% and 2.6%, respectively. To our knowledge, ours is one of the few studies that document the role of ant-associated beetles outside of a pairwise context [2,42 – 46], and the first study to demon- strate a predator sharing host/prey cues with a parasitoid to gain access to a prey item that would otherwise be unavailable.

Increasingly, it is becoming apparent that investigating the ecological complexity within a system provides instructive examples of how organisms can change their behaviour or morphology in response to challenges from other organisms, and subsequently, how these changes can have cascading effects throughout a network of interacting species [8,9].

Current literature on the role of beetles within ant societies tells us that these beetles are exceedingly common and behav- iourally diverse, but few ant – beetle associations have been examined in depth. Further investigation into both the roles of these beetles and their ant hosts, as well as within the network of organisms surrounding ant societies is crucial to understanding how social insect colonies function within the community as a whole.

Data accessibility. All data are available from the Dryad data repository:

http://dx.doi.org/10.5061/dryad.g7599. Authors’ contributions. K.A.M. conceived, designed and performed the experiments. K.A.M. and N.D.T. analysed the data and wrote the manuscript. Competing interests. We declare we have no competing interests. Funding. We acknowledge USDA National Institute of Food and Agri- culture and Hatch project CA-B-INS-0087-H for support of the Tsutsui Laboratory. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship pro- gramme (DGE 1106400), National Institutes of Health (award no.

K12GM000708), the Robert Van den Bosch Fellowship, the UC Mexus Research Fellowship, the Edna & Yoshinori Tanada Fellowship and the Margaret C. Walker fund. Acknowledgements. We thank Finca Irlanda for allowing us to conduct research on the farm. We thank SEMARNAT (Secretaria de Medio Ambiente y Recursos Naturales) for permission to collect and export samples, and J. Rojas and E. Chame ´ Vasquez for facilitating the process of acquiring permits. References 1. Kistner DH. 1979 Social and evolutionary significance of social insect symbionts. Social Insects 1, 339 – 413.

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