The farming ant Sericomyrmex amabilis nutritionally manages its fungal symbiont and its social parasite

1. When parasites exploit mutualisms involving food exchange, they can destabilise the partnership with costs to interacting partners. For instance, the ant Sericomyrmex amabilis farms fungal symbionts to produce food, but, in so doing, attracts parasitic Megalomyrmex symmetochus guest ants that infiltrate fungus‐farming ant societies and live with their hosts their entire lives. 2. The present study examined whether host foraging in parasitised colonies shifts towards nutritional requirements of the parasitic guest ants as inferred from the parasite's elemental content (%C, %N, and C:N). 3. Laboratory feeding experiments with nutritionally defined diets indicated that S. amabilis ants harvest protein‐biased substrate, and more total substrate when hosting M. symmetochus relative to when provisioning their fungus gardens and nestmates. 4. Field surveys further showed that parasitised colonies incur reductions in fungus garden nutritional quality and quantity, brood mass, and host worker body condition. And yet these costs appear manageable across growing seasons, as parasitised fungal cultivars appear to provide sufficient nutrition for stable populations of host ants. 5. The approach developed here shows how behavioural strategies for nutrient regulation can extend beyond the needs of the individual to entire fungus‐farming systems, and implies that S. amabilis dynamically adjusts collective foraging strategies when parasitised to enhance long‐term symbiotic stability.     

Disease management in two sympatric Apterostigma fungus‐growing ants for controlling the parasitic fungus Escovopsis

Antagonistic interactions between host and parasites are often embedded in networks of interacting species, in which hosts may be attacked by competing parasites species, and parasites may infect more than one host species. To better understand the evolution of host defenses and parasite counterdefenses in the context of a multihost, multiparasite system, we studied two sympatric species, of congeneric fungus‐growing ants (Attini) species and their symbiotic fungal cultivars, which are attacked by multiple morphotypes of parasitic fungi in the genus, Escovopsis. To assess whether closely related ant species and their cultured fungi are evolving defenses against the same or different parasitic strains, we characterized Escovopsis that were isolated from colonies of sympatric Apterostigma dentigerum and A. pilosum. We assessed in vitro and in vivo interactions of these parasites with their hosts. While the ant cultivars are parasitized by similar Escovopsis spp., the frequency of infection by these pathogens differs between the two ant species. The ability of the host fungi to suppress Escovopsis growth, as well as ant defensive responses toward the parasites, differs depending on the parasite strain and on the host ant species.     

Ancient Host–Pathogen Associations Maintained by Specificity of Chemotaxis and Antibiosis

Switching by parasites to novel hosts has profound effects on ecological and evolutionary disease dynamics. Switching requires that parasites are able to establish contact with novel hosts and to overcome host defenses. For most host–parasite associations, it is unclear as to what specific mechanisms prevent infection of novel hosts. Here, we show that parasitic fungal species in the genus Escovopsis, which attack and consume the fungi cultivated by fungus-growing ants, are attracted to their hosts via chemotaxis. This response is host-specific: Escovopsis spp. grow towards their natural host cultivars more rapidly than towards other closely related fungi. Moreover, the cultivated fungi secrete compounds that can suppress Escovopsis growth. These antibiotic defenses are likewise specific: in most interactions, cultivars can inhibit growth of Escovopsis spp. not known to infect them in nature but cannot inhibit isolates of their naturally infecting pathogens . Cases in which cultivars are susceptible to novel Escovopsis are limited to a narrow set of host–parasite strain combinations. Targeted chemotactic and antibiotic responses therefore explain why Escovopsis pathogens do not readily switch to novel hosts, consequently constraining long-term dynamics of host–parasite coevolution within this ancient association.     

Bacterial symbiont sharing in Megalomyrmex social parasites and their fungus‐growing ant hosts

Bacterial symbionts are important fitness determinants of insects. Some hosts have independently acquired taxonomically related microbes to meet similar challenges, but whether distantly related hosts that live in tight symbiosis can maintain similar microbial communities has not been investigated. Varying degrees of nest sharing between Megalomyrmex social parasites (Solenopsidini) and their fungus‐growing ant hosts (Attini) from the genera Cyphomyrmex, Trachymyrmex and Sericomyrmex allowed us to address this question, as both ant lineages rely on the same fungal diet, interact in varying intensities and are distantly related. We used tag‐encoded FLX 454 pyrosequencing and diagnostic PCR to map bacterial symbiont diversity across the Megalomyrmex phylogenetic tree, which also contains free‐living generalist predators. We show that social parasites and hosts share a subset of bacterial symbionts, primarily consisting of Entomoplasmatales, Bartonellaceae, Acinetobacter, Wolbachia and Pseudonocardia and that Entomoplasmatales and Bartonellaceae can co‐infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. We reconstructed in more detail the population‐level infection dynamics for Entomoplasmatales and Bartonellaceae in Megalomyrmex symmetochus guest ants and their Sericomyrmex amabilis hosts. We further assessed the stability of the bacterial communities through a diet manipulation experiment and evaluated possible transmission modes in shared nests such as consumption of the same fungus garden food, eating of host brood by social parasites, trophallaxis and grooming interactions between the ants, or parallel acquisition from the same nest environment. Our results imply that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.     

Ancient host-pathogen associations maintained by specificity of chemotaxis and antibiosis

Switching by parasites to novel hosts has profound effects on ecological and evolutionary disease dynamics. Switching requires that parasites are able to establish contact with novel hosts and to overcome host defenses. For most host–parasite associations, it is unclear as to what specific mechanisms prevent infection of novel hosts. Here, we show that parasitic fungal species in the genus Escovopsis, which attack and consume the fungi cultivated by fungus-growing ants, are attracted to their hosts via chemotaxis. This response is host-specific: Escovopsis spp. grow towards their natural host cultivars more rapidly than towards other closely related fungi. Moreover, the cultivated fungi secrete compounds that can suppress Escovopsis growth. These antibiotic defenses are likewise specific: in most interactions, cultivars can inhibit growth of Escovopsis spp. not known to infect them in nature but cannot inhibit isolates of their naturally infecting pathogens . Cases in which cultivars are susceptible to novel Escovopsis are limited to a narrow set of host–parasite strain combinations. Targeted chemotactic and antibiotic responses therefore explain why Escovopsis pathogens do not readily switch to novel hosts, consequently constraining long-term dynamics of host–parasite coevolution within this ancient association.     

Complex host-pathogen coevolution in the Apterostigma fungus-growing ant-microbe symbiosis

Background  

The fungus-growing ant-microbe symbiosis consists of coevolving microbial mutualists and pathogens. The diverse fungal lineages that these ants cultivate are attacked by parasitic microfungi of the genus Escovopsis. Previous molecular analyses have demonstrated strong phylogenetic congruence between the ants, the ants-cultivated fungi and the garden pathogen Escovopsis at ancient phylogenetic levels, suggesting coevolution of these symbionts. However, few studies have explored cophylogenetic patterns between these symbionts at the recent phylogenetic levels necessary to address whether these parasites are occasionally switching to novel hosts or whether they are diversifying with their hosts as a consequence of long-term host fidelity.     

Caste specialization in behavioral defenses against fungus garden parasites in <Emphasis Type="Italic">Acromyrmex octospinosus</Emphasis> leaf-cutting ants

Division of labor and caste specialization plays an important role in many aspects of social insect colony organization, including parasite defense. Within leaf-cutting ant colonies, worker caste specialization permeates colony tasks ranging from foraging, substrate incorporation, brood care, and chemical defenses via glandular secretions and mutualistic bacteria. Leaf-cutting ants rely on protecting a mutualistic fungus they grow for food from microfungi in the genus Escovopsis that parasitizes the ant–fungus relationship. Here, we examine whether Acromyrmex octospinosus leaf-cutter ant castes (minors and majors) display task specialization in two behavioral defenses against Escovopsis: fungus grooming (the removal of Escovopsis spores) and weeding (the removal of large pieces of Escovopsis-infected fungus garden). Using behavioral observations, we show that minors are the primary caste that performs fungus grooming, while weeding is almost exclusively performed by majors. In addition, using a sub-colony infection experimental setup, we show that at the early stages of infection, minors more efficiently remove Escovopsis spores from the fungus garden, thereby restricting Escovopsis spore germination and growth. At later stages of infection, after Escovopsis spore germination, we find that major workers are as efficient as minors in defending the fungus garden, likely due to the increased importance of weeding. Finally, we show, using SEM imaging, that the number of sensory structures is similar between minor and major workers. If these structures are invoked in recognition of the parasites, this finding suggests that both castes are able to sense Escovopsis. Our findings support that leaf-cutter ant behavioral defense tasks against Escovopsis are subject to caste specialization, likely facilitated by worker sizes being optimal for grooming and weeding by minors and majors, respectively, with important consequences for cultivar defense.     

The mitochondrial genome contribution to the phylogeny and identification of Metarhizium species and strains

The genus Metarhizium is composed of entomopathogenic fungal biological control agents (BCAs) used for invertebrate pest control. The phylogenetic relationships of species within this genus are still under scrutiny as several cryptic species can be found. In this work, the mitochondrial (mt) genome of Metarhizium brunneum ARSEF 4556 was fully sequenced and a comparative genome analysis was conducted with 7 other available mt genomes, belonging to 5 Metarhizium species: M. anisopliae, M. brunneum, M. robertsii, M. guizhouense and M. majus. Results showed that Metarhizium demonstrates greater conserved stability than other fungal mt genomes. Furthermore, this analysis located 7 diverse regions in both intergenic domains and gene fragments which were ideal for species/strain discrimination. The sequencing of these regions revealed several SNPs among 38 strains tested, 11 of which were uncharacterized. Single gene phylogenies presented variable results which may be used further for intra-species discrimination. Phylogenetic trees based on the concatenation of mt domains and the nuclear ITS1-5.8S-ITS2 region showed discrimination of the species studied and allowed the identification of uncharacterized strains. These were mostly placed within species M. anisopliae and M. brunneum. Five strains clustered together in a clade related to M. brunneum, suggesting that they comprise a cryptic species.     

Anti-pathogen protection versus survival costs mediated by an ectosymbiont in an ant host

The fitness effects of symbionts on their hosts can be context-dependent, with usually benign symbionts causing detrimental effects when their hosts are stressed, or typically parasitic symbionts providing protection towards their hosts (e.g. against pathogen infection). Here, we studied the novel association between the invasive garden ant Lasius neglectus and its fungal ectosymbiont Laboulbenia formicarum for potential costs and benefits. We tested ants with different Laboulbenia levels for their survival and immunity under resource limitation and exposure to the obligate killing entomopathogen Metarhizium brunneum. While survival of L. neglectus workers under starvation was significantly decreased with increasing Laboulbenia levels, host survival under Metarhizium exposure increased with higher levels of the ectosymbiont, suggesting a symbiont-mediated anti-pathogen protection, which seems to be driven mechanistically by both improved sanitary behaviours and an upregulated immune system. Ants with high Laboulbenia levels showed significantly longer self-grooming and elevated expression of immune genes relevant for wound repair and antifungal responses (β-1,3-glucan binding protein, Prophenoloxidase), compared with ants carrying low Laboulbenia levels. This suggests that the ectosymbiont Laboulbenia formicarum weakens its ant host by either direct resource exploitation or the costs of an upregulated behavioural and immunological response, which, however, provides a prophylactic protection upon later exposure to pathogens.     

Escovopsioides nivea is a non-specific antagonistic symbiont of ant-fungal crops

Fungus-growing attine ants maintain a mutualistic relationship with basidiomycete fungi which they cultivate for food. In addition to the fungal partner, attine ant colonies harbor a myriad of microorganisms, including the genus Escovopsis, fungal parasites of the ant crops. Because Escovopsioides nivea is phylogenetically close to Escovopsis, previous studies assumed it has a negative interaction in the ant-fungus association. Here, we present an extended phylogeny of E. nivea based on new collections from different attine ant genera found in different localities. We also carried out co-culture experiments between E. nivea with different fungal cultivars. Our results suggest E. nivea is a symbiont of attine ant colonies, which inhibits the growth of fungal crops, supporting the hypothesis it is antagonistic to the system. However, the patterns of interaction between E. nivea and fungal crops differ from those shown by Escovopsis, suggesting a different evolution from that of the parasite.     

Prevalence and impact of a virulent parasite on a tripartite mutualism

The prevalence and impact of a specialized microfungal parasite (Escovopsis) that infects the fungus gardens of leaf-cutting ants was examined in the laboratory and in the field in Panama. Escovopsis is a common parasite of leaf-cutting ant colonies and is apparently more frequent in Acromyrmex spp. gardens than in gardens of the more phylogenetically derived genus Atta spp. In addition, larger colonies of Atta spp. appear to be less frequently infected with the parasite. In this study, the parasite Escovopsis had a major impact on the success of this mutualism among ants, fungi, and bacteria. Infected colonies had a significantly lower rate of fungus garden accumulation and produced substantially fewer workers. In addition, the extent of the reduction in colony growth rate depended on the isolate, with one isolate having a significantly larger impact than two others, suggesting that Escovopsis has different levels of virulence. Escovopsis is also spatially concentrated within parts of ant fungus gardens, with the younger regions having significantly lower rates of infection as compared to the older regions. The discovery that gardens of fungus-growing ants are host to a virulent pathogen that is not related to any of the three mutualists suggests that unrelated organisms may be important but primarily overlooked components of other mutualistic associations.     

Fitness consequences of nest infiltration by the mutualist‐exploiter Megalomyrmex adamsae

1. Fungus‐growing ants are obligate mutualists. Their nutrient‐rich fungus garden provides a valuable food store that sustains the ant hosts, but can also attract social parasites. 2. The ‘guest ant' Megalomyrmex adamsae Longino parasitises the fungus‐growing Trachymyrmex zeteki Weber queen just after nest founding. The parasitic queen infiltrates the incipient nest, builds a cavity in the fungal garden, and lays eggs that develop into workers and reproductive males and females. 3. This study compared young parasitised and non‐parasitised laboratory colonies by measuring garden growth and biomass, and the number of host workers and reproductives. Host queen survival and parasite colony growth were also monitored. 4. Parasitised Trachymyrmex colonies had reduced host worker and alate numbers, as well as lower garden biomass, compared with non‐parasitised control colonies, confirming that M. adamsae is a xenobiotic social parasite. Host queen survival was not significantly different between parasitised and control colonies. 5. This is the first study that experimentally infects host colonies with a xenobiotic social parasite to measure fitness cost to the host. The natural history of M. adamsae and the fungus‐growing ant mutualism are evaluated in the context of three general predictions of (Bronstein, Ecology Letters, 4 , 277–287, 2001a) regarding the cost of mutualism exploiters.     

Maternal Exposure of a Beetle to Pathogens Protects Offspring against Fungal Disease

Maternal exposure to an immune challenge can convey enhanced immunity to invertebrate offspring in the next generation. We investigated whether maternal exposure of the Asian longhorned beetle, Anoplophora glabripennis, to two species of the fungus Metarhizium or the bacterium Serratia marcescens elicited transgenerational immune priming (TGIP). We tested specificity of this protection and whether occurrence of TGIP was dependent on maternal exposure to living versus dead pathogens. Our results show that TGIP occurred and protected offspring against Metarhizium brunneum. Maternal exposure to S. marcescens provided non-specific protection to offspring against a fungal pathogen, but TGIP in response to Metarhizium only occurred when offspring were exposed to the same fungal species that was used to prime mothers. Moreover, TGIP in response to M. brunneum occurred only after maternal exposure to living rather than dead fungus. Our findings suggest that occurrence of TGIP could be both specific and dependent on whether the pathogen was alive.     

Diversity of rhizosphere associated entomopathogenic fungi of perennial herbs, shrubs and coniferous trees

Understanding habitat selection of fungal entomopathogens is critical to improve the efficacy, persistence and cost of these fungi as microbial insecticides. This study sought to determine the prevalence of Metarhizium and Beauveria spp. isolated from the rhizosphere of strawberry, blueberry, grape and Christmas tree crops in the Willamette Valley of Oregon. Entomopathogenic fungi were assigned to thirteen species based on molecular phylogenetic criteria. Four species of Metarhizium were isolated including Metarhizium brunneum, Metarhizium guizhouense, Metarhizium robertsii, and Metarhizium flavoviride var. pemphigi. Nine Beauveria species were isolated including, Beauveria brongniartii, an undescribed species referred to as Clade C and seven phylogenetic species of Beauveria bassiana. Strawberries and blueberries were significantly associated with M. brunneum and Christmas trees with M. guizhouense and M. robertsii. Grapes were significantly associated with B. bassiana phylogenetic species Bbas-16. All of the Metarhizium isolates screened were pathogenic to Otiorhynchus sulcatus larvae in laboratory bioassays but only M. brunneum and M. robertsii caused significant levels of infection. The study results suggest that certain species of Metarhizium and Beauveria are significantly associated with the strawberry, blueberry and Christmas tree rhizosphere and could potentially provide better control of O. sulcatus.     

Nylanderia deceptrix sp. n., a new species of obligately socially parasitic formicine ant (Hymenoptera,Formicidae)

Obligately socially parasitic ants are social parasites that typically lack the sterile worker caste, and depend on the host species for survival and brood care. The genus Nylanderia has over 130 described species and subspecies, none of which, until this study, were known social parasites. Here we describe the first social parasite known in the genus, Nylanderia deceptrix. Aspects of the biology of the host species, Nylanderia parvula (Mayr 1870), and Nylanderia deceptrix are examined. The data from both the host and the parasite species are combined to better understand the host-parasite relationship.     

Choice behaviour of Myrmica rubra workers between ant larvae and larvae of their Phengaris (Maculinea) nausithous nest parasites

Larvae of Phengaris (Maculinea) butterflies are adopted by Myrmica workers and are obligate myrmecophiles. Brood recognition by Myrmica rubra workers was tested for concolonial larvae (M. rubra) versus allocolonial larvae (M. rubra and P. nausithous) to assay the mimetic efficiency of P. nausithous. In addition, we tested M. rubra ant colonies from different populations with and without the presence of Phengaris, to test for potential local adaptation in adoption behaviour. We show that M. rubra can distinguish between nest-mate and foreign larvae as well as between P. nausithous and their own larvae. Workers from the allopatric population inspected and rejected more P. nausithous larvae than workers from the sympatric population. This might reflect a local host adaptation in which the social parasite more efficiently mimes its sympatric host ants than allopatric ones.     

Virulence and community dynamics of fungal species with vertical and horizontal transmission on a plant with multiple infections

The virulence evolution of multiple infections of parasites from the same species has been modeled widely in evolution theory. However, experimental studies on this topic remain scarce, particularly regarding multiple infections by different parasite species. Here, we characterized the virulence and community dynamics of fungal pathogens on the invasive plant Ageratina adenophora to verify the predictions made by the model. We observed that A. adenophora was highly susceptible to diverse foliar pathogens with mixed vertical and horizontal transmission within leaf spots. The transmission mode mainly determined the pathogen community structure at the leaf spot level. Over time, the pathogen community within a leaf spot showed decreased Shannon diversity; moreover, the vertically transmitted pathogens exhibited decreased virulence to the host A. adenophora, but the horizontally transmitted pathogens exhibited increased virulence to the host. Our results demonstrate that the predictions of classical models for the virulence evolution of multiple infections are still valid in a complex realistic environment and highlight the impact of transmission mode on disease epidemics of foliar fungal pathogens. We also propose that seedborne fungi play an important role in structuring the foliar pathogen community from multiple infections within a leaf spot.     

Nesting biology of the arboreal fungus-growing ant <Emphasis Type="Italic">Cyphomyrmex cornutus</Emphasis> and behavioral interactions with the social-parasitic ant <Emphasis Type="Italic">Megalomyrmex mondabora</Emphasis>

We describe the extraordinary nesting habits of the fungus-growing ant Cyphomyrmex cornutus (Formicidae, Myrmicinae, Attini) and the natural history of Megalomyrmex mondabora (Formicidae, Myrmicinae, Solenopsidini), a social parasite that inhabits nests of C. cornutus and other small attine ants. The study was carried out at two sites on the Atlantic slope of Costa Rica. The C. cornutus nest is an oblong mass of accreted soil, attached to or suspended from low vegetation in wet forest understory. Less than a fourth of the nest volume has chambers and is inhabited by C. cornutus; the remainder is a semi-solid mass of accreted soil often housing a variety of arthropods, including other unspecialized commensal ant species. Five C. cornutus colonies examined were parasitized by M. mondabora. Colonies of M. mondabora inhabited chambers very near those of the host. In laboratory observations, M. mondabora and C. cornutus workers interacted with little aggression despite the consumption of C. cornutus larvae and fungi by M. mondabora. During most interactions, C. cornutus workers responded submissively, whereas M. mondabora appeared indifferent or nonresponsive. Megalomyrmex mondabora parasitizes several other attine species (Cyphomyrmex costatus, Cyphomyrmex salvini, and Apterostigma goniodes), and it appears therefore a relatively unspecialized social parasite with broad attine hostassociation. The size of M. mondabora workers vary with host species, suggesting M. mondabora sensu lato comprises either cryptic species or the host environment affects worker size. Received 14 September 2006; revised 1 February 2007; accepted 6 February 2007.