Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.     

Density of mushroom body synaptic complexes limits intraspecies brain miniaturization in highly polymorphic leaf-cutting ant workers

Hymenoptera possess voluminous mushroom bodies (MBs), brain centres associated with sensory integration, learning and memory. The mushroom body input region (calyx) is organized in distinct synaptic complexes (microglomeruli, MG) that can be quantified to analyse body size-related phenotypic plasticity of synaptic microcircuits in these small brains. Leaf-cutting ant workers (Atta vollenweideri) exhibit an enormous size polymorphism, which makes them outstanding to investigate neuronal adaptations underlying division of labour and brain miniaturization. We particularly asked how size-related division of labour in polymorphic workers is reflected in volume and total numbers of MG in olfactory calyx subregions. Whole brains of mini, media and large workers were immunolabelled with anti-synapsin antibodies, and mushroom body volumes as well as densities and absolute numbers of MG were determined by confocal imaging and three-dimensional analyses. The total brain volume and absolute volumes of olfactory mushroom body subdivisions were positively correlated with head widths, but mini workers had significantly larger MB to total brain ratios. Interestingly, the density of olfactory MG was remarkably independent from worker size. Consequently, absolute numbers of olfactory MG still were approximately three times higher in large compared with mini workers. The results show that the maximum packing density of synaptic microcircuits may represent a species-specific limit to brain miniaturization.     

Ethylene in mutualistic symbioses

Ethylene (ET) is a gaseous phytohormone that participates in various plant physiological processes and essentially contributes to plant immunity. ET conducts its functions by regulating the expression of ET-responsive genes or in crosstalk with other hormones. Several recent studies have shown the significance of ET in the establishment and development of plant-microbe interactions. Therefore, it is not surprising that pathogens and mutualistic symbionts target ET synthesis or signaling to colonize plants. This review introduces the significance of ET metabolism in plant-microbe interactions, with an emphasis on its role in mutualistic symbioses.     

Symbiotic bacteria on the cuticle of the leaf-cutting ant Acromyrmex subterraneus subterraneus protect workers from attack by entomopathogenic fungi

Although only discovered in 1999, the symbiotic filamentous actinobacteria present on the integument of certain species of leaf-cutting ants have been the subject of intense research. These bacteria have been shown to specifically suppress fungal garden parasites by secretion of antibiotics. However, more recently, a wider role for these bacteria has been suggested from research revealing their generalist anti-fungal activity. Here we show, for the first time, evidence for a role of these bacteria in the defence of young worker ants against a fungal entomopathogen. Experimental removal of the bacterial bio-film using an antibiotic resulted in a significant increase in susceptibility of worker ants to infection by the entomopathogenic fungus Metarhizium anisopliae. This is the first direct evidence for the advantage of maintaining a bacterial bio-film on the cuticle as a defensive strategy of the ants themselves and not exclusively for protection of the fungus garden.     

Optimality in a partitioned task performed by social insects

Biologists have long been aware that adaptations should not be analysed in isolation from the function of the whole organism. Here, we address the equivalent issue at the scale of a social insect colony: the optimality of component behaviours in a partitioned sequence of tasks. In colonies of Atta colombica, a leaf-cutting ant, harvested leaf tissue is passed from foragers to nest workers that distribute, clean, shred and implant the tissue in fungal gardens. In four laboratory colonies of A. colombica, we found that the highest colony-wide rate of leaf tissue processing in the nest was achieved when leaf fragment sizes were suboptimal for individual delivery rate by foragers. Leaf-cutting ant colonies appear to compromise the efficiency of collecting leaf tissue in order to increase their ability to handle the material when it arrives in the nest. Such compromise reinforces the idea that behavioural adaptations, like adaptations in general, must be considered within the context of the larger entity of which they are a part.     

The police are not the army: context-dependent aggressiveness in a clonal ant

Animals often exhibit particular ‘personalities’, i.e. their behaviour is correlated across different situations. Recent studies suggest that this limitation of behavioural plasticity may be adaptive, since continuous adjustment of one''s behaviour may be time-consuming and costly. In social insects, particularly aggressive workers might efficiently take over fighting in the contexts of both nest defence and ‘policing’, i.e. the regulation of kin conflict in the society. Here, we examine whether workers who engage in aggressive policing in the ant Platythyrea punctata play a prominent role also in nest defence against intruders. The participation of individuals in policing and nest defence was highly skewed and a minority of workers exhibited most of the aggression. Workers who attacked reproductives after experimental colony fusion were less active during nest defence and vice versa. This suggests that workers show situation-dependent behavioural plasticity rather than consistently aggressive personalities.     

Diversity of entomopathogenic fungi near leaf-cutting ant nests in a neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae

We investigated the prevalence of entomopathogenic fungi associated with leaf-cutting ant colonies in a small area of tropical forest in Panama. There was a high abundance of Metarhizium anisopliae var. anisopliae near the colonies. Beauveria bassiana was also detected in the soil, Aspergillus flavus in dump material, and six Camponotus atriceps ants were found infected with Cordyceps sp. Based on a partial sequence of the IGS region, almost all of the M. anisopliae var. anisopliae isolates fell within one of the three main clades of M. anisopliae var. anisopliae, but with there still being considerable diversity within this clade. The vast majority of leaf-cutting ants collected were not infected by any entomopathogenic fungi. While leaf-cutting ants at this site must, therefore, regularly come into contact with a diversity of entomopathogenic fungi, they do not appear to be normally infected by them.     

Highly diversified fungi are associated with the achlorophyllous orchid Gastrodia flavilabella

Background

Mycoheterotrophic orchids are achlorophyllous plants that obtain carbon and nutrients from their mycorrhizal fungi. They often show strong preferential association with certain fungi and may obtain nutrients from surrounding photosynthetic plants through ectomycorrhizal fungi. Gastrodia is a large genus of mycoheterotrophic orchids in Asia, but Gastrodia species’ association with fungi has not been well studied. We asked two questions: (1) whether certain fungi were preferentially associated with G. flavilabella, which is an orchid in Taiwan and (2) whether fungal associations of G. flavilabella were affected by the composition of fungi in the environment.

Results

Using next-generation sequencing, we studied the fungal communities in the tubers of Gastrodia flavilabella and the surrounding soil. We found (1) highly diversified fungi in the G. flavilabella tubers, (2) that Mycena species were the predominant fungi in the tubers but minor in the surrounding soil, and (3) the fungal communities in the G. flavilabella tubers were clearly distinct from those in the surrounding soil. We also found that the fungal composition in soil can change quickly with distance.

Conclusions

G. flavilabella was associated with many more fungi than previously thought. Among the fungi in the tuber of G. flavilabella, Mycena species were predominant, different from the previous finding that adult G. elata depends on Armillaria species for nutritional supply. Moreover, the preferential fungus association of G. flavilabella was not significantly influenced by the composition of fungi in the environment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1422-7) contains supplementary material, which is available to authorized users.     

Unusual heterostyly: style dimorphism and self-incompatibility are not tightly associated in Lithodora and Glandora (Boraginaceae)

Background and Aims

Heterostyly is a floral polymorphism characterized by the reciprocal position of stamens and stigmas in different flower morphs in a population. This reciprocal herkogamy is usually associated with an incompatibility system that prevents selfing and intra-morph fertilization, termed a heteromorphic incompatibility system. In different evolutionary models explaining heterostyly, it has been alternately argued that heteromorphic incompatibility either preceded or followed the evolution of reciprocal herkogamy. In some models, reciprocal herkogamy and incompatibility have been hypothesized to be linked together during the evolution of the heterostylous system.

Methods

We examine the incompatibility systems in species with different stylar polymorphisms from the genera Lithodora and Glandora (Boraginaceae). We then test whether evolution towards reciprocal herkogamy is associated with the acquisition of incompatibility. To this end, a phylogeny of these genera and related species is reconstructed and the morphological and reproductive changes that occurred during the course of evolution are assessed.

Key Results

Both self-compatibility and self-incompatibility are found within the studied genera, along with different degrees of intra-morph compatibility. We report for the first time extensive variability among members of the genus Glandora and related species in terms of the presence or absence of intraspecies polymorphism and heteromorphic incompatibility. Overall, our results do not support a tight link between floral polymorphism and incompatibility systems.

Conclusions

The independent evolution of stylar polymorphism and incompatibility appears to have occurred in this group of plants. This refutes the canonical view that there is strong linkage between these reproductive traits.     

Macroevolutionary assembly of ant/plant symbioses: Pseudomyrmex ants and their ant-housing plants in the Neotropics

Symbioses include some of the clearest cases of coevolution, but their origin, loss or reassembly with different partners can rarely be inferred. Here we use ant/plant symbioses involving three plant clades to investigate the evolution of symbioses. We generated phylogenies for the big-eyed arboreal ants (Pseudomyrmecinae), including 72% of their 286 species, as well as for five of their plant host groups, in each case sampling more than 61% of the species. We show that the ant-housing Vachellia (Mimosoideae) clade and its ants co-diversified for the past 5 Ma, with some species additionally colonized by younger plant-nesting ant species, some parasitic. An apparent co-radiation of ants and Tachigali (Caesalpinioideae) was followed by waves of colonization by the same ant clade, and subsequent occupation by a younger ant group. Wide crown and stem age differences between the ant-housing genus Triplaris (Polygonaceae) and its obligate ant inhabitants, and stochastic trait mapping, indicate that its domatium evolved earlier than the ants now occupying it, suggesting previous symbioses that dissolved. Parasitic ant species evolved from generalists, not from mutualists, and are younger than the mutualistic systems they parasitize. Our study illuminates the macroevolutionary assembly of ant/plant symbioses, which has been highly dynamic, even in very specialized systems.     

Plantago lanceolata and Plantago rugelii Extracts are Toxic to Meloidogyne incognita but not to Certain Microbes

Extracts from the plants Plantago lanceolata and P. rugelii were evaluated for toxicity to the root-knot nematode Meloidogyne incognita, the beneficial microbes Enterobacter cloacae, Pseudomonas fluorescens and Trichoderma virens, and the plant-pathogenic fungi Fusarium oxysporum f. sp. gladioli, Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani. Wild plants were collected, roots were excised from shoots, and the plant parts were dried and ground to a powder. One set of extracts (10% w/v) was prepared in water and another in methanol. Treatments included extract concentrations of 25%, 50%, 75% and 100%, and water controls. Meloidogyne incognita egg hatch was recorded after 7-day exposure to the treatments, and second-stage juvenile (J2) activity after 48 hours. All extracts were toxic to eggs and J2, with P. lanceolata shoot extract tending to have the most activity against M. incognita. Numbers of active J2 remained the same or decreased in a 24-hour water rinse following the 48-hour extract treatment, indicating that the extracts were lethal. When data from water- and methanol-extracted roots and shoots of both plant species were combined for analysis, J2 tended to be more sensitive than eggs to the toxic compounds at lower concentrations, while the higher concentrations (75% and 100%) were equally toxic to both life stages. The effective concentrations causing 50% reduction (EC₅₀) in egg hatch and in J2 viability were 44.4% and 43.7%, respectively. No extract was toxic to any of the bacteria or fungi in our assays.     

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.     

Dissecting the fungal biology of Bipolaris papendorfii: from phylogenetic to comparative genomic analysis

Bipolaris papendorfii has been reported as a fungal plant pathogen that rarely causes opportunistic infection in humans. Secondary metabolites isolated from this fungus possess medicinal and anticancer properties. However, its genetic fundamental and basic biology are largely unknown. In this study, we report the first draft genome sequence of B. papendorfii UM 226 isolated from the skin scraping of a patient. The assembled 33.4 Mb genome encodes 11,015 putative coding DNA sequences, of which, 2.49% are predicted transposable elements. Multilocus phylogenetic and phylogenomic analyses showed B. papendorfii UM 226 clustering with Curvularia species, apart from other plant pathogenic Bipolaris species. Its genomic features suggest that it is a heterothallic fungus with a putative unique gene encoding the LysM-containing protein which might be involved in fungal virulence on host plants, as well as a wide array of enzymes involved in carbohydrate metabolism, degradation of polysaccharides and lignin in the plant cell wall, secondary metabolite biosynthesis (including dimethylallyl tryptophan synthase, non-ribosomal peptide synthetase, polyketide synthase), the terpenoid pathway and the caffeine metabolism. This first genomic characterization of B. papendorfii provides the basis for further studies on its biology, pathogenicity and medicinal potential.     

Frontier mutualism: coevolutionary patterns at the northern range limit of the leaf-cutter ant-fungus symbiosis

Tropical leaf-cutter ants cultivate the fungus Attamyces bromatificus in a many-to-one, diffuse coevolutionary relationship where ant and fungal partners re-associate frequently over time. To evaluate whether ant-Attamyces coevolution is more specific (tighter) in peripheral populations, we characterized the host-specificities of Attamyces genotypes at their northern, subtropical range limits (southern USA, Mexico and Cuba). Population-genetic patterns of northern Attamyces reveal features that have so far not been observed in the diffusely coevolving, tropical ant-Attamyces associations. These unique features include (i) cases of one-to-one ant-Attamyces specialization that tighten coevolution at the northern frontier; (ii) distributions of genetically identical Attamyces clones over large areas (up to 81 000 km(2), approx. the area of Ireland, Austria or Panama); (iii) admixture rates between Attamyces lineages that appear lower in northern than in tropical populations; and (iv) long-distance gene flow of Attamyces across a dispersal barrier for leaf-cutter ants (ocean between mainland North America and Cuba). The latter suggests that Attamyces fungi may occasionally disperse independently of the ants, contrary to the traditional assumption that Attamyces fungi depend entirely on leaf-cutter queens for dispersal. Peripheral populations in Argentina or at mid-elevation sites in the Andes may reveal additional regional variants in ant-Attamyces coevolution. Studies of such populations are most likely to inform models of coextinctions of obligate mutualistic partners that are doubly stressed by habitat marginality and by environmental change.     

Fungal symbioses in hornworts: a chequered history

Hornworts are considered the sister group to vascular plants, but their fungal associations remain largely unexplored. The ancestral symbiotic condition for all plants is, nonetheless, widely assumed to be arbuscular mycorrhizal with Glomeromycota fungi. Owing to a recent report of other fungi in some non-vascular plants, here we investigate the fungi associated with diverse hornworts worldwide, using electron microscopy and molecular phylogenetics. We found that both Glomeromycota and Mucoromycotina fungi can form symbioses with most hornworts, often simultaneously. This discovery indicates that ancient terrestrial plants relied on a wider and more versatile symbiotic repertoire than previously thought, and it highlights the so far unappreciated ecological and evolutionary role of Mucoromycotina fungi.     

You are what you eat: diet-induced chemical crypsis in a coral-feeding reef fish

The vast majority of research into the mechanisms of camouflage has focused on forms that confound visual perception. However, many organisms primarily interact with their surroundings using chemosensory systems and may have evolved mechanisms to ‘blend in’ with chemical components of their habitat. One potential mechanism is ‘chemical crypsis'' via the sequestration of dietary elements, causing a consumer''s odour to chemically match that of its prey. Here, we test the potential for chemical crypsis in the coral-feeding filefish, Oxymonacanthus longirostris, by examining olfactory discrimination in obligate coral-dwelling crabs and a predatory cod. The crabs, which inhabit the corals consumed by O. longirostris, were used as a bioassay to determine the effect of coral diet on fish odour. Crabs preferred the odour of filefish fed their preferred coral over the odour of filefish fed a non-preferred coral, suggesting coral-specific dietary elements that influence odour are sequestered. Crabs also exhibited a similar preference for the odour of filefish fed their preferred coral and odour directly from that coral, suggesting a close chemical match. In behavioural trials, predatory cod were less attracted to filefish odour when presented alongside the coral it had been fed on, suggesting diet can reduce detectability. This is, we believe, the first evidence of diet-induced chemical crypsis in a vertebrate.     

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.