The evolution of uniparental transmission of fungal symbionts in fungus-growing termites (Macrotermitinae)

Mutualistic associations between different organisms are theoretically expected when the interests of independently reproducing units are aligned to form a single reproductive unit. This alignment does not come about easily, because models show that hosts and symbionts can be in conflict over the transmission of symbionts. Selection will favour hosts that are able to limit genetic variation of symbionts, for example by enforcing uniparental vertical transmission, while symbionts will be selected to disperse independently of the host. A crucial factor determining the evolution and elaboration of symbiotic relationships is therefore who controls the transmission of symbionts. In the fungus-growing termites (Macrotermintinae) horizontal transmission seems to be the rule as the termites normally acquire their cultivated fungus (Termitomyces) from the environment. In spite of this general pattern, uniparental, vertical transmission has evolved in two unrelated Macrotermitinae genera, where only one sex of the two primary reproductives carries asexual spores from the fungal comb of its parent colony to inoculate the new fungus comb. Remarkably, symbiont transmission is exclusively paternal in Macrotermes bellicosus, whereas symbionts are maternally inherited in all Microtermes species studied so far. Thus, in Macrotermitinae horizontal transmission is the ancestral state with two independent origins to uniparental, vertical transmission. This is in contrast to fungus-growing ants where uniparental, vertical transmission is the rule. Causes and consequences of this difference are further discussed. Despite this fundamental difference both groups evolved a similar symbiosis that is probably the key for their ecological success: the fungus-growing ants in the neotropics and the fungus-growing termites in the paleotropics.     

The origin of the chemical profiles of fungal symbionts and their significance for nestmate recognition in <Emphasis Type="Italic">Acromyrmex</Emphasis> leaf-cutting ants

Cuticular hydrocarbon profiles are essential for nestmate recognition in insect societies, and quantitative variation in these recognition cues is both environmentally and genetically determined. Environmental cues are normally derived from food or nest material, but an exceptional situation may exist in the fungus-growing ants where the symbiotic fungus garden may be an independent source of recognition compounds. To investigate this hypothesis, we quantified the chemical profiles of the fungal symbionts of 18 sympatric colonies of Acromyrmex echinatior and Acromyrmex octospinosus and evaluated the quantitative variation of the 47 compounds in a multivariate analysis. Colony-specific chemical profiles of fungal symbionts were highly distinct and significantly different between the two ant species. We also estimated the relative genetic distances between the fungal symbionts using amplified fragment length polymorphism (AFLP) and correlated these with the overall (Mahalanobis) chemical distances between the colony-specific profiles. Despite the standardized laboratory conditions, the correlations were generally weak, but a statistically significant portion of the total variation in chemical profiles could be explained by genetic differences between the fungal symbionts. However, there was no significant effect of ant species in partial analyses because genetic differences between symbionts tend to coincide with being reared by different ant species. However, compound groups differed significantly with amides, aldehydes, and methyl esters contributing to the correlations, but acetates, alkanes, and formates being unrelated to genetic variation among symbionts. We show experimentally that workers that are previously exposed to and fed with the fungal symbiont of another colony are met with less aggression when they are later introduced into that colony. It appears, therefore, that fungus gardens are an independent and significant source of chemical compounds, potentially contributing a richer and more abundant blend of recognition cues to the colony “gestalt” than the innate chemical profile of the ants alone. Freddie-Jeanne Richard and Michael Poulsen contributed equally to this work.     

Task partitioning, division of labour and nest compartmentalisation collectively isolate hazardous waste in the leafcutting ant Atta cephalotes

We studied the organisation of garbage disposal and management in the leafcutting ant Atta cephalotes. The nest of this species has an internal garbage heap to which waste from the fungus garden is taken. The transport of waste from the fungus gardens to the garbage heaps is an example of task partitioning. Ninety-four percent of the garbage loads transferred from the fungus garden to the garbage heap were transferred indirectly via a caching site just outside the garbage heap entrance. A further 3% were transferred directly from a fungus garden worker to a garbage heap worker, again just outside the heap entrance. Only 3% were taken directly to the garbage heap without task partitioning. This is the first described example of task partitioning in insect societies for work other than foraging and the first example of task partitioning occurring entirely within the nest. Furthermore, there is a strong division of labour between the fungus garden workers and the garbage heap workers, with garbage workers hardly ever leaving the heap. Division of labour is reinforced by aggressive behaviour directed towards workers contaminated with garbage. This pattern of work organisation minimises contact between garbage heap workers, who are probably contaminated with pathogens hazardous to both the ants and their symbiotic fungus, and both fungus garden workers and the fungus garden. Task partitioning, division of labour (reinforced by aggression) and nest compartmentalisation act synergistically to isolate the hazardous garbage heap from the fungus gardens.     

The patchiness of epifoliar fungi in tropical forests: host range, host abundance, and environment

Fungal symbioses affect the diversity, dynamics, and spatial patterns of trees in tropical forests. Their ecological importance is partly driven by their inherent patchiness. We used epifoliar fungi, a guild of common, benign, obligate, fungal symbionts of plants, as a model system to evaluate the relative importance of host phylogeny, host relative abundance, and microclimate on the three-dimensional distribution of plant-fungus symbioses. In parallel studies in rainforests in Panama and Australia, most epifoliar fungi were able to colonize several plant lineages but showed significant host preferences within the local plant community. More closely related plant species were not more likely to share fungal symbionts. Instead, fungal species were more likely to be shared by more abundant hosts, which supported a greater number and diversity of fungi. Environmental conditions strongly affected spatial distributions, with sites in the dark understory 2.5- to fourfold more likely to have epifoliar fungi than in the exposed forest canopy. In the understory, fungal incidence increased with canopy openness. Canopy trees supported only a subset of the fungal symbionts found in the understory, suggesting that adult trees are not reservoirs of these fungal symbionts for understory juveniles.     

Construction of chimaeric gardens through fungal intercropping: a symbiont choice experiment in the leafcutter ant Atta texana (Attini, Formicidae)

Interspecies or intraspecies cooperation can be stabilized evolutionarily if choosing partners favor beneficial partners and discriminate against non-beneficial partners. We quantified such partner choice (symbiont choice) in the leafcutter ant Atta texana (Attini, Formicidae) by presenting the ants in a cafeteria-style preference assay with genotypically distinct fungal cultivars from A. texana and Acromyrmex versicolor. Symbiont choice was measured as the ants' tendency to choose one or more cultivar(s) from several pure (axenic) cultivar fragments and convert a given fungal fragment into a garden. Microsatellite DNA fingerprinting enabled us to identify the cultivars chosen by the ants for their gardens. In 91% of the choice tests, A. texana workers combined multiple cultivars into a single intercropped, chimaeric garden, and the cultivars coexisted in such chimaeric gardens for as long as 4 months. Coexistence of distinct fungal genotypes in chimaeric gardens appears to contradict a recent model of cultivar competition postulating that each cultivar secretes incompatibility compounds harming other cultivars, which presumably would preclude the intercropped polyculture observed in our experiments. Although we found no clear evidence of novel, recombinant genotypes in the experimental chimaeric gardens, the intercropping of cultivar genotypes may occasionally lead under natural conditions to exchange of genetic material between coexisting cultivars, thus introducing novel cultivar genotypes into the leafcutter symbiosis. Symbiont choice by ants and any competition between coexisting cultivar strains in chimaeric gardens do not appear to operate fast enough in our laboratory assay to convert chimaeric gardens into the monocultures observed for A. texana under natural conditions.     

Is the alkaloid in 2spot ladybirds (Adalia bipunctata) a defence against ant predation?

Garden black ants,Lasius niger L., in a laboratory colony, attacked three species of live ladybirds found near their nest, killing the smaller two species. A second colony was offered artificial diets containing crushed ladybirds of two species, and the ants' choice of feeding site noted. Both the diets were aversive compared to control, but that containing 7spot,Coccinella septempunctata L., was more aversive than the diet containing 2spot,Adalia bipunctata L. The implications of this lesser protection for 2spots in terms of the chemical defence of the species are discussed.     

Experimental evidence for the costs and hygienic significance of the antibiotic metapleural gland secretion in leaf-cutting ants

The paired exocrine metapleural glands present in the large majority of ant species produce compounds with antibiotic properties. In the leaf-cutting ant, Acromyrmex octospinosus, the secretion consists of more than 20 different compounds and it has generally been assumed that the glands serve as a general defence against various infectious microbes of fungal and bacterial origin. We present results illuminating the direct costs and benefits of these metapleural gland defences in A. octospinosus. We show that major workers of this leaf-cutting ant experience a significant reduction in their respiration rate when the metapleural glands are experimentally closed, indicating that metapleural gland secretion incurs a substantial cost and that the production of compounds from these glands is terminated when the ants are incapable of secreting them. In another set of experiments, we show that the ability to secrete antibiotic compounds from the metapleural glands is of significant importance when ants are exposed to a general but potentially virulent insect pathogen, Metarhizium anisopliae. Infection with this fungus is lethal within a few days when ants have their metapleural glands experimentally closed, but relatively harmless when the metapleural glands are functional. These findings support experimentally the view that the metapleural glands play an important hygienic role in leaf-cutting ants.     

Host specificity of the symbiotic cyanobacterium<Emphasis Type="Italic"> Oscillatoria spongeliae</Emphasis> in marine sponges,<Emphasis Type="Italic"> Dysidea</Emphasis> spp.

Marine sponges can host a variety of cyanobacterial and bacterial symbionts, but it is often unclear whether these symbionts are generalists that occur in many host species or specialists that occur only in certain species or populations of sponges. The filamentous cyanobacterium Oscillatoria spongeliae is found in the sponges Dysidea n. sp. aff. herbacea 1A and 1B, and similar cyanobacteria are found in D. n. sp. aff. granulosa. We amplified and sequenced sponge nuclear ribosomal DNA (rDNA) and cyanobacterial 16S rDNA from specimens of these three sponges. We then used these sequences to construct phylogenies for host sponges and their symbiotic cyanobacteria. Each of these three sponge species hosts a distinct cyanobacterial clade, suggesting a high degree of host specificity and potential coevolution between symbiotic cyanobacteria and their host sponges.     

Complexity in an obligate mutualism: do fungus-gardening ants know what makes their garden grow?

Fungus gardening ants make clear choices among fungal substrates (food for their fungus). It has been proposed, but never demonstrated, that these ants are collecting the best for their symbiotic fungus and the production of ant biomass (fitness). The goal of this study was to determine whether preferred substrates lead to higher fitness in the attine, Trachymyrmex septentrionalis. Preferences exhibited by foragers were established. Colonies were fed a single substrate or a mixture of substrates during the entire course of the experiment, which ended when sexual offspring appeared in the nest. The response variables were numbers and weights of ant offspring and the chitin content of fungus gardens. Preference was not strongly related to fitness. The preferred oak catkins produced the highest amounts of ant and fungal biomass, but the ants collected much more material than needed, which indicates that forager activity is decoupled from fitness. The preferred caterpillar feces were rejected shortly after the feedings began. The unpreferred oak leaves were just as effective at producing ant and fungal biomass as catkins. Leaves are possibly unpreferred because they are expensive to cut. The unpreferred huckleberry flowers were inferior but did not cause rejection behavior. The mixed diet was just as productive as catkins or leaves. This study indicates that foragers possess a default mechanism to prefer catkins and frass, which can be quickly changed if substrates are bad. In contrast, there does not appear to be a similar mechanism causing substrates to become preferred quickly.     

Colonial recognition of fungus in the fungus-growing ant Acromyrmex subterraneus subterraneus (Hymenoptera: Formicidae)

Summary. Leaf cutting ants live in symbiosis with a basidiomycete fungus that is exploited as a source of nutrients for the ant larvae. Tests of fungus transport demonstrated that Acromyrmex subterraneus subterraneus workers discriminate concolonial fungus from alien fungus, and rejected the latter. Larvae and pupae of the ant were used as controls. Chemical analysis of the fungus revealed a great similarity between its hydrocarbon profile and that found on the ant brood. Experiments with lures showed that chemical extracts from the fungus are responsible for this discrimination process. Moreover, the presence of brood inside the fungus seemed to be important for discrimination of the fungus by workers. Resident workers accepted concolonial broodless fungus less than concolonial fungus inoculated with brood odor. Fungus seems to acquire colonial odor passively, simply by contact with the brood. The impact of fungus volume present in the nest on closure of the colony is discussed. We show here for the first time the importance of a symbiotic vegetal organism in colonial recognition in social insects. Received 14 April 2000; accepted 29 September 2000     

Insecticidal and antifungal chemicals produced by plants: a review

Leaf-cutting ants of the Attini tribe are a major pest of agricultural and forestry productions in the New World. Economic losses caused by these ants were estimated at several million dollars per year. These ants need to live in symbiosis with a basidiomycete fungus. Due to their mutualistic interaction with the symbiotic fungus, management of Attini ants can be done with insecticides or fungicides or both. So far, synthetic pesticides were the main control means, albeit with negative effects on the environment. Very few studies describe alternative methods for the control of leaf-cutting ants such as the use of insecticidal and fungicidal plant extracts. There is therefore a need of knowledge on phytochemicals and plants that could be used as insecticides and fungicides. Here, we review chemicals of plant origin and species with insecticidal and fungicidal activities. We establish a list of plants and phytochemicals that could manage leaf-cutting ants and also other insects, notably insects that use fungus-based agriculture. An exhaustive literature search of 1965 references from 1923 to 2010 was conducted using scientific databases, chemical databases, botanical databases, and books to identify published papers related to insecticidal and fungicidal chemical compounds stemmed from plant species. The major points are the following: (1) 119 and 284 chemicals have been cited in the literature for their insecticidal and fungicidal activities, respectively; (2) 656 and 1,064 plant species have significant insecticidal and fungicidal activities, respectively; (3) 3 main chemical classes were most cited for these activities: alkaloids, phenolics, and terpenoids; (4) 20 interesting chemicals with the both insecticidal and fungicidal activities were found; and (5) 305 plant species containing these chemicals were cited. To conclude, 20 chemicals: caryophyllene oxide, cinnamaldehyde, eugenol, helenalin, linalool, menthone, myristicin, pulegone, thymol, anethole, anisaldehyde, elemicin, isopimpinellin, plumbagin, podophyllotoxin, psoralen, xanthotoxin, anonaine, solamargine, and tomatine; two main plant families, Lamiaceae and Apiaceae; and 17 species of these families were particularly interesting for the management of leaf-cutting ants.     

Transitioning from unstable to stable colony growth in the desert leafcutter ant Acromyrmex versicolor

Like organisms, cohesive social groups such as insect colonies grow from a few individuals to large and complex integrated systems. Growth is driven by the interplay between intrinsic growth rates and environmental factors, particularly nutritional input. Ecologically inspired population growth models assume that this relationship remains constant until maturity, but more recent models suggest that it should be less stable at small colony sizes. To test this empirically, we monitored worker population growth and fungal development in the desert leafcutter ant, Acromyrmex versicolor, over the first 6 months of colony development. As a multitrophic, symbiotic system, leafcutter colonies must balance efforts to manage both fungus production and the growth of the ants consuming it. Both ants and fungus populations grew exponentially, but the shape of this relationship transitioned at a size threshold of 89?±?9 workers. Above this size, colony mortality plummeted and colonies shifted from hypometric to hypermetric growth, with a distinct stabilization of the relationship between the worker population and fungus. Our findings suggest that developing colonies undergo key changes in organizational structure and stability as they grow, with a resulting positive transition in efficiency and robustness.     

Biogeographic partitioning and host specialization among foraminiferan dinoflagellate symbionts (<Emphasis Type="Italic">Symbiodinium</Emphasis>; Dinophyta)

Large discoidal soritid foraminiferans (Soritinae) are abundant in coral reef ecosystems. As with the many cnidarian invertebrates that inhabit these systems, they also depend on symbiotic dinoflagellates (Symbiodinium) for their growth and survival. Several particular Symbiodinium sub-genera or clades inhabit these soritids. One of these groups, referred to as clade C, dominates corals and their relatives throughout the tropical Indo-Pacific. In contrast, the distributions of Symbiodinium spp. from clades A, B, and C are more evenly apportioned across Caribbean invertebrate communities. To explore the possibility that a similar biogeographic break exists in the symbionts harbored by soritids, we surveyed the Symbiodinium spp. from the soritid genus Sorites, collected from the Pacific and Caribbean coasts of Panama as well as from Florida. Characterization of Symbiodinium obtained from foraminiferal and cnidarian samples was conducted using restriction fragment length polymorphism and phylogenetic analyses of the nuclear internal transcribed spacer region 2 (ITS 2) and a portion of the large subunit ribosomal DNA sequences. A distinctive biogeographic break between the kinds of symbionts found in Sorites from the East Pacific and Caribbean was clearly evident. Differences between cnidarian and foraminferan symbioses in each ocean may be explained by the subjection of Caribbean communities to severer environmental conditions during the early Quarternary. Caribbean Sorites spp. harbored symbionts described from clade F (specifically sub-clade Fr4) and clade H (formally referred to as Fr1), while Sorites spp. from the eastern Pacific were dominated by a single Symbiodinium haplotype in clade C. An ITS 2 phylogeny determined that most clade C types recovered from Indo-Pacific soritids form a monophyletic sub-lineage with other clade C symbionts typically found in Pacific corals from the genus Porites. The existence of multiple Symbiodinium lineages at various taxonomic levels associated specifically with soritids indicates that symbioses with these hosts are important in driving Symbiodinium spp. evolution.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by O. Kinne, Oldendorf/Luhe     

Specificity of two temperate dinoflagellate–anthozoan associations from the north-western Pacific Ocean

Whilst many studies of symbiotic dinoflagellate diversity have focused on tropical reef environments, only a few have explored the degree and pattern of divergence of these endosymbionts at high latitudes. In this study, the genetic diversity and specificity of symbiotic dinoflagellates associated with two common anthozoan hosts in the north-western Pacific Ocean was studied in four different seasons during a period of 1 year. Partial nucleotide sequences of 28S and complete ITS1 ribosomal DNA regions were used to identify, genetically, the endosymbionts extracted from the scleractinian Alveopora japonica and the actinarian Heteractis sp. A. japonica harbours symbionts belonging to Symbiodinium of clade F, while Heteractis sp. associates with Symbiodinium of clade C. Moreover, no seasonal changes in the endosymbiont community were detected in these two associations during this study. This is the first evidence that these two temperate cnidarian–microalgae symbioses are stable. Furthermore, we tested the apparent specificity of the Heteractis sp.– Symbiodinium sp. clade C association, by performing alga-infection experiments with aposymbiotic hosts, and monitoring the uptake and persistence of homologous and heterologous symbionts. The findings confirm the association patterns detected in the field and show that Heteractis sp. only establishes a successful association with Symbiodinium cells of clade C, at least among the heterologous symbionts occurring in the study area. Our results are consistent with the idea that selective pressures in highly fluctuating temperate environments might have granted symbiosis-specificity an adaptive value.Communicated by T. Ikeda, Hakodate     

There are many ways to be a mutualist: endophytic fungus reduces plant survival but increases population growth

One of the challenges to quantifying the costs and benefits of symbiosis is that symbionts can influence different components of host fitness. To improve understanding of the ecology of inherited symbionts, we developed general theory for a perennial host-hereditary symbiont interaction, in which symbionts can have independent and potentially opposing effects on host regeneration and survival. The model showed that negative effects on one component of fitness may be outweighed by positive effects on another, leading to a net positive impact of symbiosis on population growth. Model predictions depended on the availability of suitable patches, which influenced the relative contributions of survival vs. regeneration to host fitness. We then used experimental symbiont removal to quantify effects of a hereditary, fungal endophyte on a grass host. Endophyte presence strongly reduced host survival but increased regeneration. Application of the model revealed that negative effects on plant survival were overwhelmed by beneficial effects on regeneration, resulting in stable endophyte persistence at 100% frequency, consistent with field observations. Our work demonstrates the utility of a demographic perspective for predicting the dynamics of symbioses and supports the hypothesis that symbionts function as mutualists when host and symbiont fitness are coupled through vertical transmission.     

Waste management in the leaf-cutting ant Acromyrmex echinatior: the role of worker size, age and plasticity

Division of labour is the hallmark of the success of many social animals. It may be especially important with regard to waste management because waste often contains pathogens or hazardous toxins and worker specialisation can reduce the number of group members exposed to it. Here we examine waste management in a fungus-farming, leaf-cutting ant, Acromyrmex echinatior, in which waste management is necessary to protect their vulnerable fungal crop. By marking ants with task-specific paint colours, we found clear division of labour between workers that engage in waste management and those that forage, at least during the fine timescale of the 3-day marking period. This division of labour was influenced by both age and size, with waste management workers tending to be smaller and younger than foragers. The role of preventing contaminated ants from entering the colony was fulfilled mainly by medium-sized workers. When the level of waste was experimentally increased, most of the ants that responded to remove the waste were workers previously engaged in tasks inside the nest rather than external waste workers or foragers. These responding workers tended to be young and medium-sized. Surprisingly, the responding ants were subsequently able to revert back to working within the fungus garden, but the probability of them doing so depended on their age and the length of time they were exposed to waste. The results demonstrate the importance of division of labour with regard to waste management in A. echinatior and show that this is adaptable to changing needs.     

Context-dependent effects of symbiosis: Zoanthidea colonization generally improves Demospongiae condition in native habitats

In order to reconcile existing data and hypotheses on the relationship outcomes of Caribbean Zoanthidea-Demospongiae symbioses, the context-dependent survival and growth (metrics of host sponge condition) were monitored for 8–12?months, with and without Zoanthidean symbionts, in native (coral reef) and novel (mangrove) habitats. Experiments repeated over space and time revealed significant positive effects of Zoanthidea colonization on metrics of host condition and indicated that the outcome of these symbioses can shift from mutualism to parasitism when transplanted to novel habitats. Although the precise mechanisms of the symbioses remain obscured and most of the species associations have not been examined, these experiments demonstrate an example where relationship outcomes are more conserved across their evolutionary history than host associations.     

Three beetles—three concepts. Different defensive strategies of congeneric myrmecophilous beetles

Myrmecophiles, i.e., organisms associated with ants live in a variety of ecological niches in the vicinity or inside ant colonies and employ different strategies to survive ant encounters. Because different niches are characterized by different encounter rates with host ants, strategies used to avoid ant aggressions should depend on these niches. This hypothesis was studied with three rove beetle species of the genus Pella, which are myrmecophiles of the ant Lasius fuliginosus and the non-myrmecophilous relative Drusilla canaliculata. Behavioral tests in the field revealed that Pella species are better adapted to interactions with ants than D. canaliculata, but that they use species-specific strategies in ant interactions. Pella cognata and Pella funesta avoid encounters with ants by swift movements. Chemical analyses of the defensive tergal gland secretions showed that P. cognata has replaced the aggression inducing undecane by the behaviorally neutral tridecane. P. funesta repels the ants by releasing the panic alarm pheromone sulcatone from its tergal gland resulting in an “ant free space” around the beetles. Finally, Pella laticollis uses a specific and unique appeasing behavior. Behavioral and chemical data did not reveal any indication for the mimicry of the ants' cuticular hydrocarbon profiles by any of the beetle species. It is discussed that the employed strategies correlate with the ecological niches of the beetles. P. cognata and P. funesta are living along ant trails with ample space to escape and the employed strategies are probably sufficient to escape from dangerous conflicts. In contrast, P. laticollis lives in refuse areas of ant nests with frequent ant encounters, and its appeasement strategy allows it to stay at the encounter site.     

Cortical and endosomal structure of the marine sponge Stelletta grubii

The fine structure of the marine astrophorid sponge Stelletta grubii is described for the first time. The following new data are presented: spongin is present, choanocyte chambers are diplodal, intercellular symbiotic bacteria are numerous and unequally distributed in the cortex and endosome, and collagenous fibril bundles are associated with lophocyte activity and are not elastic fibers. The cortex contains numerous fibril bundles, fewer symbionts, very few cells, and transitional zones with higher archeocyte density near the surface and endosome. Limited phagocytosis of the bacterial symbionts is observed. This species appears to be dioecious and oviparous. These observations suggest that the enigmatic species Chondrosia reniformis is closely related to S. grubii and that it should be placed within or near the astrophorids. The rhizoids of the red alga Phyllophora palmettoides penetrate the sponge tissue without eliciting the development of a structurally specialized contact zone in the sponge matrix or of a limiting epithelium.