Fungal farming in a non-social beetle

Culturing of microbes for food production, called cultivation mutualism, has been well-documented from eusocial and subsocial insects such as ants, termites and ambrosia beetles, but poorly described from solitary, non-social insects. Here we report a fungal farming in a non-social lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae), which entails development of a special female structure for fungal storage/inoculation, so-called mycangium, and also obligate dependence of the insect on the fungal associate. Adult females of D. bucculenta bore a hole on a recently-dead bamboo culm with their specialized mandibles, lay an egg into the internode cavity, and plug the hole with bamboo fibres. We found that the inner wall of the bamboo internode harboring a larva is always covered with a white fungal layer. A specific Saccharomycetes yeast, Wickerhamomyces anomalus ( = Pichia anomala), was consistently isolated from the inner wall of the bamboo internodes and also from the body surface of the larvae. Histological examination of the ovipositor of adult females revealed an exoskeletal pocket on the eighth abdominal segment. The putative mycangium contained yeast cells, and W. anomalus was repeatedly detected from the symbiotic organ. When first instar larvae were placed on culture media inoculated with W. anomalus, they grew and developed normally to adulthood. By contrast, first instar larvae placed on either sterile culture media or autoclaved strips of bamboo inner wall exhibited arrested growth at the second instar, and addition of W. anomalus to the media resumed growth and development of the larvae. These results strongly suggest a mutualistic nature of the D. bucculenta-W. anomalus association with morphological specialization and physiological dependence. Based on these results, we compare the fungal farming of D. bucculenta with those of social and subsocial insects, and discuss ecological factors relevant to the evolution of fungal farming in a non-social insect.     

Presence of weed fungus in a non‐social beetle–fungus cultivation mutualism

1. Farming by non‐human organisms has arisen independently in several animal lineages, allowing them to survive on food sources that are otherwise difficult to access. However, agricultural gardens are prone to invasion by parasites that overgrow cultivars in the absence of host animals. The presence of garden parasites and associated host adaptations are well studied in advanced fungal agriculture practised by social insects (ants, termites, and ambrosia beetles), but the impact of garden parasites in more primitive forms of agriculture is poorly known. The aim of the present study was to report the presence of weed fungi in fungal gardens of the non‐social lizard beetle Doubledaya bucculenta Lewis (Coleoptera: Erotylidae: Languriinae), which cultivates the yeast Wickerhamomyces anomalus (E.C. Hansen) Kurtzman, Robnett & Bas.‐Powers in the internodes of recently dead bamboo culms. 2. The filamentous fungi Arthrinium spp., Fusarium spp., and an unidentified species were isolated from the inner wall and insect‐made wall debris of bamboo internodes inhabited by the beetle. 3. When cultured together with the yeast in the absence of host larvae, Arthrinium sp.1 outcompeted the yeast, suggesting that this filamentous fungus can invade yeast gardens but is normally suppressed under natural conditions. 4. Rearing experiments showed that beetle larvae grew faster when grown on yeast cultures than when fed only Arthrinium sp.1. 5. These results suggest that Arthrinium sp.1 acts as a weed in the fungal gardens of D. bucculenta, inhibiting both growth of the beetle larvae and proliferation of the beneficial yeast.     

Predation on the woodwasp Tremex longicollis Konow (Hymenoptera: Siricidae) and its parasitoid Megarhyssa jezoensis (Matsumura) (Hymenoptera: Ichneumonidae) adults during oviposition

Adults of the woodwasp Tremex longicollis Konow (Hymenoptera: Siricidae) oviposit in the wood of dying Celtis sinensis trees using their ovipositors, and adults of the parasitoid Megarhyssa jezoensis (Matsumura) (Hymenoptera: Ichneumonidae) deposit eggs into the woodwasp larvae that live in the wood, also using their long ovipositors. Many T. longicollis ovipositors, both with and without abdominal segments, and a M. jezoensis ovipositor were found stuck in C. sinensis wood at two parks in Osaka Prefecture, central Japan. Field observations showed that ovipositing woodwasps and parasitoids were attacked by predators during the daytime, and their ovipositors were left behind in the wood. Woodwasps and their parasitoids, which have long ovipositors, may be vulnerable to predation during oviposition.     

Symbiotic complexity: discovery of a fifth symbiont in the attine ant-microbe symbiosis

The fungus-growing ant-microbe mutualism is a classic example of organismal complexity generated through symbiotic association. The ants have an ancient obligate mutualism with fungi they cultivate for food. The success of the mutualism is threatened by specialized fungal parasites (Escovopsis) that consume the cultivated fungus. To defend their nutrient-rich garden against infection, the ants have a second mutualism with bacteria (Pseudonocardia), which produce antibiotics that inhibit the garden parasite Escovopsis. Here we reveal the presence of a fourth microbial symbiont associated with fungus-growing ants: black yeasts (Ascomycota; Phialophora). We show that black yeasts are commonly associated with fungus-growing ants, occurring throughout their geographical distribution. Black yeasts grow on the ants' cuticle, specifically localized to where the mutualistic bacteria are cultured. Molecular phylogenetic analyses reveal that the black yeasts form a derived monophyletic lineage associated with the phylogenetic diversity of fungus growers. The prevalence, distribution, localization and monophyly indicate that the black yeast is a fifth symbiont within the attine ant-microbe association, further exemplifying the complexity of symbiotic associations.     

植菌昆虫与其共生真菌协同进化分子机制

昆虫菌业（fungiculture）是一种类似于人类种植业的昆虫种植体系,包括种植、耕作、收获和营养依赖4个过程,可分为高级的社会性昆虫如切叶蚂蚁、白蚁等和低级的非社会性昆虫如食菌小蠹虫、卷叶象甲、蜥蜴甲虫、树蜂等,它们均能种植并取食真菌。近年来随着组学及微生物组技术的发展,植菌昆虫与其共生真菌协同进化的分子机制研究方面取得了重要进展。系统发育分析阐明了植菌昆虫的起源与进化历程,并显示出与共生真菌系统发育的一致性;共生真菌细胞核数量也从双核增加到最多17个核,而染色体倍型也从单倍体增加为二倍体甚至多倍体;组学分析则揭示了植菌昆虫与其共生真菌在精氨酸、碳水化合物、木质素及几丁质合成或降解等方面显示出了高度的协同进化。本文系统综述了植菌昆虫及其共生真菌的系统进化、核进化及基因组进化进展,并探讨这种协同进化机制的生物学意义。     

Fungal nutrition allocation enhances mutualism with fungus-growing termite

Fungal nodules and aged fungus gardens are products of termite fungiculture systems, and are the diets of termites. To understand the nutrition flow in fungiculture, we quantified the number and mass of fungal nodules produced along with fungus garden maturation and analysed the α-amino acid and fatty acid compositions of fungal nodules, fungus gardens, and termite tissues of a fungus-growing termite, Odontotermes formosanus. 1 g of fungus garden produced 5,148 fungal nodules (∼68.0 mg). Approximately 7.0% of α-amino acids were allocated to the fungal nodules and the rest (∼93.0%) remained in the fungus gardens. The compositions of α-amino acids or fatty acids in aged fungus gardens and fungal nodules were more similar to that of termite tissues than fresh fungus gardens, which supports the idea that termites nutritionally depend on the fungal products. Among the 18 α-amino acids, tryptophan was an essential amino acid and was the only one missing from fresh and aged fungus gardens, but found in fungal nodules at significantly higher concentrations. Hence, termites must consume fungal nodules to obtain tryptophan for survival. Furthermore, the fungus spores incorporated in nodules, were transferred when nodules were ingested by termites. We propose that allocating tryptophan in fungal nodules is crucial to enhance the mutualism between the fungus and termite.     

Measuring the discrepancy between fecundity and lifetime reproductive success in a pollinating fig wasp

Lifetime reproductive success in female insects is often egg‐ or time‐limited. For instance in pro‐ovigenic species, when oviposition sites are abundant, females may quickly become devoid of eggs. Conversely, in the absence of suitable oviposition sites, females may die before laying all of their eggs. In pollinating fig wasps (Hymenoptera: Agaonidae), each species has an obligate mutualism with its host fig tree species [Ficus spp. (Moraceae)]. These pro‐ovigenic wasps oviposit in individual ovaries within the inflorescences of monoecious Ficus (syconia, or ‘figs’), which contain many flowers. Each female flower can thus become a seed or be converted into a wasp gall. The mystery is that the wasps never oviposit in all fig ovaries, even when a fig contains enough wasp females with enough eggs to do so. The failure of all wasps to translate all of their eggs into offspring clearly contributes to mutualism persistence, but the underlying causal mechanisms are unclear. We found in an undescribed Brazilian Pegoscapus wasp population that the lifetime reproductive success of lone foundresses was relatively unaffected by constraints on oviposition. The number of offspring produced by lone foundresses experimentally introduced into receptive figs was generally lower than the numbers of eggs carried, despite the fact that the wasps were able to lay all or most of their eggs. Because we excluded any effects of intraspecific competitors and parasitic non‐pollinating wasps, our data suggest that some pollinators produce few offspring because some of their eggs or larvae are unviable or are victims of plant defences.     

Effects of Asobara japonica venom on larval survival of host and nonhost Drosophila species

Abstract Ovipositing Asobara japonica females inject venom (containing paralysis‐inducing factors) immediately after the insertion of their ovipositors into Drosophila larvae, and lay eggs a little later. Interruption of their oviposition behaviour before egg laying causes high larval mortality in host Drosophila species, whereas normal oviposition does not. This suggests that venom of this parasitoid is toxic to larvae of these host species but its toxicity is suppressed by factor(s) provided by parasitoid females at the time of laying egg or by parasitoid embryos developing in the hosts. On the other hand, venom does not show toxicity to larvae of nonhost Drosophia species. Possible functions of venom are discussed.     

A Wickerhamomyces anomalus Killer Strain in the Malaria Vector Anopheles stephensi

The yeast Wickerhamomyces anomalus has been investigated for several years for its wide biotechnological potential, especially for applications in the food industry. Specifically, the antimicrobial activity of this yeast, associated with the production of Killer Toxins (KTs), has attracted a great deal of attention. The strains of W. anomalus able to produce KTs, called “killer” yeasts, have been shown to be highly competitive in the environment. Different W. anomalus strains have been isolated from diverse habitats and recently even from insects. In the malaria mosquito vector Anopheles stephensi these yeasts have been detected in the midgut and gonads. Here we show that the strain of W. anomalus isolated from An. stephensi, namely WaF17.12, is a killer yeast able to produce a KT in a cell-free medium (in vitro) as well as in the mosquito body (in vivo). We showed a constant production of WaF17.12-KT over time, after stimulation of toxin secretion in yeast cultures and reintroduction of the activated cells into the mosquito through the diet. Furthermore, the antimicrobial activity of WaF17.12-KT has been demonstrated in vitro against sensitive microbes, showing that strain WaF17.12 releases a functional toxin. The mosquito-associated yeast WaF17.12 thus possesses an antimicrobial activity, which makes this yeast worthy of further investigations, in view of its potential as an agent for the symbiotic control of malaria.     

Insect oviposition preference between Epichloë‐symbiotic and Epichloë‐free grasses does not necessarily reflect larval performance

Variation in plant communities is likely to modulate the feeding and oviposition behavior of herbivorous insects, and plant‐associated microbes are largely ignored in this context. Here, we take into account that insects feeding on grasses commonly encounter systemic and vertically transmitted (via seeds) fungal Epichloë endophytes, which are regarded as defensive grass mutualists. Defensive mutualism is primarily attributable to alkaloids of fungal origin. To study the effects of Epichloë on insect behavior and performance, we selected wild tall fescue (Festuca arundinacea) and red fescue (Festuca rubra) as grass–endophyte models. The plants used either harbored the systemic endophyte (E+) or were endophyte‐free (E?). As a model herbivore, we selected the Coenonympha hero butterfly feeding on grasses as larvae. We examined both oviposition and feeding preferences of the herbivore as well as larval performance in relation to the presence of Epichloë endophytes in the plants. Our findings did not clearly support the female's oviposition preference to reflect the performance of her offspring. First, the preference responses depended greatly on the grass–endophyte symbiotum. In F. arundinacea, C. hero females preferred E+ individuals in oviposition‐choice tests, whereas in F. rubra, the endophytes may decrease exploitation, as both C. hero adults and larvae preferred E? grasses. Second, the endophytes had no effect on larval performance. Overall, F. arundinacea was an inferior host for C. hero larvae. However, the attraction of C. hero females to E+ may not be maladaptive if these plants constitute a favorable oviposition substrate for reasons other than the plants' nutritional quality. For example, rougher surface of E+ plant may physically facilitate the attachment of eggs, or the plants offer greater protection from natural enemies. Our results highlight the importance of considering the preference of herbivorous insects in studies involving the endophyte‐symbiotic grasses as host plants.     

Fungus garden platforms improve hygiene during nest establishment in <Emphasis Type="Italic">Acromyrmex</Emphasis> ants (Hymenoptera,Formicidae, Attini)

Physically isolating organisms from disease agents should reduce the likelihood of disease transmission and infection, and increase survival and growth, particularly in more vulnerable, early ontogenetic stages. During nest founding in fungus-growing ants, foundresses of most genera use a garden platform to isolate the incipient fungal garden from the soil of the underground chamber. We studied nest founding in Acromyrmex octospinosus to test the hypotheses that the use of a platform (rootlets used to suspend the fungus garden): (i) reduces the likelihood that the garden will be contaminated by soil-borne microbial pathogens; (ii) results in more rapid growth of a young colony; and (iii) increases colony survival. We manipulated natural incipient nests to have gardens either in contact with or isolated from soil surrounding the chamber, and nests with and without foundresses present. We found a higher incidence of infection in gardens that were in contact with the chamber soil and without queens, compared with gardens isolated from the chamber soil with and without foundresses. The production of eggs, larvae and pupae, as well as leaf area harvested, were significantly different between nests with and without platforms, but there were no differences in the production of workers nor garden biomass. Likewise, there were no differences between treatments in colony survival rates over 8 weeks. Using smaller incipient gardens, however, gardens with and without platforms differed in survivorship rates after 24 hours. The results indicate that the use of a platform to cultivate an incipient fungal garden is an adaptation to reduce soil-borne diseases and increase colony performance. Received 28 July 2006; revised 15 November 2006; accepted 22 November 2006.     

Isolation of a Wickerhamomyces anomalus yeast strain from the sandfly Phlebotomus perniciosus,displaying the killer phenotype

The yeast Wickerhamomyces anomalus has been studied for its wide biotechnological potential, mainly for applications in the food industry. Different strains of W. anomalus have been isolated from diverse habitats and recently from insects, including mosquitoes of medical importance. This paper reports the isolation and phylogenetic characterization of W. anomalus from laboratory‐reared adults and larvae of Phlebotomus perniciosus (Diptera: Psychodidae), a main phlebotomine vector of human and canine leishmaniasis. Of 65 yeast strains isolated from P. perniciosus, 15 strains were identified as W. anomalus; one of these was tested for the killer phenotype and demonstrated inhibitory activity against four yeast sensitive strains, as reported for mosquito‐isolated strains. The association between P. perniciosus and W. anomalus deserves further investigation in order to explore the possibility that this yeast may exert inhibitory/killing activity against Leishmania spp.     

Are gardens effective in butterfly conservation? A case study with the pipevine swallowtail, <Emphasis Type="Italic">Battus philenor</Emphasis>

Gardens with nectar sources and larval host plants have been proposed to stem the decline in butterfly abundance caused by habitat loss. However, no study has provided evidence that gardens benefit butterflies. We examined the use of natural sites and gardens in the San Francisco bay area by the butterfly, Battus philenor. We found that natural sites were more likely to attract adult B. philenor, received more oviposition, and had higher juvenile survival than gardens sites. Butterflies were more likely to be present in gardens with established populations of the host plant, Aristolochia californica, growing in the sun. Battus philenor are unlikely to visit gardens with host plants planted within the past 7 years. Gardens between the ages of 8–40 years received oviposition, but did not always support completion of larval development of B. philenor. In gardens with host plants over 40 years of age, B. philenor consistently survived from egg to the adult stage. Natural enemy induced mortality of eggs did not differ between garden and natural sites, but overall egg survival was lower in gardens than at natural sites. It is unlikely that gardens serve as 'refugia' for B. philenor in years when populations in natural sites experience low survival or low fecundity. Even in gardens capable of supporting larvae to maturity, the density of eggs and survival rates were lower than in natural populations of the host plant suggesting that gardens were not optimal habitats. Therefore, without evidence that juvenile abundance and survival rates in gardens matches or exceeds that in natural sites, it is most likely that gardens act as population sinks for B. philenor.     

Parasites may help stabilize cooperative relationships

Background  

The persistence of cooperative relationships is an evolutionary paradox; selection should favor those individuals that exploit their partners (cheating), resulting in the breakdown of cooperation over evolutionary time. Our current understanding of the evolutionary stability of mutualisms (cooperation between species) is strongly shaped by the view that they are often maintained by partners having mechanisms to avoid or retaliate against exploitation by cheaters. In contrast, we empirically and theoretically examine how additional symbionts, specifically specialized parasites, potentially influence the stability of bipartite mutualistic associations. In our empirical work we focus on the obligate mutualism between fungus-growing ants and the fungi they cultivate for food. This mutualism is exploited by specialized microfungal parasites (genus Escovopsis) that infect the ant's fungal gardens. Using sub-colonies of fungus-growing ants, we investigate the interactions between the fungus garden parasite and cooperative and experimentally-enforced uncooperative ("cheating") pairs of ants and fungi. To further examine if parasites have the potential to help stabilize some mutualisms we conduct Iterative Prisoner's Dilemma (IPD) simulations, a common framework for predicting the outcomes of cooperative/non-cooperative interactions, which incorporate parasitism as an additional factor.     

Deterred oviposition response of Monochamus alternatus (Coleoptera: Cerambycidae) to oviposition scars occupied by eggs

1 The oviposition behaviour and response of Monochamus alternatus females to oviposition scars were investigated in the laboratory. 2 Prior to oviposition, females gnawed at the bark surface of Pinus densiflora bolts to make a wound. Then females turned their bodies 180° to position their ovipositors over the wounds and inserted them under the bark through the wounds. After an oviposition, a jelly was deposited while the ovipositor was still inserted. The females then withdrew their ovipositors and rubbed the oviposition scars with the tips of their abdomens. 3 When searching females encountered oviposition scars, they stopped walking and drummed the surface and inside of the oviposition scars with their maxillary and labial palpi. 4 Eighty-six percent of females left oviposition scars containing single eggs after the palpation. By contrast, when females encountered oviposition scars containing no eggs, 76% of them began to gnaw at the scars and 64% deposited single eggs. The response to artificial oviposition scars was similar to that to vacant oviposition scars made by the females. 5 The results of various observations and experiments showed that the females could recognize oviposition scars and discriminate the scars occupied by single eggs from vacant ones, and suggested that the palpation of oviposition scars was the critical discrimination behaviour, indicating mediation by chemical cues.     

Ecological aspects of the external morphology and functionality of the psychomyiid female ovipositor (Insecta,Trichoptera)

Females of most psychomyiid species bear an elongated ovipositor enabling them to oviposit their eggs into grooves and cavities in different kinds of substrates in freshwaters. Although the preference of psychomyiid species for oviposition into distinct substrates and the subsequent larval life performance in aquatic habitats is already known, the mode of oviposition and the functional morphology of the elongated ovipositor have not been described until now. In this study we present SEM photographs of some psychomyiid species with female ovipositors of different lengths and shapes, explaining their preference for oviposition into distinct substrates. Additionally, we discuss some ecological aspects of the ovipositor shapes and the mode of egg laying for psychomyiids. The female ovipositor consists of two body segments, with an elongated IXth and a distinctly shorter Xth segment, which is flexible and can be pushed up dorsally when releasing the eggs. On the basis of SEM photographs, we suppose that the opening of the channel through which the eggs were released is located ventrally near the ovipositor tip. The ovipositor itself is characterised by a ventral cleft reaching from the gonoporus of segment IX to the tip of the last segment X. We stored adult females abdomina overnight in different aqueous dilutions of ethanol leading to different stages of swelling of the ovipositors (grade of swelling in distilled water >30% ethanol >70% ethanol). Some internal membrane-like structures, normally infolded into the ovipositor, became visible by the swelling of the ovipositor. We discuss the possible rearrangement of the organisation of segment IX resulting in the development of an ovipositor on the basis of SEM photographs of the differently swollen ovipositors.     

A mid-Cretaceous ambrosia fungus, Paleoambrosia entomophila gen. nov. et sp. nov. (Ascomycota: Ophiostomatales) in Burmese (Myanmar) amber,and evidence for a femoral mycangium

An ambrosia fungus is described from filamentous sporodochia adjacent to a wood–boring ambrosia beetle (Coleoptera: Curculionidae: Platypodinae) in mid-Cretaceous Burmese amber. Yeast-like propagules and hyphal fragments of Paleoambrosia entomophila gen. nov. et sp. nov. occur in glandular sac mycangia located inside the femur of the beetle. This is the first record of a fossil ambrosia fungus, showing that symbiotic associations between wood–boring insects and ectosymbiotic fungi date back some 100 million years ago. The present finding moves the origin of fungus-growing by insects from the Oligocene to the mid-Cretaceous and suggests a Gondwanan origin.     

Acromyrmex Leaf-Cutting Ants Have Simple Gut Microbiota with Nitrogen-Fixing Potential

Ants and termites have independently evolved obligate fungus-farming mutualisms, but their gardening procedures are fundamentally different, as the termites predigest their plant substrate whereas the ants deposit it directly on the fungus garden. Fungus-growing termites retained diverse gut microbiota, but bacterial gut communities in fungus-growing leaf-cutting ants have not been investigated, so it is unknown whether and how they are specialized on an exclusively fungal diet. Here we characterized the gut bacterial community of Panamanian Acromyrmex species, which are dominated by only four bacterial taxa: Wolbachia, Rhizobiales, and two Entomoplasmatales taxa. We show that the Entomoplasmatales can be both intracellular and extracellular across different gut tissues, Wolbachia is mainly but not exclusively intracellular, and the Rhizobiales species is strictly extracellular and confined to the gut lumen, where it forms biofilms along the hindgut cuticle supported by an adhesive matrix of polysaccharides. Tetracycline diets eliminated the Entomoplasmatales symbionts but hardly affected Wolbachia and only moderately reduced the Rhizobiales, suggesting that the latter are protected by the biofilm matrix. We show that the Rhizobiales symbiont produces bacterial NifH proteins that have been associated with the fixation of nitrogen, suggesting that these compartmentalized hindgut symbionts alleviate nutritional constraints emanating from an exclusive fungus garden diet reared on a substrate of leaves.     

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.