Mycangia Of Beetles Research Articles

Fungi associated with the ambrosia beetle Xyleborus perforans (Coleoptera: Curculionidae: Scolytinae) on drought-stressed Pinus in New South Wales, Australia

Ambrosia beetles have co-evolved symbiotic relationships with an array of fungal partners. Mutualistic fungal partners are often highly successful in vertical transmission between beetle generations. These persisting relationships can alter beetle behaviour, resulting in the opportunity to occupy new ecological niches and to spread geographically. In Australia, ambrosia beetles are not currently considered a significant pest in commercial Pinus plantations, where the bark beetle Ips grandicollis is known as the primary invader of stressed trees. However, in 2019, ambrosia beetles Xyleborus perforans and X. bispinatus, co-occurring with I. grandicollis, were found to have colonised a large proportion of drought-stressed trees in commercial Pinus plantations in north-east New South Wales. In this study, X. perforans (the most prevalent of two Xyleborus spp.) was collected from infested dead and dying trees in two NSW Pinus plantations. Fungal isolates of suspected Pinus pathogens were recovered from beetle mycangia and exoskeletons as well as ambrosia beetle galleries. Morphological examination and multilocus sequence analysis identified five fungi associated with X. perforans: Fusarium parceramosum, Fusarium aff. solani, Ophiostoma ips, Raffaelea deltoideospora and Sporothrix pseudoabietina. For Australia, this is the first report of F. parceramosum, as well as the first records of O. ips, R. deltoideospora and S. pseudoabietina being vectored by Xyleborus. Pathogenicity tests were performed on seedlings of three Pinus spp., with O. ips producing significantly longer lesions than the other fungi. This study demonstrates the potential for seemingly harmless ambrosia beetles to vector plant pathogens in Australian forests, providing a mode of disease transmission that should be considered in plantation management and forest biosecurity.

Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora.

Symbiosis between insects and fungi arose multiple times during the evolution of both groups, and some of the most biologically diverse and economically important are mutualisms in which the insects cultivate and feed on fungi. Among these are bark beetles, whose ascomycetous cultivars are better known and studied than their frequently-overlooked and poorly understood basidiomycetous partners. In this study, we propose five new species of Entomocorticium, fungal mutualists in the Russulales (Basidiomycota) that are mutualistic symbionts of scolytine beetles. We have isolated these fungi from the beetle mycangia, which are structures adapted for the selective storage and transportation of fungal mutualists. Herein, we present the most complete phylogeny of the closely related genera Entomocorticium and Peniophora and provide insights into how an insect-associated taxon (Entomocorticium) evolved from within a wood-decaying, wind-dispersed lineage (Peniophora). Our results indicate that following a transition from angiosperms to gymnosperms, fungal domestication by beetles facilitated the evolution and diversification of Entomocorticium. We additionally propose four new species: Entomocorticium fibulatum Araújo, Li & Hulcr, sp. nov.; E. belizense Araújo, Li & Hulcr, sp. nov.; E. perryae Araújo, Li & Hulcr, sp. nov.; and E. macrovesiculatum Araújo, Li, Six & Hulcr, sp. nov. Our findings highlight the fact that insect-fungi associations remain an understudied field and that these associations harbor a large reservoir of novel fungal species.

Fungal mutualisms and pathosystems: life and death in the ambrosia beetle mycangia.

Ambrosia beetles and their microbial communities, housed in specialized structures termed mycangia, represent one of the oldest and most diverse systems of mutualism and parasitism described thus far. Comprised of core filamentous fungal members, but also including bacteria and yeasts, the mycangia represent a unique adaptation that allows beetles to store and transport their source of nutrition. Although perhaps the most ancient of "farmers," the nature of these interactions remains largely understudied, with the exception of a handful of emerging pathosystems, where the fungal partner acts as a potentially devastating tree pathogen. Such virulence is often seen during "invasions," where (invasive) beetles carrying the fungal symbiont/plant pathogen expand into new territories and presumably "naïve" trees. Here, we summarize recent findings on the phylogenetic relationships between beetles and their symbionts and advances in the developmental and genetic characterization of the mechanisms that underlie insect-fungal-plant interactions. Results on genomic, transcriptomic, and metabolomic aspects of these relationships are described. Although many members of the fungal Raffaelea-beetle symbiont genera are relatively harmless to host trees, specialized pathosystems including wilt diseases of laurel and oak, caused by specific subspecies (R. lauricola and R. quercus, in the USA and East Asia, respectively), have emerged as potent plant pathogens capable of killing healthy trees. With the development of genetic tools, coupled to biochemical and microscopic techniques, the ambrosia beetle-fungal symbiont is establishing itself as a unique model system to study the molecular determinants and mechanisms that underlie the convergences ofsymbioses, mutualism, parasitism, and virulence. KEY POINTS: • Fungal-beetle symbioses are diverse and ancient examples of microbial farming. • The mycangium is a specialized structure on insects that houses microbial symbionts. • Some beetle symbiotic fungi are potent plant pathogens vectored by the insect.

Patterns of coevolution between ambrosia beetle mycangia and the Ceratocystidaceae, with five new fungal genera and seven new species.

Ambrosia beetles farm specialised fungi in sapwood tunnels and use pocket-like organs called mycangia to carry propagules of the fungal cultivars. Ambrosia fungi selectively grow in mycangia, which is central to the symbiosis, but the history of coevolution between fungal cultivars and mycangia is poorly understood. The fungal family Ceratocystidaceae previously included three ambrosial genera (Ambrosiella, Meredithiella, and Phialophoropsis), each farmed by one of three distantly related tribes of ambrosia beetles with unique and relatively large mycangium types. Studies on the phylogenetic relationships and evolutionary histories of these three genera were expanded with the previously unstudied ambrosia fungi associated with a fourth mycangium type, that of the tribe Scolytoplatypodini. Using ITS rDNA barcoding and a concatenated dataset of six loci (28S rDNA, 18S rDNA, tef1-α, tub, mcm7, and rpl1), a comprehensive phylogeny of the family Ceratocystidaceae was developed, including Inodoromyces interjectus gen. & sp. nov., a non-ambrosial species that is closely related to the family. Three minor morphological variants of the pronotal disk mycangium of the Scolytoplatypodini were associated with ambrosia fungi in three respective clades of Ceratocystidaceae: Wolfgangiella gen. nov., Toshionella gen. nov., and Ambrosiella remansi sp. nov. Closely-related species that are not symbionts of ambrosia beetles are accommodated by Catunica adiposa gen. & comb. nov. and Solaloca norvegica gen. & comb. nov. The divergent morphology of the ambrosial genera and their phylogenetic placement among non-ambrosial genera suggest three domestication events in the Ceratocystidaceae. Estimated divergence dates for the ambrosia fungi and mycangia suggest that Scolytoplatypodini mycangia may have been the first to acquire Ceratocystidaceae symbionts and other ambrosial fungal genera emerged shortly after the evolution of new mycangium types. There is no evidence of reversion to a non-ambrosial lifestyle in the mycangial symbionts.

Geographic variation in mycangial communities of Xyleborus glabratus

Factors that influence fungal communities in ambrosia beetle mycangia are poorly understood. The beetle that is responsible for spreading laurel wilt in SE USA, Xyleborus glabratrus, was examined at three sites along a 500 km N–S transect in Florida, each populated by host trees in the Lauraceae. Fungal phenotypes were quantified in mycangia of individual females that were collected from a site in Miami-Dade County (MDC), 25.8N, with swamp bay (Persea palustris), one in Highlands County (HC), 27.9N, with silkbay (P. humulis) and swamp bay and another in Alachua County (AC), 29.8N, with redbay (P. borbonia). Based on combined LSU, SSU and beta-tubulin datasets the most prominent phenotypes were Raffaelea lauricola (cause of laurel wilt), R. subalba, R. subfusca, R. fusca, R. arxii and an undescribed Raffaelea sp. Mean numbers of colony forming units (CFUs) of R. lauricola varied by location (P < 0.003), and a multivariate analysis, which accounted for the presence and relative abundance of fungal species, indicated that there were significant variations in mycangial communities among the sites; thus climate and vegetation might have affected fungal diversity and the relative abundance of these fungi in the mycangia of X. glabratus. Statistically it was unlikely that any of the species influenced the presence and prevalence of another species.

Identification, pathogenicity and abundance of Paracremonium pembeum sp. nov. and Graphium euwallaceae sp. nov.—two newly discovered mycangial associates of the polyphagous shot hole borer (Euwallacea sp.) in California

Fusarium euwallaceae is a well-characterized fungal symbiont of the exotic ambrosia beetle Euwallacea sp. (polyphagous shot hole borer [PSHB]), together inciting Fusarium dieback on many host plants in Israel and California. Recent discoveries of additional fungal symbionts within ambrosia beetle mycangia suggest these fungi occur as communities. Colony-forming units of Graphium euwallaceae sp. nov. and Paracremonium pembeum sp. nov., two novel fungal associates of PSHB from California, grew from 36 macerated female heads and 36 gallery walls collected from Platanus racemosa, Acer negundo, Persea americana and Ricinus communis. Fungi were identified based on micromorphology and phylogenetic analyses of the combined internal transcribed spacer region (nuc rDNA ITS1-5.8S-ITS2 [ITS barcode]), elongation factor (EF 1-α), small subunit (18S rDNA) sequences for Graphium spp., ITS, EF 1-α, calmodulin (cmdA), large subunit of the ATP citrate lyase (acl1), β-tubulin (tub2), RNA polymerase II second largest subunit (rpb2) and large subunit (28S rDNA) sequences for Paracremonium spp. Other Graphium spp. recovered from PSHB in Vietnam, Euwallacea fornicatus in Thailand, E. validus in Pennsylvania and Paracremonium sp. recovered from PSHB in Vietnam were identified. F. euwallaceae was recovered from mycangia at higher frequencies and abundances in all hosts except R. communis, in which those of F. euwallaceae and P. pembeum were equal. P. pembeum was relatively more abundant within gallery walls of A. negundo and R. communis. In all hosts combined F. euwallaceae was relatively more abundant within PSHB heads than gallery walls. All three fungi grew at different rates and colonized inoculated excised stems of P. americana and A. negundo. P. pembeum produced longer lesions than F. euwallaceae and G. euwallaceae on inoculated avocado shoots. Results indicate PSHB is associated with a dynamic assemblage of mycangial fungal associates that pose additional risk to native and nonnative hosts in California.

Antibiotic-producing bacteria from stag beetle mycangia.

The search for new antibiotics or antifungal agents is crucial for the chemotherapies of infectious diseases. The limited resource of soil bacteria makes it difficult to discover such new drug candidate. We, therefore, focused on another bacterial resource than soil bacteria, the microbial flora of insect species. In the present study, we isolated 40 strains of bacteria and fungi from the mycangia of three species of stag beetle, Dorcus hopei binodulosus, Dorcus rectus, and Dorcus titanus pilifer. We identified those species with their ribosomal DNA sequences, and revealed that Klebsiella spp. are the most frequent symbiont in the stag beetle mycangia. We examined whether these microorganisms produce antibiotics against a Gram-negative bacterium, Escherichia coli, a Gram-positive bacterium, Staphylococcus aureus, or a fungus, Cryptococcus neoformans. Culture supernatants from 33, 29, or 18 strains showed antimicrobial activity against E. coli, S. aureus, or C. neoformans, respectively. These findings suggest that bacteria present in the mycangia of stag beetles are useful resources for screening novel antibiotics.

Genotype distribution of Raffaelea quercivora in the oak galleries and its composition in the mycangia of Platypus quercivorus

SummaryRaffaelea quercivora is the pathogenic fungus that causes Japanese oak wilt. The female monogynous ambrosia beetle, Platypus quercivorus, carries this fungus in mycangia on the pronotum. These beetles bore galleries in oak trees with their partners to produce offspring, and they deposit fungus on the gallery walls from their mycangia. The offspring mature in the gallery, before loading the fungal pathogen and flying from the gallery to other healthy trees. To investigate the unloading and loading modes of the fungus within the gallery, we developed four polymorphic microsatellite markers for R. quercivora and identified the fungal genotypes in the galleries and mycangia of the beetles. Small wood chips were sampled at 5–10‐mm intervals from the walls of five galleries in a dead Quercus serrata tree. The pronota were also sampled from five female adult beetles. The genotypes of the R. quercivora isolates from the wood chips and pronota were identified using the microsatellite makers. The genotypic analysis showed that each gallery was inhabited patchily by 5–10 genotypes of R. quercivora, and the mycangia of each beetle contained 3–6 genotypes. These results indicate that diverse R. quercivora genotypes are unloaded repeatedly from the mycangia of female beetles onto the gallery wall, which results in their patchy distribution on the walls. When the offspring leave the host tree, the fungal clones that proliferate in the walls are also loaded repeatedly into the mycangia of the mature beetles.

The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing.

Symbioses are increasingly seen as dynamic ecosystems with multiple associates and varying fidelity. Symbiont specificity remains elusive in one of the most ecologically successful and economically damaging eukaryotic symbioses: the ambrosia symbiosis of wood-boring beetles and fungi. We used multiplexed pyrosequencing of amplified internal transcribed spacer II (ITS2) ribosomal DNA (rDNA) libraries to document the communities of fungal associates and symbionts inside the mycangia (fungus transfer organ) of three ambrosia beetle species, Xyleborus affinis, Xyleborus ferrugineus and Xylosandrus crassiusculus. We processed 93 beetle samples from 5 locations across Florida, including reference communities. Fungal communities within mycangia included 14-20 fungus species, many more than reported by culture-based studies. We recovered previously known nutritional symbionts as members of the core community. We also detected several other fungal taxa that are equally frequent but whose function is unknown and many other transient species. The composition of fungal assemblages was significantly correlated with beetle species but not with locality. The type of mycangium appears to determine specificity: two Xyleborus with mandibular mycangia had multiple dominant associates with even abundances; Xylosandrus crassiusculus (mesonotal mycangium) communities were dominated by a single symbiont, Ambrosiella sp. Beetle mycangia also carried many fungi from the environment, including plant pathogens and endophytes. The ITS2 marker proved useful for ecological analyses, but the taxonomic resolution was limited to fungal genus or family, particularly in Ophiostomatales, which are under-represented in our amplicons as well as in public databases. This initial analysis of three beetle species suggests that each clade of ambrosia beetles and each mycangium type may support a functionally and taxonomically distinct symbiosis.

Broadscale specificity in a bark beetle-fungal symbiosis: a spatio-temporal analysis of the mycangial fungi of the western pine beetle.

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle-fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect's geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle's flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle-fungus interaction is discussed.

Mycangia of Ambrosia Beetles Host Communities of Bacteria

The research field of animal and plant symbioses is advancing from studying interactions between two species to whole communities of associates. High-throughput sequencing of microbial communities supports multiplexed sampling for statistically robust tests of hypotheses about symbiotic associations. We focus on ambrosia beetles, the increasingly damaging insects primarily associated with fungal symbionts, which have also been reported to support bacteria. To analyze the diversity, composition, and specificity of the beetles' prokaryotic associates, we combine global sampling, insect anatomy, 454 sequencing of bacterial rDNA, and multivariate statistics to analyze prokaryotic communities in ambrosia beetle mycangia, organs mostly known for transporting symbiotic fungi. We analyze six beetle species that represent three types of mycangia and include several globally distributed species, some with major economic importance (Dendroctonus frontalis, Xyleborus affinis, Xyleborus bispinatus-ferrugineus, Xyleborus glabratus, Xylosandrus crassiusculus, and Xylosandrus germanus). Ninety-six beetle mycangia yielded 1,546 bacterial phylotypes. Several phylotypes appear to form the core microbiome of the mycangium. Three Mycoplasma (originally thought restricted to vertebrates), two Burkholderiales, and two Pseudomonadales are repeatedly present worldwide in multiple beetle species. However, no bacterial phylotypes were universally present, suggesting that ambrosia beetles are not obligately dependent on bacterial symbionts. The composition of bacterial communities is structured by the host beetle species more than by the locality of origin, which suggests that more bacteria are vertically transmitted than acquired from the environment. The invasive X. glabratus and the globally distributed X. crassiusculus have unique sets of bacteria, different from species native to North America. We conclude that the mycangium hosts in multiple vertically transmitted bacteria such as Mycoplasma, most of which are likely facultative commensals or parasites.

A Technique for the Introduction of Fungi to Bark Beetle Mycangia

A Technique for the Introduction of Fungi to Bark Beetle Mycangia

Fungal symbionts of the ambrosia beetle Gnathotrichus sulcatus

Three species of Fungi Imperfecti associated with the ambrosia beetle Gnathotrichus sulcatus Lec. are described in culture, viz. Ambrosiella sulcati Funk sp. nov., Raffaelea sulcati Funk sp. nov., and Graphium sp. All are found in beetle mycangia and in tunnels of specimens from British Columbia, Canada.

A complex of fungi mutualistically involved in the nutrition of the ambrosia beetle Xyleborus ferrugineus

Paired oral mycangia (organs in which mutualistic fungi are perpetuated) were present in both sexes of the ambrosia beetle Xyleborus ferrugineus. The mycangia of stock culture beetles bore a complex of fungi, yeast, and bacteria. Fusarium solani was the dominant fungal symbiote, but a Cephalosporium sp., and a Graphium sp. also were isolated from 50% and 20% of the beetles, respectively. All three fungi formed “ambrosia” growth on the walls of brood tunnels when inoculated singly into tubes of medium bearing one female beetle previously freed of fungi. The fungus that was inoculated into a given tube of medium was the only fungus subsequently recovered from mycangia of adult beetles in that tube. Each fungus grew in characteristic forms in the mycangia of the beetles. Each of the three fungi provided the nutrients or concentrations of nutrients, not contained in the medium, that were essential to reproduction by the insect.

Ambrosia Fungi: Extent of Specificity to Ambrosia Beetles

Experiments with developing ambrosia beetles and fungi indicate that beetles may feed on more than one species growing in their tunnels. The yeast-like ambrosia propagules in mycangia of beetles arise from conida and ascospores picked up by callow adults during their stay in tunnels.