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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 “naive” 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 of symbioses, mutualism, parasitism, and virulence. • 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.

In the present study, we identified the fungal community, including yeasts, in the mycangium of the ambrosia beetle, Xylosandrus crassiusculus (Coleoptera: Curculionidae) and observed the fungal diversity in the mycangia at different times during the dispersal season of beetles. The results showed that the fungal community in the mycangia of X. crassiusculus was dominated by Ambrosiella roeperi, which is regarded as a primary ambrosia fungus. The fungal diversity in the mycangia was influenced by the timing of beetle sampling during the flight dispersal season, with more diverse fungal communities in the late dispersal season. The composition of auxiliary fungi differed significantly among individuals in this study as well as between this study and other studies, indicating that the auxiliary fungi were likely influenced by factors such as locality and seasons.

Female stag beetles (Lucanidae) possess internal mycangia to maintain microbial cultures. Yeasts from these mycangia may help with larval nutrition in nutrient poor woody substrates, but only a few Lucanidae taxa have been studied and all reports originate from Europe and Asia. We identify the first mycangial yeasts of a South African endemic Lucanidae beetle, Xiphodontus antilope, using nuclear ribosomal RNA and ITS DNA sequence data. In addition we identified yeasts from the larval gut, fecal matter, frass and woody substrate surrounding larvae and pupae. The mycangium of X. antilope was confined to females and is structurally similar to all other Lucanidae. Unlike most Lucanidae that seemingly associate with single species of yeast, or whose mycangia contain yeast monocultures, three yeast species were commonly isolated from X. antilope. Scheffersomyces coipomoensis was the most numerically dominant species on most substrates and in most individuals, but a second, undescribed, Scheffersomyces species was present in high numbers. A third species, also undescribed and unrelated to Scheffersomyces, was recovered from all mycangia but could not be detected in the larval gut, fecal matter, frass or woody substrates. We confirm a close association of Scheffersomyces yeasts with Lucanidae globally, but other taxa may also be involved. We show that the predominant mycangial yeasts also form the predominant yeasts within the larval gut and the woody substrates around the larvae and pupae. This combined external and internal colonization by the same yeasts may provide enhanced opportunities for nutrient acquisition, but this needs validation in future studies.

Adult females of stag beetles (Coleoptera: Lucanidae) possess an ovipositor-associated mycangium for conveying symbiotic microorganisms. In most lucanid species, their mycangium contains yeast symbionts of the genus Scheffersomyces Kurtzman & M. Suzuki, 2010 that are known for their xylose-fermenting capability. The lucanid genus Platycerus Geoffroy, 1762 is a group of small blue stag beetles, in which ten Japanese species constitute a monophyletic clade. Here we examined the evolutionary relationships of these Japanese Platycerus species and their yeast symbionts, together with a Korean Platycerus species and other lucanid species as outgroup taxa. Based on the internal transcribed spacer (ITS) and the intergenic spacer (IGS) sequences, the yeast symbionts of all Platycerus species were closely related to each other and formed a monophyletic clade. There is no variation in ITS sequences of the yeast symbionts of the Japanese Platycerus species. Based on IGS sequences, the yeast symbionts formed clusters that largely reflected the geographic distribution of the host insects, being shared by sympatric Platycerus species except for P. delicatulus Lewis, 1883 and P. viridicuprus Kubota, Kubota & Otobe, 2008. The symbiont phylogeny was globally not congruent with the host COI-based phylogeny, although some local congruences were observed. Statistically significant correlations were detected between the genetic distances of COI sequences of the host insects and those of IGS sequences of the yeast symbionts in Japan. These results suggest that, at least to some extent, the host insects and the yeast symbionts may have experienced co-evolutionary associations. While the Japanese Platycerus species formed a monophyletic clade in the COI phylogeny, the yeast symbionts of Japanese P. viridicuprus were very closely related to those of Korean P. hongwonpyoi Imura & Choe, 1989, suggesting the possibility that a recent secondary contact of the two beetle species during a marine withdrawal, e.g., in the last glacial period, might have resulted in an inter-specific horizontal transmission of the yeast symbiont.

Ambrosia beetles farm fungal cultivars (ambrosia fungi) and carry propagules of the fungal mutualists in storage organs called mycangia, which occur in various body parts and vary greatly in size and complexity. The evolution of ambrosia fungi is closely tied to the evolution and development of the mycangia that carry them. The understudied ambrosia beetle tribe Xyloterini included lineages with uncharacterized ambrosia fungi and mycangia, which presented an opportunity to test whether developments of different mycangium types in a single ambrosia beetle lineage correspond with concomitant diversity in their fungal mutualists. We collected representatives of all three Xyloterini genera (Trypodendron, Indocryphalus, and Xyloterinus politus) and characterized their ambrosia fungi in pure culture and by DNA sequencing. The prothoracic mycangia of seven Trypodendron species all yielded Phialophoropsis (Microascales) ambrosia fungi, including three new species, although these relationships were not all species specific. Indocryphalus mycangia are characterized for the first time in the Asian I. pubipennis. They comprise triangular prothoracic cavities substantially smaller than those of Trypodendron and unexpectedly carry an undescribed species of Toshionella (Microascales), which are otherwise ambrosia fungi of Asian Scolytoplatypus (Scolytoplatypodini). Xyloterinus politus has two different mycangia, each with a different ambrosia fungus: Raffaelea cf. canadensis RNC5 (Ophiostomatales) in oral mycangia of both sexes and Kaarikia abrahamsonii (Sordariomycetes, genus incertae sedis with affinity for Distoseptisporaceae), a new genus and species unrelated to other known ambrosia fungi, in shallow prothoracic mycangia of females. In addition to their highly adapted mycangial mutualists, Trypodendron and X. politus harbor a surprising diversity of facultative symbionts in their galleries, including Raffaelea. A diversity of ambrosia fungi and mycangia suggest multiple ancestral cultivar captures or switches in the history of tribe Xyloterini, each associated with unique adaptations in mycangium anatomy. This further supports the theory that developments of novel mycangium types are critical events in the evolution of ambrosia beetles and their coadapted fungal mutualists.

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.

Insects that depend on microbial mutualists evolved a variety of organs to transport the microsymbionts while dispersing. The ontogeny and variability of such organs is rarely studied, and the microsymbiont's effects on the animal tissue development remain unknown in most cases. Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae or Platypodinae) and their mutualistic fungi are an ideal system to study the animal-fungus interactions. While the interspecific diversity of their fungus transport organ-mycangia-is well-known, their developmental plasticity has been poorly described. To determine the ontogeny of the mycangium and the influence of the symbiotic fungus on the tissue development, we dissected by hand or scanned with micro-CT the mycangia in various developmental stages in five Xylosandrus ambrosia beetle species that possess a large, mesonotal mycangium: Xylosandrus amputatus, Xylosandrus compactus, Xylosandrus crassiusculus, Xylosandrus discolor, and Xylosandrus germanus. We processed 181 beetle samples from the United States and China. All five species displayed three stages of the mycangium development: (1) young teneral adults had an empty, deflated and cryptic mycangium without fungal mass; (2) in fully mature adults during dispersal, the pro-mesonotal membrane was inflated, and most individuals developed a mycangium mostly filled with the symbiont, though size and symmetry varied; and (3) after successful establishment of their new galleries, most females discharged the bulk of the fungal inoculum and deflated the mycangium. Experimental aposymbiotic individuals demonstrated that the pronotal membrane invaginated independently of the presence of the fungus, but the fungus was required for inflation. Mycangia are more dynamic than previously thought, and their morphological changes correspond to the phases of the symbiosis. Importantly, studies of the fungal symbionts or plant pathogen transmission in ambrosia beetles need to consider which developmental stage to sample. We provide illustrations of the different stages, including microphotography of dissections and micro-CT scans.

Among insect-fungus relationships, xylose-fermenting Scheffersomyces yeasts are well known for their potential in utilizing wood hemicelluloses and their association with various wood-feeding insects. However, their specificity to host insects or strain-level diversity within host species has not been clearly elucidated. In the insect family Lucanidae, larvae usually feed on decaying wood, and adult females consistently possess a fungus-storage organ, called the mycangium, near the abdominal tip. Here the authors investigated host-symbiont relationships between Scheffersomyces yeast symbionts and small blue stag beetles of the genus Platycerus (Coleoptera: Lucanidae) in East Asia by using intergenic spacer (IGS) region as a genetic marker. All yeast strains isolated from the female mycangium of three Platycerus species, P. hongwonpyoi from Korea and P. acuticollis and P. delicatulus from Japan, were allied to Scheffersomyces segobiensis based on the sequences of the nrDNA 26S and internal transcribed spacer (ITS), in which no sequence difference was observed among those strains. However, IGS regions showed clear genetic differentiation within the yeast symbionts of P. hongwonpyoi, as well as between those of Korean and Japanese Platycerus species. In the IGS sequences, nucleotide substitutions were mainly distributed in the whole stretch of IGS1 and the anterior half of IGS2, whereas nucleotide gaps were localized at IGS1 and the middle of IGS2. Despite the conserved association between the Platycerus beetles and the specific strains of S. segobiensis in East Asia, geophylogenetic divergence patterns of the yeast symbionts were not concordant with those of the insect hosts.

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co-evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the β-tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.

Ambrosiella species in the latest definition are tightly associated with ambrosia beetles in genera Anisandrus, Cnestus, and Xylosandrus within the tribe Xyleborini characterized by a mesothoracic mycangium. Eccoptopterus and Hadrodemius are another two xyleborine genera possessing mesothoracic mycangium, but their symbiotic fungi are unknown. A survey of Ambrosiella s.s. in Taiwan revealed five species, including one new species, A. catenulata, and indicated that members of Eccoptopterus and Hadrodemius are also associated with Ambrosiella s.s. It corroborates a previous hypothesis that Ambrosiella s.s. species are exclusively associated with ambrosia beetles having mesothoracic mycangium. The association appears asymmetrical: a single Ambrosiella species can be carried by multiple species of beetles, while each beetle species associates with only one Ambrosiella species. Descriptions and illustrations of species obtained in this study are provided. Identification is based on morphological and molecular analyses.

Transmission modes of symbionts with fungus-growing insects are closely related to the stability of symbioses. As compare to social fungus-farming insects, transmission modes of nonsocial fungus-farming insects need to be further investigated. Euops chinensis, a nonsocial leaf-rolling weevil, harbors a symbiotic fungus Penicillium herquei in the specialized mycangium. Previous works have indicated that P. herquei is cultivated to host plants by females during oviposition process, however, it is still unclear when (before or after oviposition) and how P. herquei is transmitted. In this study, we observed fungal cultivating behaviors and adult bodies by scanning electron microscopy (SEM), and compared isolation rates of P. herquei on ovary eggs, newly oviposited eggs, cradle leaves (leaf pieces cut before rolling cradles), cradles, and female mycangia. Fungal isolates were identified by internal transcribed spacers (ITS) and cytochrome oxidase I (COI) genes. We found the female's serrated tarsi and comb-like setae on the abdomen were specialized structures for fungal transmission. Newly oviposited eggs showed 81.11% frequency of fungal symbionts, but ovary eggs did not show any growth of fungal symbionts. Isolation rates of P. herquei on cradle leaves, mycangia and cradles were 76.67%, 77.71% and 87.72%, respectively. Analyses of ITS and COI genes showed that isolated fungal strains belong to the same species. We concluded that P. herquei was transmitted before oviposition, and the female's tarsi are newly found specialized structures for fungal transmission. This study elucidates the cultivar transmission mode with fungus-farming attelabid weevils, and might be useful to study of other fungiculture mutualisms.

Studies of symbioses have traditionally focused on explaining one-to-one interactions between organisms. In reality, symbioses are often much more dynamic. They can involve many interacting members, and change depending on context. In studies of the ambrosia symbiosis-the mutualism between wood borer beetles and fungi-two variables have introduced uncertainty when explaining interactions: imprecise symbiont identification, and disregard for anatomical complexity of the insects. The black twig borer, Xylosandrus compactus Eichhoff, is a globally invasive ambrosia beetle that infests >200 plant species. Despite many studies on this beetle, reports of its primary symbionts are conflicting. We sampled adult X. compactus and infested plant material in central Florida to characterize the fungal symbiont community using dilution series, beetle partitioning, and DNA-based identification. X. compactus was consistently associated with two fungal taxa, Fusarium spp. and Ambrosiella xylebori Multivariate analyses revealed that A. xylebori was strongly associated with the beetle mycangium while Fusarium spp. were associated with the abdomen and external surfaces. The Fusarium spp. carried by X. compactus are not members of the Ambrosia Fusarium Clade, and are probably not mutualists. Fungal community composition of the mycangium was less variable than external body surfaces, thus providing a more consistent fungal inoculum. This is the first report of spatial partitioning as a mechanism for maintenance of a multimember ambrosia fungus community. Our results provide an explanation for discrepancies among previous reports, and suggest that conflicting results are not due to differences in symbiont communities, but due to inconsistent and incomplete sampling.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) rely on a symbiosis with fungi for their nutrition. Symbiotic fungi are preserved and transported in specialized storage structures called mycangia. Although pivotal in the symbiosis, mycangia have been notoriously difficult to study, given their minute size and membranous structure. We compared the application of novel visualization methods for the study of mycangia, namely micro-computed tomography (micro-CT) and laser ablation tomography (LATscan) with traditional paraffin sectioning. Micro-CT scanning has shown the greatest promise in new organ discovery, while sectioning remains the only method with sufficient resolution for cellular visualization. All three common types of mycangia (oral, mesonotal, and pronotal) were successfully visualized and presented for different species of ambrosia beetles: Ambrosiodmus minor (Stebbing) 1909, Euplatypus compositus (Say) 1823, Premnobius cavipennis Eichhoff 1878, Scolytoplatypus raja Blandford 1893, Xylosandrus crassiusculus (Motschulsky) 1866 and X. amputatus (Blandford) 1894. A reconstruction of the mycangium and the surrounding musculature in X. amputatus is also presented. The advantages of micro-CT compared to the previously commonly used microtome sectioning include the easy visualization and recording of three-dimensional structures, their position in reference to other internal structures, the ability to distinguish natural aberrations from technical artifacts, and the unprecedented visualizations of the anatomic context of mycangia enabled by the integrated software.

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.

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.

Isolations from the granulate ambrosia beetle, Xylosandrus crassiusculus (Coleoptera: Curculionidae: Scolytinae: Xyleborini), collected in Georgia, South Carolina, Missouri and Ohio, yielded an undescribed species of Ambrosiella in thousands of colony-forming units (CFU) per individual female. Partial sequences of ITS and 28S rDNA regions distinguished this species from other Ambrosiella spp., which are asexual symbionts of ambrosia beetles and closely related to Ceratocystis spp. Ambrosiella roeperi sp. nov. produces sporodochia of branching conidiophores with disarticulating swollen cells, and the branches are terminated by thick-walled aleurioconidia, similar to the conidiophores and aleurioconidia of A. xylebori, which is the mycangial symbiont of a related ambrosia beetle, X. compactus. Microscopic examinations found homogeneous masses of arthrospore-like cells growing in the mycangium of X. crassiusculus, without evidence of other microbial growth. Using fungal-specific primers, only the ITS rDNA region of A. roeperi was amplified and sequenced from DNA extractions of mycangial contents, suggesting that it is the primary or only mycangial symbiont of this beetle in USA.

Thousands of species of ambrosia beetles excavate tunnels in wood to farm fungi. They maintain associations with particular lineages of fungi, but the phylogenetic extent and mechanisms of fidelity are unknown. We test the hypothesis that selectivity of their mycangium enforces fidelity at coarse phylogenetic scales, while permitting promiscuity among closely related fungal mutualists. We confirm a single evolutionary origin of the Xylosandrus complex—a group of several xyleborine genera that farm fungi in the genus Ambrosiella . Multi-level co-phylogenetic analysis revealed frequent symbiont switching within major Ambrosiella clades, but not between clades. The loss of the mycangium in Diuncus , a genus of evolutionary cheaters, was commensurate with the loss of fidelity to fungal clades, supporting the hypothesis that the mycangium reinforces fidelity. Finally, in vivo experiments tracked symbiotic compatibility throughout the symbiotic life cycle of Xylosandrus compactus and demonstrated that closely related Ambrosiella symbionts are interchangeable, but the probability of fungal uptake in the mycangium was significantly lower in more phylogenetically distant species of symbionts. Symbiont loads in experimental subjects were similar to wild-caught beetles. We conclude that partner choice in ambrosia beetles is achieved in the mycangium, and co-phylogenetic inferences can be used to predict the likelihood of specific symbiont switches.

The ambrosia beetle–fungus farming symbiosis is more heterogeneous than previously thought. There is not one but many ambrosia symbioses. Beetle-fungus specificity is clade dependent and ranges from strict to promiscuous. Each new origin has evolved a new mycangium. The most common relationship with host trees is colonization of freshly dead tissues, but there are also parasites of living trees, vectors of pathogenic fungi, and beetles living in rotten trees with a wood-decay symbiont. Most of these strategies are driven by fungal metabolism whereas beetle ecology is evolutionarily more flexible. The ambrosia lifestyle facilitated a radiation of social strategies, from fungus thieves to eusocial species to communities assembled by attraction to fungal scent. Although over 95% of the symbiotic pairs are economically harmless, there are also three types of pest damage: tree pathogen inoculation, mass accumulation on susceptible hosts, and structural damage. Beetles able to colonize live tree tissues are most likely to become invasive pests.

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co‐evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the β‐tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.

Part of the exoskeleton of some wood-inhabiting insects is modified to form a mycangium, which is a specialized organ used to convey fungal spores or yeasts to their offspring. Although most stag beetles (Coleoptera: Lucanidae) are known to have female-specific mycangia and associated yeast symbionts, the evolutionary origin of the mycangium in this group remains unresolved. Here, we report the presence of a mycangium and associated yeast symbionts in the European horned stag beetle Sinodendron cylindricum (L.), which belongs to an ancestral clade of the Lucanidae. The mycangium of S. cylindricum is shown to be female-specific and have the same developmental origin as that of other stag beetles. A total of five yeast strains were isolated from adult mycangia and larval gut of S. cylindricum . Of these, we suggest that SICYAM1 is an undescribed yeast with taxonomic novelty, and have identified SICYLG3 as the xylose-fermenting yeast Scheffersomyces insectosa using nuclear ribosomal RNA and ITS sequences. The remaining three yeast strains, SICYAM2, SICYLG1, and SICYLG2, were assigned to the genus Sugiyamaella . Yeast density in the adult mycangium was lower than that of the more evolutionarily advanced stag beetles, the European Lucanus cervus (L.) and Dorcus parallelipipedus (L.), which were also examined in this study. No living yeasts were isolated from the adult guts. However, a third instar larva of S. cylindricum harbored 104–106 living yeasts in each gut region, which suggests that gut yeasts play an important role in these wood-feeding larvae.

The Polyphagous Shot Hole Borer (Coleoptera: Curculeonidae: Scolytinae), Euwallacea sp. near fornicatus, is an ambrosia beetle native to Asia that has been introduced into Israel, California, and South Africa. The beetle maintains a symbiotic relationship with three species of fungi that it vectors between host trees by carrying spores within a mandibular mycangium. These ambrosial fungi are inoculated into host trees and are the sole nutritional source for the adults and the larvae. Unfortunately for forest and resource managers, one of the fungi, Fusarium euwallaceae, is a moderately virulent pathogen and is responsible for causing a dieback disease in susceptible hosts. High levels of infestation of susceptible host trees have resulted in high levels of mortality. The currently recognized host range for the beetle-fungus complex includes more than 200 tree species that can be attacked by the beetle, more than 100 species that can support growth of the fungus, and 37 species that can be used as a reproductive host by the beetles. Many of these reproductive hosts are important agricultural crops, components of the California urban forest, or components of native riparian forest communities. Management is currently focused on monitoring using visual inspections or trapping, sanitation using solarization or chipping, and direct control using contact or systemic insecticides. Future management approaches will include planting resistant or unsuitable host trees and biological control. As more information about the biology and ecology of the insect-fungus complex is developed, it may be possible to develop strategies for limiting spread within and among regions of the world with Mediterranean climates.

Insect fungus gardens consist of a community of interacting microorganisms that can have either beneficial or detrimental effects to the farmers. In contrast to fungus-farming ants and termites, the fungal communities of ambrosia beetles and the effects of particular fungal species on the farmers are largely unknown. Here, we used a laboratory rearing technique for studying the filamentous fungal garden community of the ambrosia beetle, Xyleborinus saxesenii, which cultivates fungi in tunnels excavated within dead trees. Raffaelea sulfurea and Fusicolla acetilerea were transmitted in spore-carrying organs by gallery founding females and established first in new gardens. Raffaelea sulfurea had positive effects on egg-laying and larval numbers. Over time, four other fungal species emerged in the gardens. Prevalence of one of them, Paecilomyces variotii, correlated negatively with larval numbers and can be harmful to adults by forming biofilms on their bodies. It also comprised the main portion of garden material removed from galleries by adults. Our data suggest that two mutualistic, several commensalistic and one to two pathogenic filamentous fungi are associated with X. saxesenii. Fungal diversity in gardens of ambrosia beetles appears to be much lower than that in gardens of fungus-culturing ants, which seems to result from essential differences in substrates and behaviours.

Platypus koryoensis (Murayama) (Coleoptera: Curculionidae: Platypodinae) is a minute ambrosia beetle that can cause significant damage to live oak trees (Quercus mongolica) along with its symbiotic pathogenic fungus (Raffaelea sp.). Although both sexes of the beetle have a number of cuticular depressions for carrying microorganisms, only the females have mycangia that are typical in size on the pronotum. The mycangial cavities are filled with a mass of amorphous substances including yeast-like spores. In addition, mycangial cavities are equipped with cuticular pores which release substances to support symbiotic microorganisms during storage. Small depressions of both sexes can be subdivided into two clusters according to their relative sizes and locations. Although they have a typical cuticular peg (basiconic sensillum), they also share a common morphological characteristic with the typical mycangium of female: a round outlet and an invaginated reservoir containing a mass of secretory substances. Accordingly, our fine structural data suggest that both the female mycangia and the clusters of small cuticular depressions in both sexes also have a mycangial function in common regardless of their relative size and location.

The laurel wilt pathogen Raffaelea lauricola was hypothesized to have been introduced to the southeastern USA in the mycangium of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia. To test this hypothesis adult X. glabratus were trapped in Taiwan and on Kyushu Island, Japan, in 2009, and dead beetles were sent to USA for isolation of fungal symbionts. Individual X. glabratus were macerated in glass tissue grinders, and the slurry was serially diluted and plated onto malt agar medium amended with cycloheximide, a medium semiselective for Ophiostoma species and their anamorphs, including members of Raffaelea. R. lauricola was isolated from 56 of 85 beetles in Taiwan and 10 of 16 beetles in Japan at up to an estimated 10 000 CFUs per beetle. The next most commonly isolated species was R. ellipticospora, which also has been recovered from X. glabratus trapped in the USA, as were two other fungi isolated from beetles in Taiwan, R. fusca and R. subfusca. Three unidentified Raffaelea spp. and three unidentified Ophiostoma spp. were isolated rarely from X. glabratus collected in Taiwan. Isolations from beetles similarly trapped in Georgia, USA, yielded R. lauricola and R. ellipticospora in numbers similar to those from beetles trapped in Taiwan and Japan. The results support the hypothesis that R. lauricola was introduced into the USA in mycangia of X. glabratus shipped to USA in solid wood packing material from Asia. However differences in the mycangial mycoflora of X. glabratus in Taiwan, Japan and USA suggest that the X. glabratus population established in USA originated in another part of Asia.

Ambrosia beetles, dominant wood degraders in the tropics, create tunnels in dead trees and employ gardens of symbiotic fungi to extract nutrients from wood. Specificity of the beetle-fungus relationship has rarely been examined, and simple vertical transmission of a specific fungal cultivar by each beetle species is often assumed in literature. We report repeated evolution of fungal crop stealing, termed mycocleptism, among ambrosia beetles. The mycocleptic species seek brood galleries of other species, and exploit their established fungal gardens by tunneling through the ambient mycelium-laden wood. Instead of carrying their own fungal sybmbionts, mycocleptae depend on adopting the fungal assemblages of their host species, as shown by an analysis of fungal DNA from beetle galleries. The evidence for widespread horizontal exchange of fungi between beetles challenges the traditional concept of ambrosia fungi as species-specific symbionts. Fungus stealing appears to be an evolutionarily successful strategy. It evolved independently in several beetle clades, two of which have radiated, and at least one case was accompanied by a loss of the beetles' fungus-transporting organs. We demonstrate this using the first robust phylogeny of one of the world's largest group of ambrosia beetles, Xyleborini.

Most wood-feeding insects need an association with microbes to utilize wood as food, and some have special organs to store and convey the microbes. We report here the discovery of the microbe-storage organ (mycangium) in stag beetles (Coleoptera: Lucanidae), which develop in decayed wood. The mycangium, which was discovered in the abdomen, is present in all adult females of 22 lucanid species examined in this study, but absent in adult males. By contrast, adult insects of both sexes of selected Passalidae, Geotrupidae, and Scarabaeidae, which are related to Lucanidae, lacked mycangia similar to those of the lucanid species. Yeast-like microbes were isolated from the mycangium of five lucanid species. DNA sequence analyses indicate that the microbes are closely related to the xylose-fermenting yeasts Pichia stipitis, Pichia segobiensis, or Pichia sp. known from the gut of a passalid species.

An undescribed Sporothrix (SJB 133) and a basidiomycete (SJB 122) were the predominant fungi observed in the prothoracic mycangium of the female southern pine beetle, Dendroctonus frontalis Zimm. Fresh preparations and single-spore cultures from mycangia of emerging females showed that Sporothrix sp. occurred as small clavate to elongate spores and the basidiomycete formed large “ambrosia propagules”. Both appeared to be in the state of growth. These were the predominant organisms isolated from mycangia of attacking females. Other microorganisms, such as Ceratocystis minor, Penicillium spp., and yeasts, may occasionally enter the mycangium during early adult life, only yeasts were present in mycangia of attacking beetles. These results suggest that the chemical regime of the mycangium selectively favors Sporothrix sp. and the basidiomycete. There is also evidence that the Sporothrix isolate is a highly modified anascigerous form of Ceratocystis. Zusammenfassung uber Pilz-Symbionten im prothorakalen Myzangium von Dendroctonus frontalis (Coleopt,: Scolytidae) Im prothorakalen Myzangium des ♀ Borkenkafers D. frontalis wurden 2 unbekannte Pilzarten gefunden: Sporothrix spec. (SJB 133) und Basidiomycete (SJB 122). In frisch geschlupften ♀♀ tritt Sporothrix spec, als langlich schmale Sporen und der Basidiomycete in Form groserer “Ambrosia-Fortpflanzungskorper” auf. Beide scheinen sich im Stadium des Wachstums zu befinden. Diese beiden Pilzarten waren die in den Myzangien vorherrschenden Symbionten. Andere Mikroorganismen wie Ceratocystis minor, Penicillium spp. und Hefepilze dringen gelegentlich wahrend des fruhen Imaginalstadiums in den Symbiontenbehalter ein, doch wurden von ihnen in einbohrenden Kafern nur Hefen gefunden. Dies weist darauf hin, das von den chemischen Verhaltnissen im Myzangium vor allem Sporothrix spec, und der Basidiomycete begunstigt werden. Einige Anzeichen deuten darauf hin, das es sich bei Sporothrix spec, um eine stark modifizierte Form von Ceratocystis handelt.

The southern pine beetle (Dendroctonus frontalis Zimmermann) kills all pines within its range and is among the most important forest pest species in the US. Using a specialized mycangium surrounded by gland cells in the pronotum, adult females culture, transport, and inoculate two fungi into beetle galleries during oviposition. These fungal symbionts, to varying degrees, exclude antagonistic fungi and provide nutrients to larvae. However, the mechanisms (e.g. secreted antibiotic chemicals or nutrients, proteins or pathways) by which this relationship is maintained are not known. Here we present the first global and differential proteome profile of the southern pine beetle pronotum. Two-dimensional polyacrylamide electrophoresis, tandem mass spectrometry, and database searches revealed that the majority of pronotal proteins were related to energy-yielding metabolism, contractile apparati, cell structure, and defence. The identified proteins provide important insights into the molecular and biochemical processes of, and candidates for functional genomics to understand mycangia and pronotum functions in, the southern pine beetle.

Mycangial structures of female Xylosandrus mutilatus (Blandford, 1894), an Asian ambrosia beetle recently introduced and now established in the southeastern United States, were examined. In addition to the glandular (secretory) mesonotal mycangium located below the scutellum, nonglandular pit mycangia containing fungal propagules were discovered to occur externally on the scutellum. The ultrastructure of the glandular mesonotal mycangium, which consisted of an outer secretory layer covering an inner paired cuticular sac, is described and illustrated. Both secretory and cuticular layers of the mesonotal mycangium were highly tracheolated. Tracheae and tracheoles in association with a Xyleborine mycangium are reported here for the first time. Type 1 secretory cells were identified and observed passing electron-dense material into the glandular mycangium via efferent cuticular ductules, although type 2 secretory cells were not observed. Secretory cells were not observed in association with the nonglandular pit mycangia.

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.

We review the Oriental species of Scolytoplatypus Schaufuss, and provide keys to both males and females. The importance at specific level of the characters of the male prosternum is stressed. The following new species are described: Scolytoplatypus blandfordi Gebhardt from Taiwan; S. cirratus Gebhardt from Indonesia (Sulawesi); S. curviciliosus Gebhardt from the Philippines; S. exiguus Beaver from Indonesia (Sulawesi), together with the previously unknown male of S. ruficauda Eggers from Nepal. Much of the published synonymy is confused. The following synonyms listed by Wood and Bright (1992) in their catalog of Scolytinae are reinstated as good species: Scolytoplatypus bombycinus Browne, S. brahma Blandford, S. luzonicus Eggers, S. macgregori Blackman, S. ruficauda Eggers. The following new synonymy is proposed: Scolytoplatypus daimio Blandford (= Scolytoplatypus kunala Strohmeyer); Scolytoplatypus brahma Blandford (= Scolytoplatypus paucegranulatus Eggers); Scolytoplatypus luzonicus Eggers (= Scolytoplatypus benguetus Blackman); Scolytoplatypus pubescens Hagedorn (= Scolytoplatypus pubescens kabakovi Axentjev). The synonymy of Scolytoplatypus hamatus Hagedorn and Scolytoplatypus hirsutus Blackman with Scolytoplatypus brahma is confirmed. New distributional records are given for some species. All species are ambrosia beetles which breed in a wide variety of host trees and show little specificity for particular host taxa, although they can be size-selective. Some details are given of the ambrosia fungus and mycangium of S. blandfordi. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)