Origin and evolution of fungus farming in wood-boring Coleoptera – a palaeontological perspective

Insect–fungus mutualism is one of the better-studied symbiotic interactions in nature. Ambrosia fungi are an ecological assemblage of unrelated fungi that are cultivated by ambrosia beetles in their galleries as obligate food for larvae. Despite recently increased research interest, it remains unclear which ecological factors facilitated the origin of fungus farming, and how it transformed into a symbiotic relationship with obligate dependency. It is clear from phylogenetic analyses that this symbiosis evolved independently many times in several beetle and fungus lineages. However, there is a mismatch between palaeontological and phylogenetic data. Herein we review, for the first time, the ambrosia system from a palaeontological perspective. Although largely ignored, families such as Lymexylidae and Bostrichidae should be included in the list of ambrosia beetles because some of their species cultivate ambrosia fungi. The estimated origin for some groups of ambrosia fungi during the Cretaceous concurs with a known high diversity of Lymexylidae and Bostrichidae at that time. Although potentially older, the greatest radiation of various ambrosia beetle lineages occurred in the weevil subfamilies Scolytinae and Platypodinae during the Eocene. In this review we explore the evolutionary relationship between ambrosia beetles, fungi and their host trees, which is likely to have persisted for longer than previously supposed.     

Host specificity of ambrosia and bark beetles (Col., Curculionidae: Scolytinae and Platypodinae) in a New Guinea rainforest

Abstract.  1. Bark and ambrosia beetles are crucial for woody biomass decomposition in tropical forests worldwide. Despite that, quantitative data on their host specificity are scarce.
2. Bark and ambrosia beetles (Scolytinae and Platypodinae) were reared from 13 species of tropical trees representing 11 families from all major lineages of dicotyledonous plants. Standardised samples of beetle-infested twigs, branches, trunks, and roots were taken from three individuals of each tree species growing in a lowland tropical rainforest in Papua New Guinea.
3. A total of 81 742 beetles from 74 species were reared, 67 of them identified. Local species richness of bark and ambrosia beetles was estimated at 80–92 species.
4. Ambrosia beetles were broad generalists as 95% of species did not show any preference for a particular host species or clade. Similarity of ambrosia beetle communities from different tree species was not correlated with phylogenetic distances between tree species. Similarity of ambrosia beetle communities from individual conspecific trees was not higher than that from heterospecific trees and different parts of the trees hosted similar ambrosia beetle communities, as only a few species preferred particular tree parts.
5. In contrast, phloeophagous bark beetles showed strict specificity to host plant genus or family. However, this guild was poor in species (12 species) and restricted to only three plant families (Moraceae, Myristicaceae, Sapindaceae).
6. Local diversity of both bark and ambrosia beetles is not driven by the local diversity of trees in tropical forests, since ambrosia beetles display no host specificity and bark beetles are species poor and restricted to a few plant families.     

Ophiostomatoid fungi associated with hardwood-infesting bark and ambrosia beetles in Poland: Taxonomic diversity and vector specificity

Fungi in the orders Ophiostomatales and Microascales (Ascomycota), often designated as ophiostomatoid fungi, are frequent associates of scolytine bark and ambrosia beetles that colonize hardwood and coniferous trees. Several species, e.g., Ophiostoma novo-ulmi, are economically damaging pathogens of trees. Because little is known regarding the ophiostomatoid fungi in Europe, we have explored the diversity of these fungi associated with hardwood-infesting beetles in Poland. This study aims to clarify the associations between fungi in the genera Ambrosiella, Graphium (Microascales), Graphilbum, Leptographium, Ophiostoma and Sporothrix (Ophiostomatales) and their beetle vectors in hardwood ecosystems. Samples associated with 18 bark and ambrosia beetle species were collected from 11 stands in Poland. Fungi were isolated from adult beetles and galleries. Isolates were identified based on morphology, DNA sequence comparisons for five gene regions (ITS, LSU, ßT, TEF 1-α, and CAL) and phylogenetic analyses. In total, 36 distinct taxa were identified, including 24 known and 12 currently unknown species. Several associations between fungi and bark and ambrosia beetles were recorded for the first time. In addition, associations between Dryocoetes alni, D. villosus, Hylesinus crenatus, Ernoporus tiliae, Pteleobius vittatus and ophiostomatoid fungi were reported for the first time, and Sporothrix eucastanea was reported for the first time outside of the USA. Among the species of Ophiostomatales, 14 species were in Ophiostoma s. l., two species were in Graphilbum, nine species were in Sporothrix, and seven species were in Leptographium s. l. Among the species of Microascales, three species were in Graphium, and one was in Ambrosiella. Twenty taxa were present on the beetles and in the galleries, twelve only on beetles, and four only in galleries. Bark and ambrosia beetles from hardwoods appear to be regular vectors, with ophiostomatoid fungi present in all the beetle species. Most ophiostomatoid species had a distinct level of vector/host specificity, although Ophiostoma quercus, the most frequently encountered species, also had the greatest range of beetle vectors and tree hosts. Plant pathogenic O. novo-ulmi was found mainly in association with elm-infesting bark beetles (Scolytus multistriatus, S. scolytus, and P. vittatus) and occasionally with H. crenatus on Fraxinus excelsior and with Scolytus intricatus on Quercus robur.     

Brief Review of the Associations of Xylobiont Nematodes with Bark Beetles (Coleoptera,Curculionidae: Scolytinae)

Nematode pathogens cause wilt diseases in conifers and deciduous trees. The longhorn beetles (Coleoptera: Cerambycidae) and bark beetles (Coleoptera: Curculionidae: Scolytinae) act as nematode vectors spreading the invasive juvenile stages during their maturation feeding or during oviposition on the plant hosts. There are numerous reviews of nematodes associated with bark beetles on conifers, while little attention has been paid to the nematodes of deciduous trees. The development of Dutch elm disease and ash dieback is mainly caused by fungal pathogens transmitted by bark beetles; the latter act as vectors of not only fungi but also nematodes enclosed in nematangia under their elytra, and also in the tracheae and Malpighian canals. Apart from phytopathogenic nematodes, bark beetles transmit mycophagous and bacterivorous nematodes and own parasites of bark beetles. The ecological groups of nematodes associated with Scolytinae are reviewed; the known records of associations of nematodes with bark beetles are listed for coniferous host plants of Russia and neighboring countries; the world-wide list of these associations for deciduous plant hosts is given.

     

Relationships among wood‐boring beetles,fungi, and the decomposition of forest biomass

A prevailing paradigm in forest ecology is that wood‐boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle‐accessible or inaccessible. Fungal communities were compared using culturing and high‐throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle‐associated fungi in beetle‐accessible trees, whereas some endophytes persisted as saprotrophs in beetle‐excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood‐decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle‐associated fungi reduce decay rates by competing with decay fungi. No effect of bark‐inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle‐associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community‐level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.     

Ophiostomatoid fungi isolated from Japanese red pine and their relationships with bark beetles

We isolated ophiostomatoid fungi from bark beetles infesting Pinus densiflora and their galleries at 24 sites in Japan. Twenty-one ophiostomatoid fungi, including species of Ophiostoma, Grosmannia, Ceratocystiopsis, Leptographium, and Pesotum, were identified. Among these, 11 species were either newly recorded in Japan or were previously undescribed species. Some of these fungal species were isolated from several bark beetles, but other species were isolated from only a particular beetle species. Thus, it is suggested that some ophiostomatoid fungi have specific relationships with particular beetle species. In addition, fungus-beetle biplots from redundancy analysis (RDA) summarizing the effects of beetle ecological characteristics suggested that the association patterns between bark beetles and the associated fungi seemed to be related to the niches occupied by the beetles.     

Four new Ophiostoma species associated with hardwood-infesting bark beetles in Norway and Poland

Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark and ambrosia beetles (Curculionidae: Scolytinae, Platypodinae). Fungi in the Ophiostomatales include serious tree pathogens as well as agents of timber blue-stain. Although these fungi have been extensively studied in the northern hemisphere, very little is known regarding their occurrence on hardwoods in Europe. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with bark and ambrosia beetles infesting hardwoods in Norway and Poland, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Norwegian and Polish isolates, using multigene phylogenetic analyses. Results obtained from five gene regions (ITS, LSU, β-tubulin, calmodulin, translation elongation factor 1-α) revealed four new Ophiostoma spp. These include Ophiostoma hylesinum sp. nov., O. signatum sp. nov., and O. villosum sp. nov. that phylogenetically are positioned within the Ophiostoma ulmi complex. The other new species, Ophiostoma pseudokarelicum sp. nov. reside along with Ophiostoma karelicum in a discrete, well-supported phylogenetic group in Ophiostoma s. stricto. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on hardwoods in Europe is poorly understood and that further studies are required to enrich our knowledge about these fungi.     

Exploring associations between international trade and environmental factors with establishment patterns of exotic Scolytinae

Although invasion of exotic ambrosia beetles (fungus feeders) and bark beetles (phloem feeders) (Coleoptera: Curculionidae: Scolytinae) is considered a major threat to forest health worldwide, no studies have quantitatively investigated the anthropogenic and environmental factors shaping the biogeographical patterns of invasion by these insects across large spatial scales. The primary aim of this study was to assess the relative importance of international trade and several environmental variables of the recipient region on species richness of established exotic Scolytinae. As a reference, we also evaluated the relationships between the same environmental variables and species richness of native Scolytinae. Using an information-theoretic framework for model selection and hierarchical partitioning, we evaluated the relative importance of the potential drivers of species richness of native and exotic Scolytinae in 20 European countries and the 48 contiguous continental US states. Analyses were conducted separately for ambrosia and bark beetle species. Value of imports was a strong predictor of the number of exotic Scolytinae species in both regions. In addition, in the USA, warmer and wetter climate was positively linked to increased numbers of both native and exotic ambrosia beetles. Forest heterogeneity and climatic heterogeneity and secondarily forest area were key drivers in explaining patterns of species richness for native bark beetles but not for exotic species in both regions. Our findings suggest that if current infestation levels continue on imported plants and wood packaging material, increasing international trade will likely lead to more establishments of exotic Scolytinae with concomitant negative effects on forest health in both Europe and the USA. Compared to Europe the risk of invasion appears higher in the USA, especially for ambrosia beetles in the southeastern USA where the climate appears highly suitable for exotic establishment.     

Low beta diversity of ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) in lowland rainforests of Papua New Guinea

We assessed the effect of geographical distance on insect species turnover in a situation where other major environmental factors, including host plant species, altitude, and climate, were constant. We sampled ambrosia beetles (Coleoptera, Curculionidae: Scolytinae and Platypodinae) from four tree species: Artocarpus altilis , Ficus nodosa , Leea indica and Nauclea orientalis , at three sites forming a 1000 km transect in lowland rainforests of northern Papua New Guinea. A standardized volume of wood from trunk, branches and twigs was sampled for ambrosia beetles from three individuals of the four tree species at each site. Each tree was killed standing and left exposed to beetle colonization for 20 days prior to sampling. We obtained 12 751 individuals from 84 morphospecies of ambrosia beetles. We surveyed most of the local species richness at each site, predicted by Chao 2 species richness estimates. The similarity of ambrosia beetle communities, estimated by Chao-Sorensen index, was not correlated with their geographical distance. Likelihood analysis and Q-mode analysis using Monte Carlo-generated null distribution of beetles among sites supported the hypothesis that the assemblages of ambrosia beetles at different sites are drawn from the same species pool, regardless of their geographical distance. Tree part (trunk, branch, or twig) was more important predictor of the composition of ambrosia beetle communities than was the host species or geographical location. All three variables, however, explained only a small portion of variability in ambrosia assemblages. The distribution of ambrosia beetles among tree parts, tree species and study sites was mostly random, suggesting limited importance of host specificity or dispersal limitation.     

Ophiostoma species (Ascomycetes: Ophiostomatales) associated with bark beetles (Coleoptera: Scolytinae) colonizing Pinus radiata in northern Spain

Bark beetles (Coleoptera: Scolytinae) are known to be associated with fungi, especially species of Ophiostoma sensu lato and Ceratocystis. However, very little is known about these fungi in Spain. In this study, we examined the fungi associated with 13 bark beetle species and one weevil (Coleoptera: Entiminae) infesting Pinus radiata in the Basque Country of northern Spain. This study included an examination of 1323 bark beetles or their galleries in P. radiata. Isolations yielded a total of 920 cultures, which included 16 species of Ophiostoma sensu lato or their asexual states. These 16 species included 69 associations between fungi and bark beetles and weevils that have not previously been recorded. The most commonly encountered fungal associates of the bark beetles were Ophiostoma ips, Leptographium guttulatum, Ophiostoma stenoceras, and Ophiostoma piceae. In most cases, the niche of colonization had a significant effect on the abundance and composition of colonizing fungi. This confirms that resource overlap between species is reduced by partial spatial segregation. Interaction between niche and time seldom had a significant effect, which suggests that spatial colonization patterns are rarely flexible throughout timber degradation. The differences in common associates among the bark beetle species could be linked to the different niches that these beetles occupy.     

Roles of bacteria in the bark beetle holobiont – how do they shape this forest pest?

Bark beetles are well‐known forest pests, some species inducing massive attacks on trees, resulting in the devastation of entire woodlands. Bark beetles are associated with microorganisms, forming an entity known as ‘holobiont’. Beetles and fungi are the best‐studied members of this multipartite symbiosis. However, recent studies have shown that bacteria may play important roles in the bark beetle holobiont, such as providing certain nutrients, promoting the growth of beneficial fungi, detoxifying the environment by lowering the levels of phenolic compounds synthesised by the host tree or by inhibiting the growth of antagonistic fungi whereas some bacterial symbionts have the potential to kill beetles under certain conditions. Therefore, bacteria probably greatly affect the life cycle of bark beetles; hence, more research is needed to clarify the extent to which a bacterial associate is implicated in a bacterial bark beetle symbiosis and how much it determines host's performance. This review summarises all of the known activities of bacteria present in the bark beetle holobiont, indicates some important gaps in the knowledge of this symbiosis and provides some guidance for overcoming the difficulties in investigating this relationship in future studies.     

Classification of weevils as a data-driven science: leaving opinion behind

Data and explicit taxonomic ranking criteria, which minimize taxonomic change, provide a scientific approach to modern taxonomy and classification. However, traditional practices of opinion-based taxonomy (i.e., mid-20^th century evolutionary systematics), which lack explicit ranking and naming criteria, are still in practice despite phylogenetic evidence. This paper discusses a recent proposed reclassification of weevils that elevates bark and ambrosia beetles (Scolytinae and Platypodinae) to the ranks of Family. We demonstrate that the proposed reclassification 1) is not supported by an evolutionary systematic justification because the apparently unique morphology of bark and ambrosia beetles is shared with other unrelated wood-boring weevil taxa; 2) introduces obvious paraphyly in weevil classification and hence violates good practices on maintaining an economy of taxonomic change; 3) is not supported by other taxonomic naming criteria, such as time banding. We recommend the abandonment of traditional practices of an opinion-based taxonomy, especially in light of available data and resulting phylogenies.     

Cover Caption

The ambrosia beetle, Xylosandrus discolor (Blandford) (Scolytinae: Curculionidae), is common in southeastern Asia. As this beetle bores into wood and reproduces, its only food source is the white fungi (cover the chamber in the figure) which are cultured by the adult. Xylosandrus ambrosia beetles each have a pronotal mycangia (an open void in the body) which transport ambrosia fungi from their natal gallery to their new gallery. Mycangia are dynamic and their morphological changes correspond to the phases of the symbiosis (see pages 732–742). Photo provided by You Li.     

REPEATED EVOLUTION OF CROP THEFT IN FUNGUS‐FARMING AMBROSIA BEETLES

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.     

Attraction of ambrosia and bark beetles to coast live oaks infected by Phytophthora ramorum

1 Sudden oak death is caused by the apparently introduced oomycete, Phytophthora ramorum. We investigated the role of bark and ambrosia beetles in disease progression in coast live oaks Quercus agrifolia. 2 In two Marin County, California sites, 80 trees were inoculated in July 2002 with P. ramorum and 40 were wounded without inoculation. Half of the trees in each group were sprayed with the insecticide permethrin [cyclopropanecarboxylic acid, 3‐(2,2‐dichloroethenyl)‐2,2‐dimethyl‐(3‐phenoxyphenyl) methyl ester] to prevent ambrosia and bark beetle attacks, and then were sprayed twice per year thereafter. After each treatment, sticky traps were placed on only the permethrin‐treated trees. Beetles were collected periodically in 2003. 3 Inoculated trees accounted for 95% of all beetles trapped. The ambrosia beetles Monarthrum scutellare and Xyleborinus saxeseni and the western oak bark beetle Pseudopityophthorus pubipennis were the most abundant of the seven species trapped. 4 Permethrin treatment delayed initiation of beetle attacks and significantly reduced the mean number of attacks per tree. Beetles did not attack any wounded or noncankered inoculated trees. 5 Trees with larger cankers trapped more beetles early in the disease. Once permethrin lost effectiveness, the number of beetle entrance tunnels was a more reliable predictor of subsequent trap catch than was canker size. 6 Beetles were initially attracted to P. ramorum cankers in response to kairomones generated in the host‐pathogen interaction. After beetles attacked the permethrin‐treated trees, aggregation pheromones most probably were the principal factor in beetle colonization behaviour.     

危害松树的小蠹虫与其伴生菌的相互关系

危害健康松属植物的小蠹虫经常与一些特殊的真菌相联系。在小蠹虫危害松属植物的过程中,这些真菌被小蠹虫的一些特殊结构或者体表携带到松属植物上。小蠹虫与其伴生菌的联系表明小蠹虫和其伴生菌之间是一种互惠互利的关系。伴生菌随小蠹虫扩散而被带到新的寄主树木。而伴生菌或作为小蠹虫的食物来源,但更重要的是,有些伴生菌能够通过其菌丝渗透寄主组织,释放毒素,致死寄主树木,以帮助小蠹虫降低寄主抗性。许多研究致力于探索小蠹虫/伴生菌联合体与寄主树木之间关系的特征和确定小蠹虫与其伴生菌相互关系在生态学上的意义。然而,不同小蠹虫和其伴生菌所组成的共生体系,不同小蠹虫的种群数量,和不同环境条件下同种小蠹虫与其伴生菌相互作用方式的差异使我们在研究小蠹虫和其伴生菌这个共生体系时,对它们各自在成功聚集寄主树木过程中所发挥的重要作用的概括变得非常困难。     

Novel associations between ophiostomatoid fungi,insects and tree hosts: current status—future prospects

Associations between fungal tree pathogens and insects have been recognized for at least 100 years. An important group of these fungi, termed ‘ophiostomatoid fungi’ on account of their morphological similarity, are represented by genera in the families Ceratocystidaceae and Ophiostomataceae. Associations between these fungi, tree-colonizing insects, and host trees have been actively researched since their first discovery. Human activities have led to the global movement of fungi from both families, resulting in the establishment of new and sometimes damaging associations between these fungi, insects and trees. Recent ‘black swan’ events have resulted in an unprecedented increase of ambrosia and bark beetle-associated diseases of forest and fruit trees. We revisit some of the most important emergent diseases caused by the ophiostomatoid fungi, outline the reasons behind the emergence of these diseases, and consider long-term prospects regarding the threats that they pose to forestry and agriculture.     

Ethanol‐injection induces attacks by ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) on a variety of tree species

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The sudden emergence of pathogenicity in insect-fungus symbioses threatens naive forest ecosystems

Invasive symbioses between wood-boring insects and fungi are emerging as a new and currently uncontrollable threat to forest ecosystems, as well as fruit and timber industries throughout the world. The bark and ambrosia beetles (Curculionidae: Scolytinae and Platypodinae) constitute the large majority of these pests, and are accompanied by a diverse community of fungal symbionts. Increasingly, some invasive symbioses are shifting from non-pathogenic saprotrophy in native ranges to a prolific tree-killing in invaded ranges, and are causing significant damage. In this paper, we review the current understanding of invasive insect-fungus symbioses. We then ask why some symbioses that evolved as non-pathogenic saprotrophs, turn into major tree-killers in non-native regions. We argue that a purely pathology-centred view of the guild is not sufficient for explaining the lethal encounters between exotic symbionts and naive trees. Instead, we propose several testable hypotheses that, if correct, lead to the conclusion that the sudden emergence of pathogenicity is a new evolutionary phenomenon with global biogeographical dynamics. To date, evidence suggests that virulence of the symbioses in invaded ranges is often triggered when several factors coincide: (i) invasion into territories with naive trees, (ii) the ability of the fungus to either overcome resistance of the naive host or trigger a suicidal over-reaction, and (iii) an 'olfactory mismatch' in the insect whereby a subset of live trees is perceived as dead and suitable for colonization. We suggest that individual cases of tree mortality caused by invasive insect-fungus symbionts should no longer be studied separately, but in a global, biogeographically and phylogenetically explicit comparative framework.     

Plasticity of mycangia in Xylosandrus ambrosia beetles

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 animalfungus interactions. While the interspecific diversity of their fungus transport organ一 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 promesonotal 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 fun gal 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.