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.     

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.     

Non‐destructive observation of the mycangia of Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae) using X‐ray computed tomography

X‐ray microtomography has been applied successfully to obtain reliable microstructural information of many insect species. Nonetheless, the technique has not been widely applied to ambrosia beetles. The ambrosia beetle Euwallacea interjectus (Blandford) was first recorded as a vector of plant pathogenic fungus Ceratocystis ficicola Kajitani & Masuya, which has caused serious wilt disease in many fig orchards in Japan since 1999. Previous studies of E. interjectus have not described the mycangia (fungus‐storing organ) in detail. In this study, we non‐destructively examined the internal structure of an adult female of E. interjectus through computed microtomography scans. Paired mycangia were observed on typical computed tomography cross‐sections of the head. Each mycangium, ovoid in shape, was located in tissues just posterior to emarginated notch of eyes, adjacent to pharynx. Three dimensions (length × width × depth) of the mycangia were measured on stereography. We confirmed the absence of mycangia in the other body parts, such as elytra, prothorax and coxa of legs.     

Dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus (Blandford) (Coleoptera: Scolytidae) in relation to its life history

The dynamics of the fungal symbionts in the gallery system and the mycangia of the ambrosia beetle,Xylosandrus mutilatus, were studied in relation to its life history using both isolation experiments and scanning electron microscopy (SEM). In the galleries,Ambrosiella sp. was predominant during the larval stages but its relative dominance gradually decreased during the development of the larvae. In contrast, yeasts (mainlyCandida sp.) andPaecilomyces sp. dominated continuously in the galleries after eclosion.Ambrosiella sp. was consistently stored in the mycangia in all adult stages, except in the teneral and overwintering adults when the other fungi were dominant. No fungal spores occurred in the mycangia of the adult beetles reared under aseptic conditions from the pupal stage, while onlyAmbrosiella sp. was stored in those reared from the teneral-adult stage. These results suggest that: (i) Xmutilatus is associated with at least three fungal species, among whichAmbrosiella sp. is the most essential food resource for development of the broods; (ii) immediately after eclosion, new female adults may take at least four associated fungal species, with no or incomplete selection, into their mycangia from the walls of the cradles; and (iii) conditions may well be produced in the mycangia of both matured and dispersing beetles whereby only the spores ofAmbrosiella sp. can proliferate.     

Landscape‐scale genetic differentiation of a mycangial fungus associated with the ambrosia beetle,Xylosandrus germanus (Blandford) (Curculionidae:Scolytinae) in Japan

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.     

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.     

Host switching by an ambrosia beetle fungal mutualist: Mycangial colonization of indigenous beetles by the invasive laurel wilt fungal pathogen

Ambrosia beetles require their fungal symbiotic partner as their cultivated (farmed) food source in tree galleries. While most fungal-beetle partners do not kill the host trees they inhabit, since their introduction (invasion) into the United states around ~2002, the invasive beetle Xyleborus glabratus has vectored its mutualist partner (but plant pathogenic) fungus, Harringtonia lauricola, resulting in the deaths of over 300 million trees. Concerningly, indigenous beetles have been caught bearing H. lauricola. Here, we show colonization of the mycangia of the indigenous X. affinis ambrosia beetle by H. lauricola. Mycangial colonization occurred within 1 h of feeding, with similar levels seen for H. lauricola as found for the native X. affinis-R. arxii fungal partner. Fungal mycangial occupancy was stable over time and after removal of the fungal source, but showed rapid turnover when additional fungal cells were available. Microscopic visualization revealed two pre-oral mycangial pouches of ~100–200 × 25–50 μm/each, with narrow entry channels of 25–50 × 3–10 μm. Fungi within the mycangia underwent a dimorphic transition from filamentous/blastospore growth to yeast-like budding with alterations to membrane structures. These data identify the characteristics of ambrosia beetle mycangial colonization, implicating turnover as a mechanism for host switching of H. lauricola to other ambrosia beetle species.     

Mycangia of the ambrosia beetle, Austroplatypus incompertus (Schedl) (Coleoptera: Curculionidae: Platypodinae)

Abstract  Ambrosia beetles have an obligate relationship with the ambrosia fungi that they feed on. This requires that the beetles have means to transport those fungi when they colonise new hosts. Some ambrosia beetles have special structures called mycangia to transport fungi in. This paper describes the mycangia of the ambrosia beetle Austroplatypus incompertus and illustrates how the mycangical hairs are probably used by the beetle to acquire fungal spores for transport. The mycangia and probable method of fungal acquisition of this species are compared with those of other ambrosia beetles.     

Identification of the ambrosia fungus of <Emphasis Type="Italic">Xyleborus monographus</Emphasis> and <Emphasis Type="Italic">X. dryographus</Emphasis> (Coleoptera: Curculionidae,Scolytinae)

The scolytid ambrosia beetles Xyleborus monographus and X. dryographus were investigated to identify their nutritional ambrosia fungi. The examination of the oral mycetangia of the beetles, the specialized organs for fungal transport, revealed the dominant occurrence of Raffaelea montetyi, a fungus that was also predominant in the beetle tunnels in the immediate vicinity of the feeding larvae. R. montetyi was previously known only as the ambrosia fungus of the platypodid ambrosia beetle, Platypus cylindrus. These beetle species inhabit the same habitat, mainly trunks of oaks in the Western Palaeartic. The possibility of an exchange of the symbiotic fungus between the ambrosia beetles within their common breeding place is discussed. Consequently, the previous hypothesis of a species-specific association of a single ambrosia fungus with a single beetle species is questioned. A phylogenetic analysis based on DNA sequences classified R. montetyi within the Ophiostomatales of the ascomycetes. The investigation of conidiogenesis of R. montetyi by SEM supported this taxonomic placement and showed the development of the conidia by annellidic percurrent proliferation, identical to the conidiogenesis reported for many anamorph states of the Ophiostomatales.     

Ambrosia beetle Premnobius cavipennis (Scolytinae: Ipini) carries highly divergent ascomycotan ambrosia fungus,Afroraffaelea ambrosiae gen. nov. et sp. nov. (Ophiostomatales)

Ambrosia beetles and fungi represent an interesting and economically important symbiosis, but the vast majority of ambrosia fungi remain unexplored, hindering research, management of pathogens, and mitigation of invasive species. Beetles in the subtribe Premnobiini are one example of an entire beetle lineage whose fungal symbionts have never been studied. Here, we identify one dominant fungal symbiont of Premnobius cavipennis by using fungus culturing, community sequencing, microtome sectioning and micro-CT scanning of mycangia. Phylogenetic analyses of combined 18S and 28S rDNA and β-tubulin sequences revealed a highly divergent fungal lineage within Ophiostomatales, Afroraffaelea ambrosiae gen. nov. et sp. nov. The newly described fungal lineage represents another origin of the symbiosis within the Kingdom Fungi, adding to our understanding of the geographic ancestry of ambrosia fungi. P. cavipennis possesses pharyngeal mycangia which appear restrictive in fungus selection. This ambrosia beetle-fungus association has remained stable even after invasions into non-native regions.     

Integrating repellent and attractant semiochemicals into a push–pull strategy for ambrosia beetles (Coleoptera: Curculionidae)

Non‐native ambrosia beetles (Coleoptera: Curculionidae), especially Xylosandrus compactus (Eichhoff), Xylosandrus crassiusculus (Motschulsky) and Xylosandrus germanus (Blandford), are destructive wood‐boring pests of trees in ornamental nurseries and tree fruit orchards. Previous studies have demonstrated the adults are repelled by verbenone and strongly attracted to ethanol. We tested a “push–pull” semiochemical strategy in Ohio, Virginia and Mississippi using verbenone emitters to “push” beetles away from vulnerable trees and ethanol lures to “pull” them into annihilative traps. Container‐grown trees were flood‐stressed to induce ambrosia beetle attacks and then deployed in the presence or absence of verbenone emitters and a perimeter of ethanol‐baited interception traps to achieve the following treatment combinations: (a) untreated control, (b) verbenone only, (c) ethanol only, and (d) verbenone plus ethanol. Verbenone and ethanol did not interact to reduce attacks on the flooded trees, nor did verbenone alone reduce attacks. The ethanol‐baited traps intercepted enough beetles to reduce attacks on trees deployed in Virginia and Mississippi in 2016, but not in 2017, or in Ohio in 2016. Xylosandrus germanus, X. crassiusculus and both Hypothenemus dissimilis Zimmermann and X. crassiusculus were among the predominant species collected in ethanol‐baited traps deployed in Ohio, Virginia and Mississippi, respectively. Xylosandrus germanus and X. crassiusculus were also the predominant species dissected from trees deployed in Ohio and Virginia, respectively. While the ethanol‐baited traps showed promise for helping to protect trees by intercepting ambrosia beetles, the repellent “push” component (i.e., verbenone) and attractant “pull” component (i.e., ethanol) will need to be further optimized in order to implement a “push–pull” semiochemical strategy.     

光滑足距小蠹虫道真菌的组成和变化

本文同时采用分离和电镜扫描技术研究了光滑足距小蠹Xylosandrus germanus(Blan dford)不同发育阶段坑道和贮菌器内真菌种类的组成和变化。Ambrosiella hartigii是光滑足距小蠹扩散、蛀孔、越冬和体壁新硬化的成虫贮菌器内唯一分离到的真菌,而刚羽化的雌成虫贮菌器中没有分离到任何真菌种类,体壁新硬化的成虫贮菌器中真菌的分离频率最低。坑道内共分离到4种真菌：A. hartigii,Acremonium kiliense和2种镰刀菌Fusarium spp.。从卵期到蛹期的坑道中,A. hartigii总是能被分离到,占分离物总数的百分率较高,达40％～60％;而在成虫期坑道中,A. kiliense和Fusarium sp.1被分离率较高,都为34.6％。扫描电镜观察表明,A. hartigii为卵期到蛹期坑道中的优势种,而镰刀菌Fusarium spp.则是成虫期坑道中的优势种。这些结果均表明,Ambrosiella hartigii,Acremonium kiliense和2种镰刀菌Fusarium spp.是光滑足距小蠹的虫道真菌,而且这些真菌种类组成和优势种类是随着蠹虫的发育阶段而变化的。真菌种类的组成和变化可 能与幼虫和成虫的营养需求以及虫道真菌的生长特性有关。     

Ability of stress‐related volatiles to attract and induce attacks by Xylosandrus germanus and other ambrosia beetles

• 1 Xylosandrus germanus typically colonizes physiologically‐stressed deciduous hosts but it is increasingly being recognized as a key pest of ornamental nursery stock. We tested the attractiveness of common plant stress‐related volatiles to ambrosia beetles occupying the nursery agroecosystem, as well as their ability to induce attacks on selected trees. Experiments were conducted in Ohio, U.S.A.
• 2 Stress volatile attractiveness was first assessed by positioning traps baited with acetaldehyde, acetone, ethanol and methanol in ornamental nurseries. Cumulative trap counts confirmed that ethanol was the most attractive stress‐related volatile to X. germanus. Methanol‐baited traps were slightly attractive to X. germanus, whereas traps baited with acetaldehyde and acetone were not attractive to any ambrosia beetle.
• 3 A series of tree injection experiments were also conducted to determine the ability of these volatiles to induce attacks by ambrosia beetles under field conditions. Injection of ethanol into Magnolia virginiana induced the largest number of attacks, whereas injection of acetaldehyde induced more attacks than methanol or acetone. Xylosandrus germanus was the most predominant species emerging from M. virginiana injected with each of the stress‐related volatiles. No attacks by wood‐boring beetles were observed on water injected or uninjected control trees.
• 4 Solid‐phase microextraction–gas chromatography–mass spectrometry confirmed the emission of acetaldehyde, acetone, ethanol and methanol after their injection into M. virginiana.
• 5 Xylosandrus germanus has an efficient olfactory‐based mechanism for differentiating among host volatile cues. Injecting select trees with stress‐related volatiles, particularly ethanol, shows promise as a trap tree strategy for X. germanus and other ambrosia beetles.

     

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.     

Ultrastructure of the mycangium of the southern pine beetle,Dendroctonus frontalis (Coleoptera: Curculionidae,Scolytinae): complex morphology for complex interactions

Yuceer, C, Hsu, C.‐Y., Erbilgin, N and Klepzig, K.D. 2011. Ultrastructure of the mycangium of the southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae, Scolytinae): complex morphology for complex interactions. —Acta Zoologica (Stockholm) 92 : 216–224. The southern pine beetle (SPB) (Dendroctonus frontalis Zimmermann) is the most economically important pest of southern pine forests. Beetles carry fungal cells within specialised cuticular structures, called mycangia. Little is known about the mycangia ultrastructure or function. We used cryo‐fracturing and scanning electron microscopy to examine the ultrastructural features of SPB mycangia and surrounding tissues. Mycangia, one on each side of anterior portion of the prothorax, are terminated on the dorsal side at a ‘mycangial bridge’. This sclerotised mycangial bridge does not appear to provide a passage between the two mycangia, suggesting that each mycangium functions independently. Mycangia are surrounded by abundant tracheoles connecting the structures to the outside via openings within the prothorax. Previously unknown pits overlying the mycangial gland cells were also observed in both the inner wall and anterior fold of prothorax. We hypothesise that these openings and pits may play roles in determining which fungi enter, and grow within, the mycangium.     

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.     

Microstructure of the prothoracic mycangia in the ambrosia beetle Platypus koryoensis (Coleoptera: Curculionidae: Platypodinae)

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.     

Fungal pathogen and ethanol affect host selection and colonization success in ambrosia beetles

1. Ambrosia beetles exhibit broad host ranges but a narrow preference based on the condition of the host. Tissues infected by pathogens or containing ethanol can facilitate attacks by ambrosia beetles, although it still remains unclear how these factors interact.
2. The present study aimed to examine how (i) chestnut logs infected with the fungal pathogen Cryphonectria parasitica and treated with ethanol (i.e. baited with ethanol lure, soaked in ethanol or untreated) and (ii) hornbeam logs soaked in different ethanol concentrations (3–12.5%) affect host selection and colonization success of ambrosia beetles.
3. Ethanol‐soaked logs were more attractive to Anisandrus dispar than ethanol‐baited logs or untreated logs, although this difference was more evident in uninfected than infected logs. Increasing ethanol concentration in host tissues was differentially attractive to Xyleborinus saxesenii and Xylosandrus germanus. A nonlinear relationship was also documented between ethanol concentration and emergence of X. germanus adults.
4. Overall, the results obtained suggest that the presence of C. parasitica in chestnut logs can affect host selection in ambrosia beetles. In addition, the ethanol concentration in tree tissues affects host selection and colonization success, although the effect varies depending on the beetle species. This contrasting response could be a niche‐partitioning mechanism based on ethanol within host tissues.

     

Ambrosia beetle response to ethanol concentration and host tree species

Ethanol emitted by stressed trees is an olfactory cue used by ambrosia beetles (Coleoptera; Curculionidae; Scolytinae) to locate susceptible hosts to colonize. In addition, ethanol enhances the growth of ambrosia beetle fungal symbionts, improving colonization. Whether host selection and colonization are affected also by the amount of ethanol produced by stressed trees and by tree species is unclear. To investigate these mechanisms, we induced attacks by ambrosia beetles in bolts of eight tree species by coring and filling them with either 5% or 90% ethanol solutions in water. For each ethanol concentration, bolts of the eight different tree species were replicated six times in a randomized complete block design. Entry holes were used as a proxy for host selection whereas gallery development stage was used as a proxy for colonization. Ethanol concentration differentially affected host selection of the three ambrosia beetles that were active during this study. Anisandrus dispar Fabricius preferentially attacked bolts with 90% ethanol concentration, Xylosandrus crassiusculus (Motschulsky) preferentially attacked bolts with 5% ethanol concentration, and Xyleborinus saxesenii (Ratzeburg) attacked bolts irrespective of ethanol concentration. Colonization of X. crassiusculus reflected the same pattern observed for entry holes. The effect of host tree species on host selection was most prominent for X. saxesenii, while X. crassiusculus established a higher number of developed galleries in Ostrya carpinifolia Scopoli bolts than on five of the other tested tree species. Our results suggest that ethanol concentration and host tree species may influence ecological niche partitioning among ambrosia beetle species.     

Suitability of California bay laurel and other species as hosts for the non‐native redbay ambrosia beetle and granulate ambrosia beetle

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