Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts

During a survey of insect gut micro-organisms, we consistently isolated Pichia stipitis-like yeasts (Fungi: Ascomycota, Saccharomycetes) from the wood-ingesting beetles, Odontotaenius disjunctus and Verres sternbergianus (Coleoptera: Passalidae). The yeasts were isolated from passalid beetles over a wide area, including the eastern and midwestern USA and Panama. Phylogenetic analyses of the nuclear encoded small and large subunit rRNA gene (rDNA) sequences distinguished a well-supported clade consisting of the passalid yeasts and Pichia stipitis, P. segobiensis, Candida shehatae and C. ergatensis. Members of this clade have the ability to ferment and assimilate xylose or to hydrolyse xylan, major components of the polysaccharide, hemicellulose. Sexual reproduction was present in the passalid isolates but was rare among the gut yeasts of other beetles to which they were compared. Minor genetic and phenotypic variation among some of the passalid yeasts was detected using markers from the internal transcribed spacer region of the rDNA repeat unit, morphology, and in vitro metabolic tests. The consistent association of xylose-fermenting yeasts of almost identical genotypes with passalid beetles across a broad geographical distribution, suggests a significant symbiotic association.     

Microbial symbiotes of the ambrosia beetleXyloterinus politus

Progression in the understanding of the microecology of ambrosia beetles and their associated microorganisms is briefly reviewed. Between the 1840s and the early 1960s the concept of one ambrosial fungus per ambrosia beetle was emphasized. Some subsequent research has supported the view that each ambrosia beetle plus several associated microorganisms constitute a highly co-evolved symbiotic community. It was hypothesized in this study that such a community of symbiotic microbial species, not just one ambrosial fungus, is actively cultivated and perpetuated by the ambrosia beetleXyloterinus politus. Experimental results indicated that bacteria, yeasts, a yeastlike fungus, and ambrosial fungi compose such a symbiotic microbial complex in association withX. politus. The microecology of the ectosymbiotic microorganisms in relation to this insect is discussed.     

The gut of Guatemalan passalid beetles: a habitat colonized by cellobiose- and xylose-fermenting yeasts

The gut of insects is a productive environment for discovering undescribed species of yeasts, and the gut of wood-feeding insects of several families is especially rich in yeasts that carry out the fermentation of cellobiose and xylose. Passalid beetles (Passalidae, Coleoptera) live in dead wood that they ingest as their primary food source. We report the isolation, molecular identification and physiological characterization of 771 yeast cultures isolated from the gut of 16 species of passalids collected in nine localities in Guatemala. Ascomycete yeasts were present in the gut of every passalid studied, and the xylose-fermenting (X-F) yeasts Scheffersomyces shehatae and Scheffersomyces stipitis were the most abundant taxa isolated. The gut of the beetles also contained undescribed cellobiose-fermenting and X-F species in the Lodderomyces, Scheffersomyces and Spathaspora, and undescribed species in Sugiyamaella clades as well as rare yeast species in the Phaffomyces and Spencermartinsiella clades. Basidiomycete yeasts in the genera Cryptococcus and Trichosporon were also common. The yeast species richness was influenced by the host species and the substrate, and gut-inhabiting yeasts have the ability to survive the differing physiological conditions of several gut compartments.     

Microorganisms in the gut of beetles: evidence from molecular cloning

We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

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.     

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.     

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.     

纵坑切梢小蠹携带云南半帚孢研究

云南半帚孢 (Leptogramphyunnanensis)是纵坑切梢小蠹Tomicuspiniperda重要的共生真菌 ,在纵坑切梢小蠹危害寄主树木过程中发挥着重要作用。研究揭示 ,纵坑切梢小蠹主要通过与受到感染云南半帚孢的韧皮组织的接触携带上云南半帚孢的。纵坑切梢小蠹卵、幼虫和蛹对云南半帚孢的带菌率较高 ,均大于 90 % ,而成虫的带菌率较低。纵坑切梢小蠹的体表和体内均携带有云南半帚孢 ,但体表带菌是纵坑切梢小蠹带菌的主要途径。通过对纵坑切梢小蠹成虫头、足、翅和腹部带菌率的研究发现 ,云南半帚孢在纵坑切梢小蠹各部位的分布大体相同 ,揭示纵坑切梢小蠹没有携带云南半帚孢的特化构造或器官。     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.     

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.     

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.     

In Vitro Interactions Between Yeasts and Bacteria and the Fungal Symbionts of the Mountain Pine Beetle (Dendroctonus ponderosae)

Multi-trophic interactions between prokaryotes, unicellular eukaryotes, and ecologically intertwined metazoans are presumably common in nature, yet rarely described. The mountain pine beetle, Dendroctonus ponderosae, is associated with two filamentous fungi, Grosmannia clavigera and Ophiostoma montium. Other microbes, including yeasts and bacteria, are also present in the phloem, but it is not known whether they interact with the symbiotic fungi or the host beetle. To test whether such interactions occur, we performed a suite of in vitro assays. Overall, relative yield of O. montium grown with microbes isolated from larval galleries was significantly greater than when the fungus was grown alone. Conversely, the yield of G. clavigera grown with these same microbes was less than or equal to when it was grown alone, suggesting that O. montium, and at least some microbes in larval galleries, have a mutualistic or commensal relationship, while G. clavigera and those same microbes have an antagonistic relationship. A bacterium isolated from phloem not colonized by beetles was found to inhibit growth of both G. clavigera and O. montium and appears to be an antagonist to both fungi. Our results suggest that bacteria and yeasts likely influence the distribution of mycangial fungi in the host tree, which, in turn, may affect the fitness of D. ponderosae.     

Behaviours of phoretic mites (Acari) associated with Ips pini and Ips grandicollis (Coleoptera: Curculionidae) during host‐tree colonization

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Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.     

On the functions of stridulation by the passalid beetle Odontotaenius disjunctus (Coleoptera: Passalidae)

Crows (Corvus brachyrhynchos) are more hesitant to continue attacking and take longer to kill and eat stridulating passalid beetles (Odontotaenius disjunctus) than silenced beetles. This is an experimental demonstration of the deterrent capability of disturbance stridulation by an insect that has no active defences against the predator. Stridulation is not necessary for congregation by colony members. Silenced beetles congregate as effectively as controls. There is no evidence that the disturbance stridulation causes predictable responses in other colony members of this presocial beetle.     

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.     

Midgut and fat body bacteriocytes in neotropical cerambycid beetles (Coleoptera: Cerambycidae)

Xylophagous insects derive nutrients from intractable substrates by producing or ingesting cellulolytic enzymes, or by maintaining associations with symbiotic microbes. Wood-boring cerambycid beetle larvae sometimes house maternally-transmitted endosymbiotic yeasts that are presumed to provide their hosts with nutritional benefits. These are thought to be absent from species in the large subfamily Lamiinae; nevertheless yeasts have been repeatedly isolated from the guts of neotropical lamiines. The objective of this study was to conduct transmission electron microscopy (TEM) studies of cerambycid larval midgut tissues to determine if gut yeasts were intracellular, or simply present in the gut lumen. Nine cerambycid larvae were harvested from two trees in the Brazil nut family (Lecythidaceae) in the rain forest of SE Peru; seven were identified using mtDNA sequence data and processed for TEM. Yeasts cultured from larval frass or exuvia, and identified with rDNA sequence data, were identical or similar to yeasts previously isolated from beetles. In TEM analyses yeast cells were found only in the gut lumens, sometimes associated with fragments of thick-walled xylem cells. Apparent bacteriocytes were found in either midgut or fat body tissue of three larval specimens, including two lamiines. This is the first report of a potential fat body symbiosis in a cerambycid beetle. Future studies of cerambycid symbiosis should distinguish the identities and potential roles of free-living organisms in the gut lumen from those of organisms harbored within gut epithelial or fat body tissue.     

Identification of a xylulokinase catalyzing xylulose phosphorylation in the xylose metabolic pathway of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> NBRC1777

Xylulokinase is one of the key enzymes in xylose metabolism and fermentation, and fine-tuned expression of xylulokinase can improve xylose fermentation in yeast. To improve the efficiency of xylose fermentation in Kluyveromyces marxianus, the gene KmXYL3, which encodes a d-xylulokinase (E.C. 2.7.1.17), was isolated from K. marxianus NBRC1777. KmXYL3 was expressed in Escherichia coli BL21 (DE3) cells, and the specific activity of the resulting recombinant purified xylulokinase was 23.5 mU/mg. Disruption of KmXYL3 resulted in both loss of xylitol utilization and marked decrease in xylose utilization, proving that KmXYL3 encodes a xylulokinase that catalyzes the reaction from xylulose to xylulose 5-phosphate in the xylose metabolic pathway. The slow assimilation of xylose observed in the KmXYL3-disrupted strain indicates that KmXYL3 is critical for xylose and xylitol utilization; however, K. marxianus utilizes a bypass pathway for xylose assimilation, and this pathway does not involve xylitol or xylulose.