Dietary benefits of fungal associates to an eruptive herbivore: potential implications of multiple associates on host population dynamics

We used the mountain pine beetle (Dendroctonus ponderosae Hopkins) and its two fungal associates, Grosmannia clavigera and Ophiostoma montium, to study potential nutritional benefits of fungi to bark beetles. We tested for potential effects of feeding on phloem colonized by fungi on beetle performance in field and laboratory studies. The fungi increased nitrogen levels in the phloem of attacked trees by 40%, indicating that it may be an important source of dietary nitrogen for mountain pine beetles. However, nitrogen levels of phloem inoculated with fungi in the laboratory were similar to uncolonized phloem, indicating that the fungi may redistribute nitrogen from the sapwood to the phloem rather than increase absolute levels of nitrogen. Beetles emerging from attacked trees carrying G. clavigera were larger than beetles carrying O. montium, which in turn were larger than beetles lacking fungi. Results of experimental laboratory studies varied, likely because of differences in the growth and sporulation of fungi under artificial conditions. Results indicate that the two fungi may offer complementary benefits to the mountain pine beetle because larvae preferentially fed on phloem colonized by both fungi together over phloem colonized by one fungus or uncolonized phloem. Teneral adults preemergence fed on spores in pupal chambers when they were produced and consumed little phloem before emerging. Teneral adults mined extensively in the phloem before emerging when spores were not produced in the pupal chamber. Our results provide evidence for a nutritional role of fungi in the diet of bark beetles and show that multiple associates may differentially affect beetle performance, which could have important implications for bark beetle population dynamics.     

The impact of phloem nutrients on overwintering mountain pine beetles and their fungal symbionts

In the low nutrient environment of conifer bark, subcortical beetles often carry symbiotic fungi that concentrate nutrients in host tissues. Although bark beetles are known to benefit from these symbioses, whether this is because they survive better in nutrient-rich phloem is unknown. After manipulating phloem nutrition by fertilizing lodgepole pine trees (Pinus contorta Douglas var. latifolia), we found bolts from fertilized trees to contain more living individuals, and especially more pupae and teneral adults than bolts from unfertilized trees at our southern site. At our northern site, we found that a larger proportion of mountain pine beetle (Dendroctonus ponderosae Hopkins) larvae built pupal chambers in bolts from fertilized trees than in bolts from unfertilized trees. The symbiotic fungi of the mountain pine beetle also responded to fertilization. Two mutualistic fungi of bark beetles, Grosmannia clavigera (Rob.-Jeffr. & R. W. Davidson) Zipfel, Z. W. de Beer, & M. J. Wingf. and Leptographium longiclavatum Lee, S., J. J. Kim, & C. Breuil, doubled the nitrogen concentrations near the point of infection in the phloem of fertilized trees. These fungi were less capable of concentrating nitrogen in unfertilized trees. Thus, the fungal symbionts of mountain pine beetle enhance phloem nutrition and likely mediate the beneficial effects of fertilization on the survival and development of mountain pine beetle larvae.     

An invasive beetle–fungus complex is maintained by fungal nutritional-compensation mediated by bacterial volatiles

Mutualisms between symbiotic microbes and animals have been well documented, and nutritional relationships provide the foundation for maintaining beneficial associations. The well-studied mutualism between bark beetles and their fungi has become a classic model system in the study of symbioses. Despite the nutritional competition between bark beetles and beneficial fungi in the same niche due to poor nutritional feeding substrates, bark beetles still maintain mutualistic associations with beneficial fungi over time. The mechanism behind this phenomenon, however, remains largely unknown. Here, we demonstrated the bark beetle Dendroctonus valens LeConte relies on the symbiotic bacterial volatile ammonia, as a nitrogen source, to regulate carbohydrate metabolism of its mutualistic fungus Leptographium procerum to alleviate nutritional competition, thereby maintaining the stability of the bark beetle–fungus mutualism. Ammonia significantly reduces competition of L. procerum for carbon resources for D. valens larval growth and increases fungal growth. Using stable isotope analysis, we show the fungus breakdown of phloem starch into d-glucose by switching on amylase genes only in the presence of ammonia. Deletion of amylase genes interferes with the conversion of starch to glucose. The acceleration of carbohydrate consumption and the conversion of starch into glucose benefit this invasive beetle–fungus complex. The nutrient consumption–compensation strategy mediated by tripartite beetle–fungus–bacterium aids the maintenance of this invasive mutualism under limited nutritional conditions, exacerbating its invasiveness with this competitive nutritional edge.Subject terms: Microbial ecology, Microbial ecology     

Temporal variation in mycophagy and prevalence of fungi associated with developmental stages of Dendroctonus ponderosae (Coleoptera: Curculionidae)

Mycophagy by bark beetles is widespread. However, little is known regarding which developmental stages of bark beetles actually feed on fungi. To study this question, we sampled fungi associated with Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae) throughout development in naturally attacked trees. Isolations of fungi were made from phloem adjacent to brood and from brood exoskeletons and guts. Overall, the incidence of fungi with individual brood increased as brood development progressed. Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingf. and Ophiostoma montium (Rumbold) von Arx exhibited generally opposing trends in prevalence. G. clavigera was most likely to be found in phloem adjacent to prewintering third- and postwintering fourth-instar larvae. O. montium was most likely to be found in phloem adjacent to eggs, first-instar larvae, pupae, and teneral adults. In contrast to isolations made from phloem, fungi isolated from brood guts and exoskeletons were not observed to shift in prevalence. First- and third-instar larvae were often observed migrating to older portions of their galleries, indicating that they do not spend all of their time feeding at, and extending, the apex of the gallery. Our results suggest that not only are D. ponderosae brood in contact with and feeding on fungi throughout development, but also, that during development, contact of brood with a particular fungus is likely to change. Such temporal shifts in fungal symbionts may be environmentally driven and have important implications in how these fungi interact with their hosts within and across generations.     

Mutual interactions between an invasive bark beetle and its associated fungi

Interactions between invasive insects and their fungal associates have important effects on the behavior, reproductive success, population dynamics and evolution of the organisms involved. The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Scolytinae), an invasive forest pest in China, is closely associated with fungi. By carrying fungi on specialized structures in the exoskeleton, RTB inoculates fungi in the phloem of pines (when females dig galleries for egg laying and when males join them for mating). After eggs hatch, larvae gregariously feed on the phloem colonized by the fungi. We examined the effects of five isolates of RTB associated fungi (two from North America, Leptographium terebrantis and L. procerum, and three from China, Ophiostoma minus, L. sinoprocerum and L. procerum) on larval feeding activity, development and mortality. We also studied the effects of volatile chemicals produced in the beetle hindgut on fungal growth. Ophiostoma minus impaired feeding activity and reduced weight in RTB larvae. Leptographium sinoprocerum, L. terebrantis and L. procerum did not dramatically influence larval feeding and development compared to fungi-free controls. Larval mortality was not influenced by any of the tested fungi. Hindgut volatiles of RTB larvae, verbenol, myrtenol and myrtenal, inhibited growth rate of all the fungi. Our results not only show that D. valens associated fungus, O. minus, can be detrimental to its larvae; but, most importantly, they also show that these notorious beetles have an outstanding adaptive response evidenced by the ability to produce volatiles that inhibit growth of harmful fungus.     

Factors influencing bark beetle outbreaks after forest fires on the Iberian Peninsula

Fires are among the most globally important disturbances in forest ecosystems. Forest fires can be followed by bark beetle outbreaks. Therefore, the dynamic interactions between bark beetle outbreaks and fire appear to be of general importance in coniferous forests throughout the world. We tested three hypotheses of how forest fires in pine ecosystems (Pinus pinaster Alton and P. radiata D. Don) in Spain could alter the population dynamics of bark beetles and influence the probability of further disturbance from beetle outbreaks: fire could affect the antiherbivore resin defenses of trees, change their nutritional suitability, or affect top-down controls on herbivore populations. P. radiata defenses decreased immediately after fire, but trees with little crown damage soon recovered with defenses higher than before. Fire either reduced or did not affect nutritional quality of phloem and either reduced or had no effect on the abundance, diversity, and relative biomass of natural enemies. After fire, bark beetle abundance increased via rapid aggregation of reproductive adults on scorched trees. However, our results indicate that for populations to increase to an outbreak situation, colonizing beetles must initiate attacks before tree resin defenses recover, host trees must retain enough undamaged phloem to facilitate larval development, and natural enemies should be sufficiently rare to permit high beetle recruitment into the next generation. Coincidence of these circumstances may promote the possibility of beetle populations escaping to outbreak levels.     

Patterns of functional enzyme activity in fungus farming ambrosia beetles

ABSTRACT: INTRODUCTION: In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. RESULTS: We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-beta-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-beta-1,4-xylanase activity was exclusively detected in larvae. CONCLUSION: Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles do not degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae and not in adults indicates that larvae (pre-) digest plant cell wall structures exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi). This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.     

Anatomical defences against bark beetles relate to degree of historical exposure between species and are allocated independently of chemical defences within trees

Conifers possess chemical and anatomical defences against tree‐killing bark beetles that feed in their phloem. Resins accumulating at attack sites can delay and entomb beetles while toxins reach lethal levels. Trees with high concentrations of metabolites active against bark beetle‐microbial complexes, and more extensive resin ducts, achieve greater survival. It is unknown if and how conifers integrate chemical and anatomical components of defence or how these capabilities vary with historical exposure. We compared linkages between phloem chemistry and tree ring anatomy of two mountain pine beetle hosts. Lodgepole pine, a mid‐elevation species, has had extensive, continual contact with this herbivore, whereas high‐elevation whitebark pines have historically had intermittent exposure that is increasing with warming climate. Lodgepole pine had more and larger resin ducts. In both species, anatomical defences were positively related to tree growth and nutrients. Within‐tree constitutive and induced concentrations of compounds bioactive against bark beetles and symbionts were largely unrelated to resin duct abundance and size. Fewer anatomical defences in the semi‐naïve compared with the continually exposed host concurs with directional differences in chemical defences. Partially uncoupling chemical and morphological antiherbivore traits may enable trees to confront beetles with more diverse defence permutations that interact to resist attack.     

Competition and Coexistence In a Multi-partner Mutualism: Interactions Between two Fungal Symbionts of the Mountain Pine Beetle In Beetle-attacked Trees

Despite overlap in niches, two fungal symbionts of the mountain pine beetle (Dendroctonus ponderosae), Grosmannia clavigera and Ophiostoma montium, appear to coexist with one another and their bark beetle host in the phloem of trees. We sampled the percent of phloem colonized by fungi four times over 1 year to investigate the nature of the interaction between these two fungi and to determine how changing conditions in the tree (e.g., moisture) affect the interaction. Both fungi colonized phloem at similar rates; however, G. clavigera colonized a disproportionately larger amount of phloem than O. montium considering their relative prevalence in the beetle population. High phloem moisture appeared to inhibit fungal growth shortly after beetle attack; however, by 1 year, low phloem moisture likely inhibited fungal growth and survival. There was no inverse relationship between the percent of phloem colonized by G. clavigera only and O. montium only, which would indicate competition between the species. However, the percent of phloem colonized by G. clavigera and O. montium together decreased after 1 year, while the percent of phloem from which no fungi were isolated increased. A reduction in living fungi in the phloem at this time may have significant impacts on both beetles and fungi. These results indicate that exploitation competition occurred after a year when the two fungi colonized the phloem together, but we found no evidence of strong interference competition. Each species also maintained an exclusive area, which may promote coexistence of species with similar resource use.     

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.     

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.     

Death of vigorous trees benefits bark beetles

Bark beetles (Coleoptera: Scolytidae) are commonly associated with live host trees that are stressed, a relationship that has been attributed to lower host defenses or greater nutritional quality of these trees. However, most bark beetle species commonly inhabit freshly dead trees where induced host defenses are absent. In this study, we investigate the role of tree vigor at the time of death for pine engraver bark beetles, Ips pini (Say), breeding in freshly dead jack pine, Pinus banksiana Lamb. As indices of tree vigor, we considered tree size, phloem thickness, and several measures of recent growth rate (last year's growth increment, mean annual increment and basal area increment in the past 5 and 10 years, and periodic growth ratio). We examined the relationship between these indices in three stands, aged 60, 77, and 126 years, and found that phloem thickness, previously shown to have a strong positive effect on bark beetle reproduction, was only weakly associated with tree growth rate and inconsistently related to tree size among the three stands. To examine the effects of tree vigor on pine engraver reproduction, we felled 20 trees of various sizes from the 77-year-old stand, and experimentally established breeding males and females in 25-cm-long sections. Offspring were collected and characteristics of breeding galleries were measured. Using stepwise regression, we consistently found that indices associated with tree growth rate best explained beetle reproductive performance, as they were positively related to parental male and female establishment on logs, female reproductive success, length of egg galleries, proportion of eggs resulting in emerged offspring, and negatively related to the length of the post-egg gallery. Surprisingly, phloem thickness had no unique effect on pine engraver reproduction, except for a weak negative effect on the establishment success of parental females. The strong effect of tree vigor observed in this study suggests that substantial mortality of vigorous trees, such as caused by windthrow, can contribute to significant increases in bark beetle populations that could trigger outbreaks in living trees. Received: 3 February 1999 / Accepted: 27 April 1999     

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.     

Function of Sitka spruce stone cells as a physical defence against white pine weevil

Stone cells are a physical defence of conifers against stem feeding insects such as weevils and bark beetles. In Sitka spruce, abundance of stone cells in the cortex of apical shoot tips is associated with resistance to white pine weevil. However, the mode of action by which stone cells interfere with growth and development of weevil larvae is unknown. We developed a bioassay system for testing potential effects of stone cells, which were isolated from resistant trees, on weevil larvae. Bioassays using artificial diet and controlled amounts of stone cells focused on physical defence. We evaluated the effects of stone cells on establishment of neonate larvae, mandible wear and changes in relative growth rates of third instar larvae. Establishment of neonates and relative growth rates of third instars were significantly reduced by stone cells. Stone cells appeared to be indigestible by weevil larvae. Our results suggest that stone cells affect weevil establishment and development by forming a physical feeding barrier against neonate larvae at the site of oviposition, and by reducing access to nutrients in the cortex of resistant trees, which contain an abundance of stone cells in place of a more nutrient rich tissue in susceptible trees.     

Ophiostomatoid fungi can emit the bark beetle pheromone verbenone and other semiochemicals in media amended with various pine chemicals and beetle-released compounds

Fungal volatile compounds can mediate fungal-insect interactions. Whether fungi can emit insect pheromones and how volatile chemicals change in response to chemicals the fungi naturally encounter is poorly understood. We analyzed volatiles emitted by Grosmannia clavigera (symbiont of the mountain pine beetle) and Ophiostoma ips (symbiont of the pine engraver beetle) growing in liquid media amended with compounds that the fungi naturally encounter: (−)-α-pinene, (+)-α-pinene, (−)-trans-verbenol, verbenone, or ipsdienol. Nine volatile compounds were identified among the fungal and amendment treatments. Volatiles qualitatively and quantitatively differed between fungal species and among amendment treatments. The bark beetle anti-aggregation pheromone (−)-verbenone was detected from both fungi growing in (−)-trans-verbenol-amended media. G. clavigera and O. ips can emit beetle pheromones and other beetle semiochemicals, suggesting that ophiostomatoid fungi could contribute to the chemical ecology of bark beetles. However, such contributions could be modulated by the presence of other environmental chemicals.     

The effect of larval predators Thanasimus dubius (Coleoptera: Cleridae), produced by an improved system of rearing, against the southern pine beetle Dendroctonus frontalis (Coleoptera: Curculionidae)

The southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae) (SPB), is known to be a major bark beetle pest of pines throughout the southeastern United States. A common predator of bark beetles, Thanasimus dubius (Coleoptera: Cleridae), has been suggested to play a prevalent role on SPB dynamics. Evaluations of T. dubius have been limited by rearing methods; an artificial diet for larval T. dubius exists, and preservatives such as sorbic acid could help to maximize diet shelf-life and enhance the efficiency of the rearing system. The effects of sorbic acid at different concentrations (0%, 0.1% and 0.2%) in the larval diet for T. dubius were measured, and the effects of increased feeding time intervals (2-3 vs. 5 days) on predator performance evaluated. In addition, an experimental bioassay was conducted where newly hatched T. dubius larvae were released at four densities (0, 50, 100, and 200 per log) on pine logs infested by SPB. Sorbic acid in the diet reduced female fecundity (by 20-40%), but did not affect adult T. dubius size or longevity. However, using this preservative may not be necessary because it had no effect on the overall efficiency of the rearing system, while refreshing the larval diet every 5 days (compared with 2-3 days) did improve its efficiency, even without sorbic acid. The release of larval T. dubius resulted in a highly significant effect on the SPB ratio of increase (RI). This experiment was facilitated by the improvements in our rearing methods.     

Nutritional and developmental factors in larval growth and retrogression of Trogoderma glabrum

Diet is an important determinant of the developmental pattern of Trogoderma glabrum (Herbst). Two nutritionally adequate diets were compared. The average weight of larvae of the same age was greater when cultured on meridic diet than on standard crude diet. The expected pattern of instars, as determined by head capsule widths, was observed for larvae reared on standard diet. On meridic diet, head capsules of each instar were significantly wider, and both sexes pupated after 5 stadia.Survival during retrogression correlated positively with weight at the time of isolation. Larvae reared on meridic diet survived longer than larvae of equal culture age or weight from standard diet. Survival and maintenance of weight were greatly improved by direct contact with nutritional media, some of which were incomplete (powdered cellulose plus 10% lactalbumin, fructose, or glucose). Cellulose alone, however, was more detrimental to survival than no medium.A moult after transfer to isolation tubes produced 3 morphologically distinct forms, determined by the extent of maturation. Immature larvae retromoulted to smaller larvae; fully mature larvae pupated; and larvae that had initiated but not completed pupal differentiation became prothetelic.     

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.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

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.