Entomocorticium Sp Research Articles

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.

Temperature effects on growth of fungal symbionts of the western pine beetle, Dendroctonus brevicomis

Abstract Differences in temperature ranges and optima among poikilothermic partners in symbioses can have profound effects on their interactions and stability. In this study, we investigated how the two mutualist mycangial fungi (Ceratocystiopsis brevicomi and Entomocorticium sp. B) associated with the western pine beetle, Dendroctonus brevicomis, respond to temperature in vitro. Little variability in growth rate at the various temperatures tested occurred among isolates of C. brevicomi either within or among sites. In contrast, E. sp. B exhibited highly variable responses to mid-range temperatures among sites and within some sites, and, unlike C. brevicomi, grew not at all or only very poorly at the highest and lowest temperatures tested. This variability affected both optimal temperature and maximum growth rate. The high variability in response to some temperatures among isolates of E. sp. B in some populations indicates that the ability to capture spatial and nutritional resources can vary greatly within this species which may have considerable impact on the outcome of both inter- and intra-specific competition among the fungi within trees and the short- and long-term dynamics of the fungi with the host beetle.

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.

Plant secondary chemistry mediates the performance of a nutritional symbiont associated with a tree‐killing herbivore

Many herbivores consume microbial food sources in addition to plant tissues for nutrition. Despite the ubiquity of herbivore-microbe feeding associations, few studies examine how host plant phenotypes affect microbial symbionts of herbivores. We tested the hypothesis that chemical polymorphism in a plant population mediates the performance of nutritional microbial symbionts. We surveyed the composition of ponderosa pine resin in northern Arizona, USA, for variation in six monoterpenes, and we approximated four chemical phenotypes. We reared populations of an herbivorous tree-killing beetle (Dendroctonus brevicomis) in ponderosa pine host material, controlling for three monoterpene compositions representing an alpha-pinene to delta-3-carene gradient. Beetles were reared in host material where the dominant monoterpene was alpha-pinene, delta-3-carene, or a phenotype that was intermediate between the two. We isolated nutritional fungal symbionts (Entomocorticium sp. B) from beetle populations reared in each phenotype and performed reciprocal growth experiments in media amended to represent four "average" monoterpene compositions. This allowed us to test the effects of natal host phenotype, chemical polymorphism, and the interaction between natal host phenotype and chemical polymorphism on a nutritional symbiont. Three important findings emerged: (1) fungal isolates grew 25-32% faster when acquired from beetles reared in the intermediate phenotype; (2) the mean growth rate of nutritional fungi varied up to 44% depending on which monoterpene composition media was amended with; and (3) fungal isolates uniformly performed best in the intermediate phenotype regardless of the chemical composition of their natal host. The performance of nutritional fungi related to both the chemical "history" of their associated herbivore and the chemical phenotypes they are exposed to. However, all fungal isolates appeared adapted to a common chemical phenotype. These experiments argue in favor of the hypothesis that chemical polymorphism in plant populations mediates growth of nutritional symbionts of herbivores. Intraspecific chemical polymorphism in plants contributes indirectly to the regulation of herbivore populations, and our experiments demonstrate that the ecological effects of plant secondary chemistry extend beyond the trophic scale of the herbivore-plant interaction.

Interactions Between the Yeast Ogataea pini and Filamentous Fungi Associated with the Western Pine Beetle

Ecologically important microbes other than filamentous fungi can be housed within the fungal-transport structures (mycangia) of Dendroctonus bark beetles. The yeast Ogataea pini (Saccharomycetales: Saccharomycetaceae) was isolated from the mycangia of western pine beetle (Dendroctonus brevicomis) populations in northern Arizona (USA) with a frequency of 56%. We performed a series of in vitro assays to test whether volatile organic compounds produced by O. pini affected radial growth rates of mutualistic and antagonistic species of filamentous fungi that are commonly found in association with the beetle including Entomocorticium sp. B, Ophiostoma minus, Beauvaria bassiana, and an Aspergillus sp. We determined the compounds O. pini produced when grown on 2% malt extract agar using a gas chromatography/mass spectrometry (GC/MS) analysis of headspace volatiles. Volatiles produced by O. pini on artificial media significantly enhanced the growth of the mutualistic Entomocorticium sp. B, and inhibited growth of the entomopathogenic fungus B. bassiana. GC/MS revealed that O. pini produced ethanol, carbon disulfide (CS(2)), and Δ-3-carene in headspace. The results of these studies implicate O. pini as an important component in D. brevicomis community ecology, and we introduce multiple hypotheses for future tests of the effects of yeasts in the symbiont assemblages associated with Dendroctonus bark beetles.

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase. In time course analyses of cellulase production in batch cultures, Entomocorticium sp. A showed maximum activity of 0.109 U/ml and 0.141 U/ml for total cellulase and endoglucanase activity respectively. Polyphenol oxidase production was simultaneous with fungal growth. Characterization of polyphenol oxidase by activity staining suggests that the enzyme is a tyrosinase/catechol oxidase. Enzyme assays in the presence of polyphenol oxidase inhibitors support the results of the activity staining.

Effects Of Tree Phytochemistry On The Interactions Among Endophloedic Fungi Associated With The Southern Pine Beetle

We examined the interaction between host trees and fungi associated with a tree-killing bark beetle, Dendroctonus frontalis. We evaluated (1) the response of four Pinus species to fungal invasion and (2) the effects of plant secondary metabolites on primary growth of and secondary colonization of three consistent fungal associates. Two of these fungi, Entomocorticium sp. A and Ophiostoma ranaculosum, are obligate mutualists with D. frontalis, and the third associate is a blue-staining fungus, O. minus, that is commonly introduced by beetles and phoretic mites. O. minus negatively affects beetle larvae and in high abundance can impact D. frontalis population dynamics. Size of lesions formed and quantity of secondary metabolites produced in response to fungal inoculations varied significantly among Pinus species. However, monoterpene composition within infected tissue did not significantly vary across treatments. While all eight tested metabolites negatively affected the growth rate of O. minus, only 4-allylanisole, p-cymene, and terpinene reduced the growth of the mycangial fungi. Surprisingly, growth rates of mycangial fungi increased in the presence of several secondary metabolite volatiles. O. minus out-competed both mycangial fungi, but the presence of secondary metabolites altered the outcome slightly. O. ranaculosum out-performed E. sp. A in the presence of dominant conifer monoterpenes, such as alpha- and beta-pinene. Volatiles from the mycangial fungi, particularly E. sp. A, had a negative effect on O. minus growth. In general, phloem phytochemistry of particular Pinus species appeared to alter the relative growth and competitiveness of mutualistic and non-mutualistic fungi associated with D. frontalis. The outcome of interactions among these fungi likely has important consequences for the population dynamics of D. frontalis.

Effects of available water on growth and competition of southern pine beetle associated fungi

Competitive, interactions among bark beetle associated fungi are potentially influenced by abiotic factors. Water potential, in particular, undergoes marked changes over the course of beetle colonization of tree hosts. To investigate the impact of water potential on competition among three southern pine beetle associated fungi, Ophiostoma minus, Entomocorticium sp. A and Ceratocystiopsis ranaculosus, we utilized artificial media with water potentials of 0, -5, -10, and -20 MPa. Growth of all three fungi, when grown alone, decreased on media with lower water potentials. Growth rates of all three fungi were likewise reduced in competition experiments. At -5 to -10 MPa, C. ranaculosus (a fungus with beneficial effects toward southern pine beetle) was nearly equal in competitive ability to O. minus (a fungus with antagonistic effects towards southern pine beetle). This was not true on control media, nor at other water potentials tested. The range of water potentials used in our assays was similar to the range of water potentials we measured in loblolly pines within a southern pine beetle infestation. This study indicates that water potential may alter the outcome of competitive interactions among bark beetle-associated fungi in ways that favour bark beetle success.

Biology, demography and community interactions of Tarsonemus (Acarina: Tarsonemidae) mites phoretic on Dendroctonus frontalis (Coleoptera: Scolytidae)

Summary1 Dendroctonus frontalis, the southern pine beetle, is associated with a diverse community of fungi and mites that are phoretic on the adult beetles. Tarsonemus ips, T. kranzti and T. fusarii (Acarina: Tarsonemidae) may interact within this community in ways that link the population dynamics of D. frontalis, the mites and three dominant species of fungi. We explored species associations by comparing the dietary suitability of different fungi for Tarsonemus spp.2 All three mite species fed and reproduced at high rates when feeding on the bluestain fungus, Ophiostoma minus, which is an antagonist of D. frontalis larvae.3 Mites also had positive population growth rates when feeding upon Ceratocystiopsis ranaculosus, one of the mycangial fungi, but could barely reproduce when feeding upon Entomocorticium sp. A, the mycangial fungus that is most suitable for D. frontalis.4 During the time from colonization of a tree by D. frontalis adults until departure from the tree of their progeny (≈ 40 d at 30 °C), mite populations feeding upon O. minus can increase by factors of up to 209 (T. fusarii), 173 (T. ips) or 384 (T. krantzi). These high growth rates are allowed by rapid development (age of first reproduction = 8–9 d), high fecundity (≈ 1 egg/d) and high longevity (> 28 d).5 Precocious mating increases the chance that females are mated prior to colonizing a new tree and arrhenotokous parthenogenesis permits reproduction by unmated females.6 Tarsonemus mites may introduce negative feedback into D. frontalis population dynamics by generating indirect interactions between D. frontalis and O. minus.

Competition between a biological control fungus, Ophiostoma piliferum, and symbionts of the southern pine beetle

A colorless isolate of O. piliferum was paired in a series of competitive interactions with three fungal symbionts of Dendroctonus frontalis, the southern pine beetle. Two of these fungi, Ceratocystiopsis ranaculosus and Entomocorticium sp. A, are considered to be mutualists of the southern pine beetle. The third fungal symbiont, O. minus, is considered to be an antagonist. I found strong evidence of differential competition between O. piliferum and all three symbionts. In primary and secondary resource capture contests on an artificial medium O. piliferum outcompeted all three fungi. In inoculations of natural substrate, O. piliferum outcompeted the two mutualists but did not outcompete O. minus. The ability of O. piliferum to outcompete beetle mutualists on both artificial and natural substrates indicates promise for this fungus as a biological control agent of the southern pine beetle. However, it may not be able to always prevent colonization by O. minus and the resultant discoloration of colonized wood.

Competitive Interactions among Symbiotic Fungi of the Southern Pine Beetle

The southern pine beetle, a damaging pest of conifers, is intimately linked to three symbiotic fungi. Two fungi, Ceratocystiopsis ranaculosus and Entomocorticium sp. A, are transported within specialized structures (mycangia) in the beetle exoskeleton and are mutualists of the beetle. A third fungus, Ophiostoma minus, is transported externally on the beetle exoskeleton (phoretically) and is an antagonist of the beetle. This study examined competitive interactions among these three fungi. The results of de Wit replacement series and primary and secondary resource capture assays with these fungi provide strong evidence for differential competition between the phoretic and mycangial fungi. O. minus was the most able to capture both uncolonized and colonized resources. Entomocorticium sp. A and C. ranaculosus, although equal to one another in competitive abilities, differed in their ability to compete with O. minus. Entomocorticium sp. A was able to maintain space free of O. minus to a much greater degree than was C. ranaculosus. The outcome of such competitive interactions may have significant impacts on the biology of this ecologically and economically important beetle.