Management of Western North American Bark Beetles with Semiochemicals.

We summarize the status of semiochemical-based management of the major bark beetle species in western North America. The conifer forests of this region have a long history of profound impacts by phloem-feeding bark beetles, and species such as the mountain pine beetle (Dendroctonus ponderosae) and the spruce beetle (D. rufipennis) have recently undergone epic outbreaks linked to changing climate. At the same time, great strides are being made in the application of semiochemicals to the integrated pest management of bark beetles. In this review, we synthesize and interpret these recent advances in applied chemical ecology of bark beetles for scientists and land managers.

A Blend of Ethanol and (-)-α-Pinene were Highly Attractive to Native Siricid Woodwasps (Siricidae, Siricinae) Infesting Conifers of the Sierra Nevada and the Allegheny Mountains.

Woodwasps in Sirex and related genera are well-represented in North American conifer forests, but the chemical ecology of native woodwasps is limited to a few studies demonstrating their attraction to volatile host tree compounds, primarily monoterpene hydrocarbons and monoterpene alcohols. Thus, we systematically investigated woodwasp-host chemical interactions in California's Sierra Nevada and West Virginia's Allegheny Mountains. We first tested common conifer monoterpene hydrocarbons and found that (-)-α-pinene, (+)-3-carene, and (-)-ß-pinene were the three most attractive compounds. Based on these results and those of earlier studies, we further tested three monoterpene hydrocarbons and four monoterpene alcohols along with ethanol in California: monoterpene hydrocarbons caught 72.3% of all woodwasps. Among monoterpene hydrocarbons, (+)-3-carene was the most attractive followed by (-)-ß-pinene and (-)-α-pinene. Among alcohols, ethanol was the most attractive, catching 41.4% of woodwasps trapped. Subsequent tests were done with fewer selected compounds, including ethanol, 3-carene, and ethanol plus (-)-α-pinene in both Sierra Nevada and Allegheny Mountains. In both locations, ethanol plus (-)-α-pinene caught more woodwasps than other treatments. We discussed the implications of these results for understanding the chemical ecology of native woodwasps and invasive Sirex noctilio in North America. In California, 749 woodwasps were caught, representing five species: Sirex areolatus Cresson, Sirex behrensii Cresson, Sirex cyaneus Fabricius, Sirex longicauda Middlekauff, and Urocerus californicus Norton. In West Virginia 411 woodwasps were caught representing four species: Sirex edwardsii Brullé, Tremex columba Linnaeus, Sirex nigricornis F., and Urocerus cressoni Norton.

Efficacy of verbenone and green leaf volatiles for protecting whitebark and limber pines from attack by mountain pine beetle (Coleoptera: Curculionidae: Scolytinae).

To develop safe and effective methods to protect whitebark pines, Pinus albicaulis Engelmann, and limber pines, Pinus flexilis James, from attack by mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), we compared verbenone and verbenone plus green leaf volatiles (GLVs) for prevention of beetle attack. We used two strategies: area-wide protection where semiochemical-releasing flakes are dispersed over the forest floor, and individual tree tests where flakes are applied to tree trunks. The area-wide bioassays were conducted by applying verbenone- and GLV-releasing flakes without stickers to the forest floor on 0.81-ha plots dominated by whitebark pines in the State of Washington with four replicates. We conducted individual tree bioassays by applying the same formulations with stickers to whitebark and limber pines in Montana and Colorado, respectively. In all three situations, both verbenone-alone and verbenone plus GLVs significantly increased the proportion of trees escaping mass attack by beetles, but the two formulations were not significantly different from one another. Despite a lack of significance at a Bonferroni-adjusted α = 0.05, adding GLVs gave slightly greater absolute levels of tree protection in most cases. Monitoring traps placed in the area-wide treatments in Washington showed similar outcomes for numbers of beetles trapped: both treatments had significantly fewer beetles than controls, and they were not significantly different from one another. At peak flight, however, plots with GLVs combined with verbenone had roughly 40% fewer beetles than plots with verbenone alone. GLVs are considerably cheaper than verbenone, so tests of higher application rates may be warranted to achieve enhanced tree protection at reasonable cost.

Large shift in symbiont assemblage in the invasive red turpentine beetle.

Changes in symbiont assemblages can affect the success and impact of invasive species, and may provide knowledge regarding the invasion histories of their vectors. Bark beetle symbioses are ideal systems to study changes in symbiont assemblages resulting from invasions. The red turpentine beetle (Dendroctonus valens) is a bark beetle species that recently invaded China from its native range in North America. It is associated with ophiostomatalean fungi in both locations, although the fungi have previously been well-surveyed only in China. We surveyed the ophiostomatalean fungi associated with D. valens in eastern and western North America, and identified the fungal species using multi-gene phylogenies. From the 307 collected isolates (147 in eastern North America and 160 in western North America), we identified 20 species: 11 in eastern North America and 13 in western North America. Four species were shared between eastern North America and western North America, one species (Ophiostoma floccosum) was shared between western North America and China, and three species (Grosmannia koreana, Leptographium procerum, and Ophiostoma abietinum) were shared between eastern North America and China. Ophiostoma floccosum and O. abietinum have worldwide distributions, and were rarely isolated from D. valens. However, G. koreana and L. procerum are primarily limited to Asia and North America respectively. Leptographium procerum, which is thought to be native to North America, represented >45% of the symbionts of D. valens in eastern North America and China, suggesting D. valens may have been introduced to China from eastern North America. These results are surprising, as previous population genetics studies on D. valens based on the cytochrome oxidase I gene have suggested that the insect was introduced into China from western North America.

Red turpentine beetle: innocuous native becomes invasive tree killer in China.

The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae), is a secondary pest of pines in its native range in North and Central America. Outbreaks and tree mortality attributed to RTB alone are rare in its native range. RTB was introduced into China in the early 1980s and spread rapidly from Shanxi Province to four adjacent provinces; it has infested over 500,000 ha of pine forest and has caused extensive tree mortality since 1999. We provide a historical background on RTB outbreaks, explanations for its invasive success, management options, and economic impacts of RTB in China. Genetic variation in RTB fungal associates, interactions between RTB and its associated fungi, behavioral differences in Chinese RTB, and other factors favoring RTB outbreaks are considered in an effort to explain the invasiveness of RTB in China. The promise of semiochemicals as a management tool is also discussed.

The push-pull tactic for mitigation of mountain pine beetle (Coleoptera: Curculionidae) damage in lodgepole and whitebark pines.

In an attempt to improve semiochemical-based treatments for protecting forest stands from bark beetle attack, we compared push-pull versus push-only tactics for protecting lodgepole pine (Pinus contorta Douglas ex Loudon) and whitebark pine (Pinus albicaulis Engelm.) stands from attack by mountain pine beetle (Dendroctonus ponderosae Hopkins) in two studies. The first was conducted on replicated 4.04-ha plots in lodgepole pine stands (California, 2008) and the second on 0.81-ha plots in whitebark pine stands (Washington, 2010). In both studies, D. ponderosae population levels were moderate to severe. The treatments were 1) push-only (D. ponderosae antiaggregant semiochemicals alone); 2) push-pull (D. ponderosae antiaggregants plus perimeter traps placed at regular intervals, baited with four-component D. ponderosae aggregation pheromone); and 3) untreated controls. We installed monitoring traps baited with two-component D. ponderosae lures inside each plot to assess effect of treatments on beetle flight. In California, fewer beetles were collected in push-pull treated plots than in control plots, but push-only did not have a significant effect on trap catch. Both treatments significantly reduced the rate of mass and strip attacks by D. ponderosae, but the difference in attack rates between push-pull and push-only was not significant. In Washington, both push-pull and push-only treatments significantly reduced numbers of beetles caught in traps. Differences between attack rates in treated and control plots in Washington were not significant, but the push-only treatment reduced attack rates by 30% compared with both the control and push-pull treatment. We conclude that, at these spatial scales and beetle densities, push-only may be preferable for mitigating D. ponderosae attack because it is much less expensive, simpler, and adding trap-out does not appear to improve efficacy.

Do novel genotypes drive the success of an invasive bark beetle-fungus complex? Implications for potential reinvasion.

Novel genotypes often arise during biological invasions, but their role in invasion success has rarely been elucidated. Here we examined the population genetics and behavior of the fungus, Leptographium procerum, vectored by a highly invasive bark beetle, Dendroctonus valens, to determine whether genetic changes in the fungus contributed to the invasive success of the beetle-fungal complex in China. The fungus was introduced by the beetle from the United States to China, where we identified several novel genotypes using microsatellite markers. These novel genotypes were more pathogenic to Chinese host seedlings than were other genotypes and they also induced the release of higher amounts of 3-carene, the primary host attractant for the beetle vector, from inoculated seedlings. This evidence suggests a possible mechanism, based on the evolution of a novel genotype during the two or three decades since its introduction, for the success of the beetle-fungal complex in its introduced region.

Complex interactions among host pines and fungi vectored by an invasive bark beetle.

*Recent studies have investigated the relationships between pairs or groups of exotic species to illustrate invasive mechanisms, but most have focused on interactions at a single trophic level. *Here, we conducted pathogenicity tests, analyses of host volatiles and fungal growth tests to elucidate an intricate network of interactions between the host tree, the invasive red turpentine beetle and its fungal associates. *Seedlings inoculated with two strains of Leptographium procerum isolated from Dendroctonus valens in China had significantly longer lesions and higher mortality rates than seedlings inoculated with other fungal isolates. These two strains of L. procerum were significantly more tolerant of 3-carene than all other fungi isolated there, and the infection of Chinese pine (Pinus tabuliformis) seedlings by these two strains enhanced the production and release of 3-carene, the main attractant for D. valens, by the seedlings. *Our results raise the possibility that interactions among the fungal associates of D. valens and their pine hosts in China may confer advantages to these strains of L. procerum and, by extension, to the beetles themselves. These interactions may therefore enhance invasion by the beetle-fungal complex.

Geographic variation in bacterial communities associated with the red turpentine beetle (Coleoptera: Curculionidae).

Bacterial communities are known to play important roles in insect life histories, yet their consistency or variation across populations is poorly understood. Bacteria associated with the bark beetle Dendroctonus valens LeConte from eight populations, ranging from Wisconsin to Oregon, were evaluated and compared. We used the culture-independent technique of denaturing gradient gel electrophoresis to visualize bacterial diversity, or individual operational taxonomic units (OTUs), from individual beetles. One-way analysis of similarities was used to test for differences of bacterial communities between sites. Analysis of community profiles showed that individual beetles on average contained 10 OTUs, with frequency of association from 2 to 100% of beetles. OTU sequences most closely matched beta- and gamma-proteobacteria, and one each matched Bacilli and Actinobacteria. Several OTUs were particularly abundant, most notably an Actinobacterium from 100% and two Proteobacteria from 60% of beetles sampled. Some OTUs were similar to previously described bacteria with known biochemical capabilities and ecological functions, suggesting that some bacterial associates of D. valens may contribute to its ability to exploit a resource low in nutrients and high in defensive compounds. There were significant differences of bacterial communities between sites. The strength of these differences was positively correlated with distance between sites, although additional unexplained factors also contribute to the variation.

Mitochondrial phylogeny of pine cone beetles (Scolytinae, Conophthorus) and their affiliation with geographic area and host.

Pine cone beetles (Conophthorus spp.) feed and kill immature cones of Pinus species, thereby reducing seed production and seriously impairing reforestation of forest ecosystems. Population variation of Conophthorus reproductive behavior has hampered the development of semiochemical control of these pests. This difficulty is compounded by a lack of taxonomic knowledge and species diagnostic characters. Researchers and managers rely, in part, on host associations and geographic locality for species identifications and these have arguable taxonomic utility. However, host use and/or geographic separation may influence Conophthorus lineage diversification. To improve Conophthorus taxonomy and understand the association of host and geography with lineage diversification, a phylogeny of 43 individuals, including all valid species and a robust sample of C. ponderosae from different hosts, is reconstructed using 785 nucleotides of the 3'-end of the mitochondrial cytochrome oxidase I gene. Thirty trees were recovered in a parsimony analysis and the strict consensus was well resolved and supported by branch support measures. Conophthorus was monophyletic but mitochondrial polyphyly was uncovered for several species. The data also suggested an underestimation of species diversity. Phylogenetically related Conophthorus lineages were significantly associated with geographic proximity but not with host, as indicated by comparisons of character optimized geographic distributions and host associations against randomized distributions of these attributes on the parsimony tree. These results suggest that geographic separation better explains the mode of Conophthorus lineage diversification than does host specialization. Based on these results, researchers and managers of Conophthorus should consider populations as potentially different evolutionary entities until species boundaries are delineated via a robust phylogenetic revision of Conophthorus.

Safety and systemic absorption of pulmonary delivered human IFN-beta1a in the nonhuman primate: comparison with subcutaneous dosing.

Safety and bioavailability of pulmonary delivered interferon-beta 1a (IFN-beta1a, AVONEX, Biogen, Inc., Cambridge, MA) was evaluated in the nonhuman primate. Pulmonary bioavailability following intratracheal (i.t.) instillation of 50 microg/kg IFN-beta1a to rhesus macaques was approximately 10%. To evaluate pulmonary safety, IFN-beta1a was administered intrabronchially to rhesus and cynomolgus macaques at a dose of 60 microg/dose one, three, or seven times per week for 4 weeks. At scheduled termination, lungs were evaluated for gross and histomorphologic changes. IFN-beta1a or vehicle (human serum albumin [HSA] in phosphate-buffered saline [PBS]) treatment resulted in minimal to mild subchronic alveolitis, located primarily near the instillation sites. These responses were considered nonspecific and consistent with either instillation of a foreign protein or minor injury associated with the instillation procedure. In one rhesus macaque treated every day for 4 weeks, IFN-beta1a induced mild to moderate eosinophilic alveolitis, considered possibly an isolated type I hypersensitivity response to HSA or IFN-beta1a. Partial resolution of pulmonary lesions was seen in all recovery animals killed 2 weeks after cessation of treatment. In conclusion, this study shows that pulmonary administration of human IFN-beta1a is safe and that the pulmonary route of administration is a possible alternate route for the systemic delivery of IFN-beta1a.