Role of 3-Carene in Host Location and Colonization by Dendroctonus pseudotsugae (Coleoptera: Curculionidae).

The Douglas-fir beetle (Dendroctonus pseudotsugae Hopkins) occasionally colonizes western larch [Larix occidentalis Nutt. (Pinales: Pinaceae)] growing in close proximity to its primary host, Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco (Pinales: Pinaceae)], but brood have never been found to survive in live western larch. Western larch produces the monoterpene 3-carene in higher concentrations than Douglas-fir. In this study, the toxicity and repellency of 3-carene to Douglas-fir beetle was evaluated in a series of laboratory and field tests. In a laboratory bioassay, 3-carene was one of the most toxic monoterpenes to Douglas-fir beetles among those tested. In a field trial, addition of 3-carene to multiple-funnel traps baited with frontalin (the primary component of Douglas-fir beetle aggregation pheromone) or frontalin and α-pinene significantly reduced the number of Douglas-fir beetles collected. In another field study, live western larch, felled western larch, live Douglas-fir, felled Douglas-fir, and live Douglas-fir surrounded by 3-carene releasers were baited with Douglas-fir beetle aggregation pheromones. There were significantly fewer Douglas-fir beetle entrance holes and egg galleries excavated on both live western larch and live Douglas-fir surrounded by 3-carene compared with live Douglas-fir. Most egg galleries excavated in live western larch were heavily impregnated with resin and no eggs hatched. There were no significant differences in egg galleries excavated or eggs hatched between felled western larch and felled Douglas-fir. Collectively, these data support the hypothesis that 3-carene slows the colonization process in live western larch allowing more time for host trees to respond to a colonization attempt and a higher likelihood of successfully resisting infestation.

Temporal Asynchrony of Adult Emergence Between Leucopis argenticollis and Leucopis piniperda (Diptera: Chamaemyiidae), Predators of the Hemlock Woolly Adelgid (Hemiptera: Adelgidae), with Implications for Biological Control.

Two species of silver fly, Leucopis argenticollis (Zetterstedt) and Leucopis piniperda (Malloch) (Diptera: Chamaemyiidae), from the Pacific Northwest region of North America have been identified as potential biological control agents of hemlock woolly adelgid (Hemiptera: Adelgidae: Adelges tsugae Annand) in eastern North America. The two predators are collectively synchronized with A. tsugae development. To determine whether adult emergence of the two species of silver fly are also synchronized with one another, we collected adult Leucopis which emerged from A. tsugae-infested western hemlock [Pinaceae: Tsuga heterophylla (Raf.) Sarg.] from four sites in the Pacific Northwest over a 29-d period. Specimens were collected twice daily in the laboratory and identified to species using DNA barcoding. The study found that more adult Leucopis were collected in the evening than the morning. Additionally, the daily emergences of adults over the 29-d sampling period exhibited sinusoidal-like fluctuations of peak abundance of each species, lending evidence to a pattern of temporal partitioning. This pattern could have logistical implications for their use as biological control agents in eastern North America, namely the need to release both species for maximum efficacy in decreasing A. tsugae populations.

Coexistence of three specialist predators of the hemlock woolly adelgid in the Pacific Northwest USA.

The hemlock woolly adelgid (Hemiptera: Adelgidae: Adelges tsugae Annand) is an invasive insect, introduced from Japan to eastern North America, where it causes decline and death of hemlock trees. There is a closely related lineage of A. tsugae native to western North America. To inform classical biological control of A. tsugae in the eastern USA, the density and phenology of three native western adelgid specialist predators, Leucopis argenticollis (Zetterstedt), Le. piniperda (Malloch) (Diptera: Chamaemyiidae), and Laricobius nigrinus Fender (Coleoptera: Derodontidae), were quantified in the Pacific Northwest. Infested branches were collected from western hemlock (Pinaceae: Tsuga heterophylla (Raf.) Sarg.) at four sites around the Puget Sound, Washington and three sites in Oregon. Immature Leucopis were identified to species using DNA barcodes. Leucopis argenticollis was roughly twice as abundant as Le. piniperda. Laricobius nigrinus larvae were more abundant than the two species of Leucopis during the egg stage of the first adelgid generation, but Leucopis were present as feeding larvae during the second adelgid generation when La. nigrinus was aestivating in the soil, resulting in Leucopis being more abundant than La. nigrinus across the entire sampling period. Adelges tsugae and La. nigrinus densities were not correlated, while A. tsugae and Leucopis spp. densities were positively correlated. Leucopis spp. and La. nigrinus densities were negatively correlated. These results support the complementary use of La. nigrinus and the two Leucopis species for biological control of A. tsugae in the eastern USA, and point to the need for further investigation of spatial and temporal niche partitioning among the three predator species.

Residual survival and local dispersal drive reinfestation by Triatoma dimidiata following insecticide application in Guatemala.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted by triatomine insect vectors. In Guatemala, insecticide spraying is an integral part of management of the main vector, Triatoma dimidiata. Spraying typically has low efficacy, which may be due to incomplete elimination from infested houses, within-village dispersal, or influx from other villages or sylvan environments. To evaluate how these mechanisms contribute to reinfestation, we conducted a time-course analysis of T. dimidiata infestation, abundance and household genetic structure in two nearby villages in Jutiapa, Guatemala; houses in the first village were surveyed, treated with insecticide if infested and then re-surveyed at eight and 22 months following spraying, while the second village served as an untreated control to quantify changes associated with seasonal dispersal. Insects were genotyped at 2-3000 SNP loci for kinship and population genetic analyses. Insecticide application reduced overall infestation and abundance, while the untreated village was stable over time. Nevertheless, within two years 35.5% of treated houses were reinfested and genetic diversity had largely recovered. Insects collected from reinfested houses post-spraying were most closely related to pre-spray collections from the same house, suggesting that infestations had not been fully eliminated. Immigration by unrelated insects was also detected within a year of spraying; when it occurred, dispersal was primarily local from neighboring houses. Similar dispersal patterns were observed following the annual dispersal season in the untreated village, with high-infestation houses serving as sources for neighboring homes. Our findings suggest that the efficacy of pyrethroid application is rapidly diminished by both within-house breeding by survivors and annual cycles of among-house movement. Given these patterns, we conclude that house structural improvements, an integral part of the Ecohealth approach that makes houses refractory to vector colonization and persistence, are critical for long-term reduction of T. dimidiata infestation.

Racemic Pheromone Blends Disrupt Mate Location in the Invasive Swede Midge, Contarinia nasturtii.

Swede midge, Contarinia nasturtii Kieffer, is an invasive cecidomyiid pest that causes serious losses of Brassica oilseed and vegetable crops in the Northeastern U.S. and Canada. Currently, few alternatives to systemic insecticides exist for its management. Because a single feeding larva can render heading Brassica crops unmarketable, management strategies that prevent oviposition are needed urgently. Pheromone-mediated mating disruption is a promising management approach for swede midge because it prevents mating and subsequent crop damage. While the swede midge pheromone has been identified, one of the major barriers to using it in mating disruption is the high cost of synthesis. Racemic blends, consisting of natural and non-natural stereoisomers, could be useful for mating disruption because they are cheaper to produce. However, it is not clear whether racemic pheromone blends attract males and/or prevent them from locating and mating with females. Here, we studied the behavior of male swede midge in Y-tube and wind tunnel bioassays to pheromone blends. Specifically, we tested whether males: (1) are attracted to different doses of pheromone, (2) discriminate between blends comprising natural stereospecific or racemic components, or a combination thereof, and (3) are able to locate and copulate with females in pheromone-permeated olfactometers. We found that picogram amounts of pheromone attracted males and prevented them from locating females in y-tube olfactometers. While males were more attracted to stereospecific blends, compared to racemic blends, all blends tested prevented nearly all males mating with females. Therefore, low dose racemic blends may be promising for pheromone-mediated mating disruption.

Uncovering vector, parasite, blood meal and microbiome patterns from mixed-DNA specimens of the Chagas disease vector Triatoma dimidiata.

Chagas disease, considered a neglected disease by the World Health Organization, is caused by the protozoan parasite Trypanosoma cruzi, and transmitted by >140 triatomine species across the Americas. In Central America, the main vector is Triatoma dimidiata, an opportunistic blood meal feeder inhabiting both domestic and sylvatic ecotopes. Given the diversity of interacting biological agents involved in the epidemiology of Chagas disease, having simultaneous information on the dynamics of the parasite, vector, the gut microbiome of the vector, and the blood meal source would facilitate identifying key biotic factors associated with the risk of T. cruzi transmission. In this study, we developed a RADseq-based analysis pipeline to study mixed-species DNA extracted from T. dimidiata abdomens. To evaluate the efficacy of the method across spatial scales, we used a nested spatial sampling design that spanned from individual villages within Guatemala to major biogeographic regions of Central America. Information from each biotic source was distinguished with bioinformatics tools and used to evaluate the prevalence of T. cruzi infection and predominant Discrete Typing Units (DTUs) in the region, the population genetic structure of T. dimidiata, gut microbial diversity, and the blood meal history. An average of 3.25 million reads per specimen were obtained, with approximately 1% assigned to the parasite, 20% to the vector, 11% to bacteria, and 4% to putative blood meals. Using a total of 6,405 T. cruzi SNPs, we detected nine infected vectors harboring two distinct DTUs: TcI and a second unidentified strain, possibly TcIV. Vector specimens were sufficiently variable for population genomic analyses, with a total of 25,710 T. dimidiata SNPs across all samples that were sufficient to detect geographic genetic structure at both local and regional scales. We observed a diverse microbiotic community, with significantly higher bacterial species richness in infected T. dimidiata abdomens than those that were not infected. Unifrac analysis suggests a common assemblage of bacteria associated with infection, which co-occurs with the typical gut microbial community derived from the local environment. We identified vertebrate blood meals from five T. dimidiata abdomens, including chicken, dog, duck and human; however, additional detection methods would be necessary to confidently identify blood meal sources from most specimens. Overall, our study shows this method is effective for simultaneously generating genetic data on vectors and their associated parasites, along with ecological information on feeding patterns and microbial interactions that may be followed up with complementary approaches such as PCR-based parasite detection, 18S eukaryotic and 16S bacterial barcoding.

Neighbour tolerance, not suppression, provides competitive advantage to non-native plants.

High competitive ability has often been invoked as a key determinant of invasion success and ecological impacts of non-native plants. Yet our understanding of the strategies that non-natives use to gain competitive dominance remains limited. Particularly, it remains unknown whether the two non-mutually exclusive competitive strategies, neighbour suppression and neighbour tolerance, are equally important for the competitive advantage of non-native plants. Here, we analyse data from 192 peer-reviewed studies on pairwise plant competition within a Bayesian multilevel meta-analytic framework and show that non-native plants outperform their native counterparts due to high tolerance of competition, as opposed to strong suppressive ability. Competitive tolerance ability of non-native plants was driven by neighbour's origin and was expressed in response to a heterospecific native but not heterospecific non-native neighbour. In contrast to natives, non-native species were not more suppressed by hetero- vs. conspecific neighbours, which was partially due to higher intensity of intraspecific competition among non-natives. Heterogeneity in the data was primarily associated with methodological differences among studies and not with phylogenetic relatedness among species. Altogether, our synthesis demonstrates that non-native plants are competitively distinct from native plants and challenges the common notion that neighbour suppression is the primary strategy for plant invasion success.

Behavioral Responses of Laricobius spp. and Hybrids (Coleoptera: Derodontidae) to Hemlock Woolly Adelgid and Adelgid Host Tree Odors in an Olfactometer.

The predatory species Laricobius nigrinus (Fender) and Laricobius osakensis (Shiyake and Montgomery) (Coleoptera: Derodontidae) have been released for biological control of hemlock woolly adelgid (Adelges tsugae; Hemiptera: Adelgidae) in eastern North America. L. osakensis is native to Japan, whereas L. nigrinus is endemic to the Pacific Northwest of the United States and Canada. After release, L. nigrinus was found to hybridize with the native eastern species, Laricobius rubidus (LeConte). The purpose of this study is to observe prey location behaviors of these three Laricobius species and L. nigrinus × L. rubidus (Ln × Lr) hybrids. Olfactometer bioassays were used to test response to host odors of adelgid-infested eastern hemlock, uninfested eastern hemlock, and uninfested eastern white pine. Predators reacted in the olfactometer more quickly when adelgid-infested foliage was included as a choice. L. nigrinus preferred infested eastern hemlock over uninfested eastern white pine, and L. rubidus preferred uninfested eastern white pine over uninfested eastern hemlock. Laricobius hybrids did not show a preference for foliage types known to be primary adelgid hosts (eastern hemlock and eastern white pine). Unequal preference by species of Laricobius for host trees of different adelgid prey could therefore be maintaining Laricobius species barriers despite hybridization. L. osakensis for this study were reared in the laboratory, whereas other species in this study were collected from the field, yet still were attracted to infested and uninfested eastern hemlock. This species also responded most quickly in the olfactometer, which is encouraging for successful biological control with this species.

Influence of experimental snow removal on root and canopy physiology of sugar maple trees in a northern hardwood forest.

Due to projected increases in winter air temperatures in the northeastern USA over the next 100 years, the snowpack is expected to decrease in depth and duration, thereby increasing soil exposure to freezing air temperatures. To evaluate the potential physiological responses of sugar maple (Acer saccharum Marsh.) to a reduced snowpack, we measured root injury, foliar cation and carbohydrate concentrations, woody shoot carbohydrate levels, and terminal woody shoot lengths of trees in a snow manipulation experiment in New Hampshire, USA. Snow was removed from treatment plots for the first 6 weeks of winter for two consecutive years, resulting in lower soil temperatures to a depth of 50 cm for both winters compared to reference plots with an undisturbed snowpack. Visibly uninjured roots from trees in the snow removal plots had significantly higher (but sub-lethal) levels of relative electrolyte leakage than trees in the reference plots. Foliar calcium: aluminum (Al) molar ratios were significantly lower, and Al concentrations were significantly higher, in trees from snow removal plots than trees from reference plots. Snow removal also reduced terminal shoot growth and increased foliar starch concentrations. Our results are consistent with previous research implicating soil freezing as a cause of soil acidification that leads to soil cation imbalances, but are the first to show that this translates into altered foliar cation pools, and changes in soluble and structural carbon pools in trees. Increased soil freezing due to a reduced snowpack could exacerbate soil cation imbalances already caused by acidic deposition, and have widespread implications for forest health in the northeastern USA.

Genetic variation of lodgepole pine, Pinus contorta var. latifolia, chemical and physical defenses that affect mountain pine beetle, Dendroctonus ponderosae, attack and tree mortality.

Plant secondary chemistry is determined by both genetic and environmental factors, and while large intraspecific variation in secondary chemistry has been reported frequently, the levels of genetic variation of many secondary metabolites in forest trees in the context of potential resistance against pests have been rarely investigated. We examined the effect of tree genotype and environment/site on the variation in defensive secondary chemistry of lodgepole pine, Pinus contorta var. latifolia, against the fungus, Grosmannia clavigera (formerly known as Ophiostoma clavigerum), associated with the mountain pine beetle, Dendroctonus ponderosae. Terpenoids were analyzed in phloem samples from 887, 20-yr-old trees originating from 45 half-sibling families planted at two sites. Samples were collected both pre- and post-inoculation with G. clavigera. Significant variation in constitutive and induced terpenoid compounds was attributed to differences among families. The response to the challenge inoculation with G. clavigera was strong for some individual compounds, but primarily for monoterpenoids. Environment (site) also had a significant effect on the accumulation of some compounds, whereas for others, no significant environmental effect occurred. However, for a few compounds significant family x environment interactions were found. These results suggest that P. c. latifolia secondary chemistry is under strong genetic control, but the effects depend on the individual compounds and whether or not they are expressed constitutively or following induction.

Prey suitability and phenology of Leucopis spp. (Diptera: Chamaemyiidae) associated with hemlock woolly adelgid (Hemiptera: Adelgidae) in the Pacific Northwest.

Leucopis spp. (Diptera: Chamaemyiidae) from the Pacific Northwest previously were identified as potential biological control agents for the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), in the eastern United States. We collected Leucopis spp. larvae from A. tsugae infested western hemlocks in Oregon and Washington and reared them on an unidentified Pineus spp., Pineus strobi (Hartig), Adelges cooleyi (Gillette), Adelges piceae (Ratzeburg), and A. tsugae in three no-choice tests. Leucopis spp. survival on A. tsugae was significantly higher than on A. piceae during the 2010 progrediens generation test and significantly higher than on P. strobi and A. cooleyi during the 2010 sistens generation test. However, across all three tests, some larvae completed development to adult on all four of the alternative adelgid species. Larvae that survived to the adult stage were identified as Leucopis argenticollis Zetterstedt and Leucopis piniperda Malloch. These results suggest that populations of L. argenticollis and L. piniperda in the Pacific Northwest may not be specific to A. tsugae. We also studied the phenology of Leucopis spp. on fourteen A. tsugae infested western hemlock trees in Oregon and Washington over a period of 14 mo. Leucopis spp. larvae were collected year-round, but highest densities coincided with the presence of progrediens and sistens eggs and adults of A. tsugae. There was a positive correlation between Leucopis spp. and A. tsugae abundance.

Orientation behavior of the predator Laricobius nigrinus (Coleoptera: Derodontidae) to hemlock woolly adelgid and host tree odors in a multi-chambered olfactometer.

We studied the adult ambulatory response of the predator, Laricobius nigrinus Fender (Coleoptera: Derodontidae), to odors from its prey, Adelges tsugae Annand, the hemlock woolly adelgid, and foliage of hemlock woolly adelgid, host hemlocks (Tsuga spp.), and other conifers. Both the predator and hemlock woolly adelgid are apparently native to western North America, but the predator is being released in the eastern United States, which has different hemlock species, for biological control of a lineage of hemlock woolly adelgid inadvertently introduced from Japan. L. nigrinus responded to odors from hemlock woolly adelgid host trees, but not to odors from hemlock woolly adelgid. L. nigrinus collected from hemlock woolly adelgid-infested western hemlock were more strongly attracted to odors from western hemlock [Tsuga heterophylla (Rafinesque) Sargent] than eastern hemlock [Tsuga canadensis (L.) Carrière] in most trials. Odors from western white pine (Pinus monticola Douglas ex D. Don) and white spruce [Picea glauca (Moench) Voss] were as attractive as western hemlock odors whereas odors from Douglas-fir [Pseudotsuga menziesii variety menziesii (Mirbel)] and ponderosa pine (Pinus ponderosa Douglas ex Lawson) were avoided. L. nigrinus reared on hemlock woolly adelgid-infested eastern hemlock in the laboratory were lethargic and were not attracted to either eastern or western hemlock odors. Predators collected in the field and tested monthly from December to March responded similarly each month, except February, when they flew rather than walked in the olfactometer, suggesting a period of dispersal or mate finding at that time of year. The implications of these results for programs to release L. nigrinus in the eastern United States for control of hemlock woolly adelgid are discussed.

Phylogeography of spruce beetles (Dendroctonus rufipennis Kirby) (Curculionidae: Scolytinae) in North America.

Tree-feeding insects that are widespread in north temperate regions are excellent models for studying how past glaciations have impacted differentiation and speciation. We used mitochondrial DNA (mtDNA) sequences and allele frequencies at nine microsatellite loci to examine genetic population structure across the current range of the spruce beetle (Dendroctonus rufipennis), an economically important insect in North America. Two major haplotype groups occur across northern North America, from Newfoundland to Alaska, on white spruce (Picea glauca), and a third distinctive haplotype group occurs throughout the Rocky Mountains on Engelmann spruce (Picea engelmannii). The two mtDNA lineages found in northern populations are 3-4% divergent from each other and from the lineages found in the Rocky Mountains. Analyses of microsatellite data also suggest the existence of major population groupings associated with different geographical regions. In the Pacific Northwest, concordant contact zones for genetically distinct populations of spruce beetles and their principal hosts appear to reflect recent secondary contact. Although we could detect no evidence of historical mtDNA gene flow between allopatric population groups, patterns of variation in the Pacific Northwest suggest recent hybridization and introgression. Together with the pollen record for spruce, they also suggest that beetles have spread from at least three glacial refugia. A minimum estimate of divergence time between the Rocky Mountain and northern populations was 1.7 Myr (million years), presumably reflecting the combined effects of isolation during multiple glacial cycles.