Use of a Monitoring System to Evaluate Pesticide Efficacy and Residual Activity Against Two Pine Root Weevils, \u3ci\u3eHylobius Pales\u3c/i\u3e and \u3ci\u3ePachylobius Picivorus\u3c/i\u3e (Coleoptera: Curculionidae), in Christmas Tree Farms

Hylobius pales, the pales weevil, and Pachylobius picivorus, the pitch-eating weevil, comprise part of a weevil complex which affects plantation pine production in the Lake States. Common control practices on Christmas tree farms include calendar applications of persistent insecticides. The resulting environmental risks could be minimized by repeating applications only when needed. A sampling method using ethanol-and turpentine-baited pitfall traps was used here to assess the efficacy and monitor persistence of chemical sprays. Trap catch in lindane-treated field plots and untreated controls were compared over two years. Unsprayed controls had significantly higher weevil populations than treated plots. Spray efficacy continued for three years following tion. Weevil population growth in relation to insecticide efficacy and degradation is discussed

Validation of Reference Genes for Quantitative PCR in the Forest Pest, \u3ci\u3eIps calligraphus\u3c/i\u3e

The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality. Biotic and abiotic disturbances weaken trees, creating breeding substrate that promotes rapid population growth. Management historically relied on silvicultural practices, but as forests become increasingly stressed, innovative management is needed. Manipulation of the cellular RNA interference (RNAi) pathway to induce gene silencing is an emerging means of insect suppression, and is effective for some bark beetles. Quantitative PCR (qPCR) is a powerful tool for analysis of gene expression, and is essential for examining RNAi. To compare gene expression among individuals, stably expressed reference genes must be validated for qPCR. We evaluated six candidate reference genes (18s, 16s, 28s, ef1a, cad, coi) for stability under biotic (beetle sex, developmental stage, and host plant), and abiotic (temperature, photoperiod, and dsRNA exposure) conditions. We used the comprehensive RefFinder tool to compare stability rankings across four algorithms. These algorithms identified 18s, 16s, and 28s as the most stably expressed. Overall, 16s and 28s were selected as reference genes due to their stability and moderate expression levels, and can be used for I. calligraphus gene expression studies using qPCR, including those evaluating RNAi

Emerald Ash Borer Specific Gene Silencing Has No Effect on Non-Target Organisms

The sequence complementarity of the RNA interference (RNAi) pathway allows for targeted suppression of genes essential for insect survival, and enables development of pest management strategies specific to a given species while reducing the likelihood of adversely impacting non-target organisms (NTOs). The feasibility of manipulating the RNAi pathway to cause mortality in the highly invasive emerald ash borer (EAB) has been demonstrated. Here the spectrum of activity of three double stranded RNAs (dsRNAs) targeting the genes hsp, shi, and sn-rnp in EAB was evaluated in model insects representing five functional guilds including herbivore, predator, detritivore, pollinator, parasitoid; the last represented by the classical biological control agents currently deployed for EAB management in North America. All NTOs were exposed to EAB-specific dsRNAs in diet bioassays that measured potential lethal effects. Gene expression and in silico analysis were also assessed on NTOs for which gene sequences were publicly available. Bioassays demonstrated no lethal effects on our model insects, suggesting a narrow spectrum of activity for the three EAB-specific dsRNAs evaluated. The gene expression and in silico analyses suggest potential sublethal effects on our model pollinator; however we found no effects on insect survival. Overall, our results suggest no adverse effects of the RNAi strategy targeting EAB genes on the survival of the selected non-target organisms we evaluated. The results from this study provide guidance for future RNAi risk analyses that will allow this technology to move forward to a deployment stage

Benthic Collector and Grazer Communities Are Threatened by Hemlock Woolly Adelgid-Induced Eastern Hemlock Loss

Eastern hemlock (Tsuga canadensis (L.) is a foundation species in eastern North America where it is under threat from the highly invasive, exotic hemlock woolly adelgid (Adelges tsugae). Eastern hemlock is especially important in riparian areas of Central and Southern Appalachia, so we compared the spatial and temporal composition of benthic collector-gatherers, collector-filterers, and grazers in headwater streams with hemlock-dominated riparian vegetation to those with deciduous tree-dominated riparian vegetation to evaluate the extent to which adelgid-induced hemlock loss could influence composition and abundance of these two functional feeding groups. We found differences in benthic invertebrate abundance and family-level diversity based on riparian vegetation and sampling approach, and, often, riparian vegetation significantly interacted with location or season. Collector-gatherers and grazers were more abundant in eastern hemlock streams in the summer, when hemlock litter is readily available and deciduous litter is relatively sparse. Riparian eastern hemlock appears to exert considerable influence on benthic invertebrate functional feeding group composition in headwater stream communities, as expected with a foundation species. With the loss of eastern hemlock due to adelgid-induced mortality, we should expect to see alterations in spatial and temporal patterns of benthic invertebrate abundance and diversity, with potential consequences to both benthic and terrestrial ecosystem function

Healthy Trees – Healthy People: A Model for Engaging Citizen Scientists in Exotic Pest Detection in Urban Parks

Healthy Trees – Healthy People (HT-HP) is an outreach and education program created to enlist and train participants in exotic pest detection while providing them with structured opportunities to connect with urban nature and increase their physical and emotional health. HT- HP creates infrastructure to increase engagement by the urban populace in the urban tree canopy. The program solicits participants to engage in an 8-week intervention designed to expand capacity to detect non-native insect pests and pathogens, while increasing physical activity, raising awareness of healthy lifestyle choices, and improving the health of participants. Program participants were trained in tree and tree pest identification to provide ‘eyes on the ground’ that can contribute to exotic pest detection, reducing the risk of pest establishment, and increasing the sustainability of urban tree canopies. Participants in this pilot program reported significant improvements in their knowledge of tree identification and tree care, an awareness of, and ability to identify selected exotic invasive tree pests, and improved mindfulness. Healthy Trees – Healthy People is nimble, easily adaptable, and serves as a unique model that could include tailored tree identification and pest detection. The program can engage citizen scientists in exotic pest detection while increasing usage of urban parks

Oral Ingestion of Bacterially Expressed dsRNA Can Silence Genes and Cause Mortality in a Highly Invasive, Tree-Killing Pest, the Emerald Ash Borer

RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since its discovery in 2002, prompting the need for innovative management strategies. In our study, bacterial expression and synthesis of dsRNA were performed with E. coli strain HT115 using the L4440 expression vector. EAB-specific dsRNAs (shi and hsp) over-expressed in E. coli were toxic to neonate EAB after oral administration, successfully triggering gene silencing and subsequent mortality; however, a non-specific dsRNA control was not included. Our results suggest that ingestion of transformed E. coli expressing dsRNAs can induce an RNAi response in EAB. To our knowledge, this is the first example of an effective RNAi response induced by feeding dsRNA-expressing bacteria in a forest pest

Coleopteran Communities Associated with Forests Invaded by Emerald Ash Borer

Extensive ash mortality caused by the non-native emerald ash borer alters canopy structure and creates inputs of coarse woody debris as dead and dying ash fall to the forest floor; this affects habitat heterogeneity; resource availability; and exposure to predation and parasitism. As EAB-induced (emerald ash borer-induced) disturbance progresses the native arthropod associates of these forests may be irreversibly altered through loss of habitat; changing abiotic conditions and altered trophic interactions. We documented coleopteran communities associated with EAB-disturbed forests in a one-year study to evaluate the nature of these changes. Arthropods were collected via ethanol-baited traps on five sites with varying levels of EAB-induced ash mortality from May to September; captured beetles were identified to the family level and assigned to feeding guilds (herbivore; fungivore; xylophage; saprophage; predator; or parasite). Over 11,700 Coleoptera were identified in 57 families. In spite of their abundance; herbivores comprised a relatively small portion of coleopteran family richness (8 of 57 families). Conversely, coleopteran fungivore richness was high (23 families), and fungivore abundance was low. Herbivores and fungivores were more abundant at sites where ash decline was most evident. The predatory Trogossitidae and Cleridae were positively correlated with ash decline, suggesting a positive numerical response to the increased prey base associated with EAB invasion. Ash forests are changing, and a deeper understanding of arthropod community responses will facilitate restoration

Biomechanical Properties of Hemlocks: A Novel Approach to Evaluating Physical Barriers of the Plant-Insect Interface and Resistance to a Phloem-Feeding Herbivore

Micromechanical properties that help mediate herbivore access may be particularly important when considering herbivorous insects that feed with piercing-sucking stylets. We used microindentation to quantify the micromechanical properties of hemlock, Tsuga spp., to quantify the hardness of the feeding site of the invasive hemlock woolly adelgid, Adelges tsugae. We measured hardness of the hemlock leaf cushion, the stylet insertion point of the adelgid, across four seasons in a 1 y period for four hemlock species growing in a common garden, including eastern, western, mountain, and northern Japanese hemlocks. Leaf cushion hardness was highest in the fall and winter and lowest in summer for all species. Northern Japanese hemlock had relatively greater hardness than the remaining species. Our data contributes an additional perspective to the existing framework within which greater susceptibility and subsequent mortality of eastern hemlocks is observed. The potential application of microindentation to understanding the nature and relevance of plant mechanical defenses in plant–herbivore interactions is also demonstrated and highlighted

RNA Interference and Validation of Reference Genes for Gene Expression Analyses Using qPCR in Southern Pine Beetle, \u3cem\u3eDendroctonus frontalis\u3c/em\u3e

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion