Pests and pathogens are a continuous threat to the health of Western honey bee Apis mellifera L. Monitoring honey bee colonies for arthropod pests, disease-causing bacteria and fungi, and early detection of new invasions is essential to maintain the pollination services provided by honey bees. We investigated the feasibility of using eDNA metabarcoding to detect pests and pathogens in bee hives and across their foraging environment. We sampled 13 sources for eDNA within and outside hives from our test apiary to determine where the most informative eDNA could be obtained, with most sources sampled thrice. This resulted in 61 samples, 20 negative controls, and three positive controls. Furthermore, we compared two eDNA collection techniques-wiping surfaces with moistened forensic swabs and using a spray/wash technique that aggregated surface eDNA into a container, before collecting the eDNA on a filter. We used DNA metabarcoding with universal primer sets to target arthropod, bacterial, and fungal communities. Our results showed that most sources yielded sufficient eDNA and that results of the swab and spray/wash methods were similar when they could be applied to the same surface. We detected DNA from honey bee bacterial symbionts, mycotoxin-producing fungi and Brachymyrmex sp. rover ants. Common pests and pathogens of concern to honey bees (i.e., [small hive beetle (Aethina tumida), Varroa destructor, and Melissococcus plutonius]) were detected. This matched our visual observations of clinical signs of these pests and pathogens in the hives we tested. DNA from some species was source specific, which has implications for using eDNA as a monitoring tool. Collectively, our data show that eDNA metabarcoding can accurately detect DNA from arthropods and microbes honey bees contact and can be used as a comprehensive molecular predictor tool for colony health surveys.

Local-scale studies have shown that an overabundance of Cervidae species (deer, elk, moose) impacts forest bird communities. Through meta-analysis, we provide a generalized estimate of the overall direction and magnitude of the indirect effects overabundant cervids have on avian species. We conducted 2 distinct meta-analyses that synthesized data on 130 bird species collected from 17 publications. These analyses compared bird species’ population abundance and/or species richness at sites with overabundant cervids to sites with lower cervid abundance or without cervids. We evaluated whether the impacts of overabundant cervids are generally in the same direction (positive, negative) across avian species and locations and if effects vary in magnitude according to avian nesting location and foraging habitat. We found that where cervids were overabundant, there was a significant decrease in mean bird population abundance and species richness. Species that nest in trees, shrubs, and on the ground showed the largest decreases in abundance, as did species whose primary habitat is forest and open woodland and species that are primarily insectivores or omnivores. We did not find significant decreases in abundance for avian species that nest in cavities, whose primary habitat is grassland or scrub, nor for species that mainly eat seeds. Our results indicate that overabundant cervids, likely through their direct effects on vegetation and indirect effects on insects and forest birds, negatively impact individual bird populations and decrease overall avian species richness.

Abstract Environmental DNA (eDNA) analyses have become invaluable for detecting and monitoring aquatic and terrestrial species and assessing site biodiversity within aquatic environments or soil. Recent studies have extended these techniques by using eDNA to identify the presence of aboveground terrestrial arthropods directly from vegetative surfaces. However, while the dynamics of eDNA state, transport, and fate (its “ecology”) have been explored within aquatic environments and soil, they have yet to be explored within aboveground terrestrial systems. Here, we explore the ecology of terrestrial eDNA deposited by fluid‐feeding arthropods on leaf surfaces. We carried out a series of experiments to evaluate the optimal filter pore size for intracellular eDNA collection, how eDNA is affected by rain events, and its degradation rate under different solar radiation conditions. We found that the captured concentration of intracellular eDNA was not significantly affected by an increase in filter pore size, suggesting a wide range of viable pore size options exist for targeting intracellular eDNA. We also found extracellular eDNA from fluid excrement degrades more rapidly than intracellular when exposed to solar radiation, indicating the latter is a more viable target for collection. Finally, we identified that rainfall or mist will remove most terrestrial eDNA present on vegetation surfaces. We provide researchers and environmental managers key insights into successfully designing and carrying out aboveground terrestrial arthropod eDNA surveys that maximize detection probability.

The use of environmental DNA (eDNA) surveys to monitor terrestrial species has been relatively limited, with successful implementations still confined to sampling DNA from natural or artificial water bodies and soil. Sampling water for eDNA depends on proximity to or availability of water, whereas eDNA from soil is limited in its spatial scale due to the large quantities necessary for processing and difficulty in doing so. These challenges limit the widespread use of eDNA in several systems, such as surveying forests for invasive insects. We developed two new eDNA aggregation approaches that overcome the challenges of above-ground terrestrial sampling and eliminate the dependency on creating or utilizing pre-existing water bodies to conduct eDNA sampling. The first, "spray aggregation," uses spray action to remove eDNA from surface substrates and was developed for shrubs and other understorey vegetation, while the second, "tree rolling," uses physical transfer via a roller to remove eDNA from the surface of tree trunks and large branches. We tested these approaches by surveying for spotted lanternfly, Lycorma delicatula, a recent invasive pest of northeastern USA that is considered a significant ecological and economic threat to forests and agriculture. We found that our terrestrial eDNA surveys matched visual surveys, but also detected L. delicatula presence ahead of visual surveys, indicating increased sensitivity of terrestrial eDNA surveys over currently used methodology. The terrestrial eDNA approaches we describe can be adapted for use in surveying a variety of forest insects and represent a novel strategy for surveying terrestrial biodiversity.

Environmental DNA (eDNA) analysis has become a valuable tool for detecting aquatic and terrestrial species for monitoring efforts and site biodiversity assessments. However, if aboveground terrestrial eDNA surveys are to be widely adopted, it is necessary to first understand how terrestrial conditions affect the state, transport, and ultimate fate (or ‘ecology’) of terrestrially deposited eDNA. Many of the processes that affect the state, transport, and fate of eDNA in aquatic environments may not be applicable in aboveground systems, warranting an exploration of the terrestrial processes that likely do affect eDNA. Here we explore the ecology of aboveground terrestrial eDNA through a series of experiments evaluating the optimal filter pore size for intracellular eDNA collection, how eDNA is affected by rain events, and its degradation rate under different solar radiation conditions. We found that the captured concentration of intracellular eDNA was not significantly affected by an increase in filter pore size, suggesting there is a wide range of viable pore size options for targeting intracellular eDNA. We also found extracellular eDNA degrades more rapidly than intracellular forms when exposed to solar radiation, indicating the latter is a more viable target for collection. Finally, we identified that rainfall or mist will remove most terrestrial eDNA present on vegetation substrate. This study provides researchers and managers key insights into successfully designing and carrying out terrestrial eDNA surveys that maximize detection probability and minimize false positive results.

The number of exotic species invasions has increased over recent decades, as have the ecological harm and economic burdens they impose. Rapid‐response eradication of nascent exotic populations is a viable approach to minimizing damage, but implementation is limited by the difficulty of detecting such species during the early stages of infestation due to their small numbers. The use of environmental DNA (eDNA) has helped address this issue in aquatic ecosystems, but to the best of our knowledge has not been trialed for surveillance of exotic species in terrestrial systems. Using a high‐resolution, real‐time (quantitative) polymerase chain reaction assay, we developed a highly efficient protocol to survey agricultural fields for the invasive non‐native brown marmorated stink bug (BMSB; Halyomorpha halys). We compared results using eDNA to those for conventional monitoring traps and documented substantially higher sensitivity and detection effectiveness. Our methodology is transferable to situations in which the DNA of terrestrial target species can be accumulated into a single substrate, suggesting that eDNA‐based approaches could transform our ability to detect exotic insects in non‐aquatic settings.

There is increasing evidence that exotic populations may rapidly differentiate from those in their native range and that differences also arise among populations within the exotic range. Using morphological and DNA-based analyses, we document the extent of trait divergence among native North American and exotic Hawaiian populations of northern cardinal (Cardinalis cardinalis). Furthermore, using a combination of historical records and DNA-based analyses, we evaluate the role of founder effects in producing observed trait differences. We measured and compared key morphological traits across northern cardinal populations in the native and exotic ranges to assess whether trait divergence across the Hawaiian Islands, where this species was introduced between 1929 and 1931, reflected observed variation across native phylogeographic clades in its native North America. We used and added to prior phylogenetic analyses based on a mitochondrial locus to identify the most likely native source clade(s) for the Hawaiian cardinal populations. We then used Approximate Bayesian Computation (ABC) to evaluate the role of founder effects in producing the observed differences in body size and bill morphology across native and exotic populations. We found cardinal populations on the Hawaiian Islands had morphological traits that diverged substantially across islands and overlapped the trait space of all measured native North American clades. The phylogeographic analysis identified the eastern North American clade (C.cardinalis cardinalis) as the most likely and sole native source for all the Hawaiian cardinal populations. The ABC analyses supported written accounts of the cardinal's introduction that indicate the original 300 cardinals shipped to Hawaii were simultaneously and evenly released across Hawaii, Kauai, and Oahu. Populations on each island likely experienced bottlenecks followed by expansion, with cardinals from the island of Hawaii eventually colonizing Maui unaided. Overall, our results suggest that founder effects had limited impact on morphological trait divergence of exotic cardinal populations in the Hawaiian archipelago, which instead reflect postintroduction events.

Human mediated transportation into novel habitats is a prerequisite for the establishment of non-native species that become invasive, so knowledge of common sources may allow prevention. The brown marmorated stink bug (BMSB, Halyomorpha halys) is an East Asian species now established across North America and Europe, that in the Eastern United States of America (US) and Italy is causing significant economic losses to agriculture. After US populations were shown to originate from Northern China, others have tried to source BMSB populations now in Canada, Switzerland, Italy, France, Greece, and Hungary. Due to selection of different molecular markers, however, integrating all the datasets to obtain a broader picture of BMSB's expansion has been difficult. To address this limitation we focused on a single locus, the barcode region in the cytochrome oxidase I mitochondrial gene, and analyzed representative BMSB samples from across its current global range using an Approximate Bayesian Computation approach. We found that China is the likely source of most non-native populations, with at least four separate introductions in North America and three in Europe. Additionally, we found evidence of one bridgehead event: a likely Eastern US source for the central Italy populations that interestingly share enhanced pest status.

Invasive insect pests cost the agricultural industry billions of dollars annually in crop losses. Timely detection of pests is critical for management efficiency. Innovative pest detection strategies, such as environmental DNA (eDNA) techniques, combined with efficient predators, maximize sampling resolution across space and time and may improve surveillance. We tested the hypothesis that temperate insectivorous bats can be important sentinels of agricultural insect pest surveillance. Specifically, we used a new high-sensitivity molecular assay for invasive brown marmorated stink bugs (Halyomorpha halys) to examine the extent to which big brown bats (Eptesicus fuscus) detect agricultural pests in the landscape. We documented consistent seasonal predation of stink bugs by big brown bats. Importantly, bats detected brown marmorated stink bugs 3-4 weeks earlier than the current standard monitoring tool, blacklight traps, across all sites. We highlight here the previously unrecognized potential ecosystem service of bats as agents of pest surveillance (or chirosurveillance). Additional studies examining interactions between other bat and insect pest species, coupled with comparisons of detectability among various conventional monitoring methods, are needed to verify the patterns extracted from this study. Ultimately, robust economic analyses will be needed to assess the cost-effectiveness of chirosurveillance as a standard strategy for integrated pest management.

BACKGROUNDEarly detection before establishment and identification of key predators are time-honored strategies towards effective eradication or control of invasive species. The brown marmorated stink bug (BMSB), Halyomorpha halys, is a recent exotic pest of several important crops in North America and Europe. Resulting widespread applications of insecticides have countered years of careful integrated pest management and are leading to the resurgence of other agricultural pests. Environmental DNA (eDNA) has been used effectively to detect aquatic invasives.RESULTSWe developed a real-time PCR (qPCR) assay for BMSB in a conserved region of the ribosomal DNA interspacer 1 (ITS1). We validated this assay on worldwide populations of BMSB and tested its specificity and sensitivity against other US Pentatomidae species and on guano of big brown bat, Eptesicus fuscus, which we confirmed is a BMSB predator in New Jersey. We also detected BMSB DNA after rapid (and inexpensive) HotSHOT DNA extractions of soiled paper from cages briefly holding BMSB, as well as from discarded exuviae.CONCLUSIONGiven the high sensitivity of our assay to BMSB environmental DNA (eDNA) in terrestrial samples, this tool should become a cost-effective approach for using eDNA to detect terrestrial invasive species and their key predators. © 2016 Society of Chemical Industry.