Comparing modern identification methods for wild bees: Metabarcoding and image-based morphological taxonomic assignment.

With the decline of bee populations worldwide, studies determining current wild bee distributions and diversity are increasingly important. Wild bee identification is often completed by experienced taxonomists or by genetic analysis. The current study was designed to compare two methods of identification including: (1) morphological identification by experienced taxonomists using images of field-collected wild bees and (2) genetic analysis of composite bee legs (multiple taxa) using metabarcoding. Bees were collected from conservation grasslands in eastern Iowa in summer 2019 and identified to the lowest taxonomic unit using both methods. Sanger sequencing of individual wild bee legs was used as a positive control for metabarcoding. Morphological identification of bees using images resulted in 36 unique taxa among 22 genera, and >80% of Bombus specimens were identified to species. Metabarcoding was limited to genus-level assignments among 18 genera but resolved some morphologically similar genera. Metabarcoding did not consistently detect all genera in the composite samples, including kleptoparasitic bees. Sanger sequencing showed similar presence or absence detection results as metabarcoding but provided species-level identifications for cryptic species (i.e., Lasioglossum). Genus-specific detections were more frequent with morphological identification than metabarcoding, but certain genera such as Ceratina and Halictus were identified equally well with metabarcoding and morphology. Genera with proportionately less tissue in a composite sample were less likely to be detected using metabarcoding. Image-based methods were limited by image quality and visible morphological features, while genetic methods were limited by databases, primers, and amplification at target loci. This study shows how an image-based identification method compares with genetic techniques, and how in combination, the methods provide valuable genus- and species-level information for wild bees while preserving tissue for other analyses. These methods could be improved and transferred to a field setting to advance our understanding of wild bee distributions and to expedite conservation research.

Conserved grasslands support similar pollinator diversity as pollinator-specific practice regardless of proximal cropland and pesticide exposure.

Pollinator diversity and abundance are declining globally. Cropland agriculture and the corresponding use of agricultural pesticides may contribute to these declines, while increased pollinator habitat (flowering plants) can help mitigate them. Here we tested whether the relative effect of wildflower plantings on pollinator diversity and counts were modified by proportion of nearby agricultural land cover and pesticide exposure in 24 conserved grasslands in Iowa, USA. Compared with general grassland conservation practices, wildflower plantings led to only a 5% increase in pollinator diversity and no change in counts regardless of the proportion of cropland agriculture within a 1 km radius. Pollinator diversity increased earlier in the growing season and with per cent flower cover. Unexpectedly, neither insecticide nor total pesticide concentrations on above-ground passive samplers were related to pollinator diversity. However, pollinator community composition was most strongly related to date of sampling, total pesticide concentration, and forb or flower cover. Our results indicate very little difference in pollinator diversity between grassland conservation practices with and without wildflower plantings. Given the relatively high economic costs of wildflower plantings, our research provides initial evidence that investment in general grassland conservation may efficiently conserve pollinator diversity in temperate regions of intensive cropland agriculture.

Wild Bee Exposure to Pesticides in Conservation Grasslands Increases along an Agricultural Gradient: A Tale of Two Sample Types.

Conservation efforts have been implemented in agroecosystems to enhance pollinator diversity by creating grassland habitat, but little is known about the exposure of bees to pesticides while foraging in these grassland fields. Pesticide exposure was assessed in 24 conservation grassland fields along an agricultural gradient at two time points (July and August) using silicone band passive samplers (nonlethal) and bee tissues (lethal). Overall, 46 pesticides were detected including 9 herbicides, 19 insecticides, 17 fungicides, and a plant growth regulator. For the bands, there were more frequent/higher concentrations of herbicides in July (maximum: 1600 ng/band in July; 570 ng/band in August), while insecticides and fungicides had more frequent/higher concentrations in August (maximum: 110 and 65 ng/band in July; 1500 and 1700 ng/band in August). Pesticide concentrations in bands increased 16% with every 10% increase in cultivated crops. The bee tissues showed no difference in detection frequency, and concentrations were similar among months; maximum concentrations of herbicides, insecticides, and fungicides in July and August were 17, 27, and 180 and 19, 120, and 170 ng/g, respectively. Pesticide residues in bands and bee tissues did not always show the same patterns; of the 20 compounds observed in both media, six (primarily fungicides) showed a detection-concentration relationship between the two media. Together, the band and bee residue data can provide a more complete understanding of pesticide exposure and accumulation in conserved grasslands.

Exploring the amphibian exposome in an agricultural landscape using telemetry and passive sampling.

This is the first field study of its kind to combine radio telemetry, passive samplers, and pesticide accumulation in tissues to characterize the amphibian exposome as it relates to pesticides. Understanding how habitat drives exposure in individuals (i.e., their exposome), and how that relates to individual health is critical to managing species in an agricultural landscape where pesticide exposure is likely. We followed 72 northern leopard frogs (Lithobates pipiens) in two agricultural wetlands for insight into where and when individuals are at high risk of pesticide exposure. Novel passive sampling devices (PSDs) were deployed at sites where telemetered frogs were located, then moved to subsequent locations as frogs were radio-tracked. Pesticide concentration in PSDs varied by habitat and was greatest in agricultural fields where frogs were rarely found. Pesticide concentrations in frogs were greatest in spring when frogs were occupying wetlands compared to late summer when frogs occupied terrestrial habitats. Our results indicate that habitat and time of year influence exposure and accumulation of pesticides in amphibians. Our study illustrates the feasibility of quantifying the amphibian exposome to interpret the role of habitat use in pesticide accumulation in frogs to better manage amphibians in agricultural landscapes.

Exposure of native bees foraging in an agricultural landscape to current-use pesticides.

The awareness of insects as pollinators and indicators of environmental quality has grown in recent years, partially in response to declines in honey bee (Apis mellifera) populations. While most pesticide research has focused on honey bees, there has been less work on native bee populations. To determine the exposure of native bees to pesticides, bees were collected from an existing research area in northeastern Colorado in both grasslands (2013-2014) and wheat fields (2014). Traps were deployed bi-monthly during the summer at each land cover type and all bees, regardless of species, were composited as whole samples and analyzed for 136 current-use pesticides and degradates. This reconnaissance approach provides a sampling of all species and represents overall pesticide exposure (internal and external). Nineteen pesticides and degradates were detected in 54 composite samples collected. Compounds detected in >2% of the samples included: insecticides thiamethoxam (46%), bifenthrin (28%), clothianidin (24%), chlorpyrifos (17%), imidacloprid (13%), fipronil desulfinyl (7%; degradate); fungicides azoxystrobin (17%), pyraclostrobin (11%), fluxapyroxad (9%), and propiconazole (9%); herbicides atrazine (19%) and metolachlor (9%). Concentrations ranged from 1 to 310 ng/g for individual pesticides. Pesticides were detected in samples collected from both grasslands and wheat fields; the location of the sample and the surrounding land cover at the 1000 m radius influenced the pesticides detected but because of a small number of temporally comparable samples, correlations between pesticide concentration and land cover were not significant. The results show native bees collected in an agricultural landscape are exposed to multiple pesticides, these results can direct future research on routes/timing of pesticide exposure and the design of future conservation efforts for pollinators.

Pesticide concentrations in frog tissue and wetland habitats in a landscape dominated by agriculture.

Habitat loss and exposure to pesticides are likely primary factors contributing to amphibian decline in agricultural landscapes. Conservation efforts have attempted to restore wetlands lost through landscape modifications to reduce contaminant loads in surface waters and providing quality habitat to wildlife. The benefits of this increased wetland area, perhaps especially for amphibians, may be negated if habitat quality is insufficient to support persistent populations. We examined the presence of pesticides and nutrients in water and sediment as indicators of habitat quality and assessed the bioaccumulation of pesticides in the tissue of two native amphibian species Pseudacris maculata (chorus frogs) and Lithobates pipiens (leopard frogs) at six wetlands (3 restored and 3 reference) in Iowa, USA. Restored wetlands are positioned on the landscape to receive subsurface tile drainage water while reference wetlands receive water from overland run-off and shallow groundwater sources. Concentrations of the pesticides frequently detected in water and sediment samples were not different between wetland types. The median concentration of atrazine in surface water was 0.2 µg/L. Reproductive abnormalities in leopard frogs have been observed in other studies at these concentrations. Nutrient concentrations were higher in the restored wetlands but lower than concentrations thought lethal to frogs. Complex mixtures of pesticides including up to 8 fungicides, some previously unreported in tissue, were detected with concentrations ranging from 0.08 to 1,500 µg/kg wet weight. No significant differences in pesticide concentrations were observed between species, although concentrations tended to be higher in leopard frogs compared to chorus frogs, possibly because of differences in life histories. Our results provide information on habitat quality in restored wetlands that will assist state and federal agencies, landowners, and resource managers in identifying and implementing conservation and management actions for these and similar wetlands in agriculturally dominated landscapes.