Addressing Detection Uncertainty in Bombus affinis (Hymenoptera: Apidae) Surveys Can Improve Inferences Made From Monitoring.

The U.S. Fish and Wildlife Service developed national guidelines to track species recovery of the endangered rusty patched bumble bee [Bombus affinis Cresson (Hymenoptera: Apidae)] and to investigate changes in species occupancy across space and time. As with other native bee monitoring efforts, managers have specifically acknowledged the need to address species detection uncertainty and determine the sampling effort required to infer species absence within sites. We used single-season, single-species occupancy models fit to field data collected in four states to estimate imperfect detection of B. affinis and to determine the survey effort required to achieve high confidence of species detection. Our analysis revealed a precipitous, seasonal, decline in B. affinis detection probability throughout the July through September sampling window in 2021. We estimated that six, 30-min surveys conducted in early July are required to achieve a 95% cumulative detection probability, whereas >10 surveys would be required in early August to achieve the same level of confidence. Our analysis also showed B. affinis was less likely to be detected during hot and humid days and at patches of reduced habitat quality. Bombus affinis was frequently observed on Monarda fistulosa (Lamiales: Lamiaceae), followed by [Pycnanthemum virginianum Rob. and Fernald (Lamiales: Lamiaceae)], Eutrochium maculatum Lamont (Asterales: Asteraceae), and Veronicastrum virginicum Farw. (Lamiales: Plantaginaceae). Although our research is focused on B. affinis, it is relevant for monitoring other bumble bees of conservation concern, such as B. occidentalis Greene (Hymenoptera: Apidae) and B. terricola Kirby (Hymenoptera: Apidae) for which monitoring efforts have been recently initiated and occupancy is a variable of conservation interest.

Patch utilization and flower visitations by wild bees in a honey bee-dominated, grassland landscape.

Understanding habitat needs and patch utilization of wild and managed bees has been identified as a national research priority in the United States. We used occupancy models to investigate patterns of bee use across 1030 transects spanning a gradient of floral resource abundance and richness and distance from apiaries in the Prairie Pothole Region (PPR) of the United States. Estimates of transect use by honey bees were nearly 1.0 during our 3.5-month sampling period, suggesting honey bees were nearly ubiquitous across transects. Wild bees more frequently used transects with higher flower richness and more abundant flowers; however, the effect size of the native flower abundance covariate ( ß ^ native  = 3.90 ± 0.65 [1SE]) was four times greater than the non-native flower covariate ( ß ^ n o n - n a t i v e  = 0.99 ± 0.17). We found some evidence that wild bee use was lower at transects near commercial apiaries, but the effect size was imprecise ( ß ^ distance  = 1.4 ± 0.81). Honey bees were more frequently detected during sampling events with more non-native flowers and higher species richness but showed an uncertain relationship with native flower abundance. Of the 4039 honey bee and flower interactions, 85% occurred on non-native flowers, while only 43% of the 738 wild bee observations occurred on non-native flowers. Our study suggests wild bees and honey bees routinely use the same resource patches in the PPR but often visit different flowering plants. The greatest potential for resource overlap between honey bees and wild bees appears to be for non-native flowers in the PPR. Our results are valuable to natural resource managers tasked with supporting habitat for managed and wild pollinators in agroecosystems.

Honey bee (Apis mellifera) colonies benefit from grassland/ pasture while bumble bee (Bombus impatiens) colonies in the same landscapes benefit from non-corn/soybean cropland.

Agriculturally important commercially managed pollinators including honey bees (Apis mellifera L., 1758) and bumble bees (Bombus impatiens Cresson, 1863) rely on the surrounding landscape to fulfill their dietary needs. A previous study in Europe demonstrated that managed honey bee foragers and unmanaged native bumble bee foragers are associated with different land uses. However, it is unclear how response to land use compares between managed honey bees and a managed native bumble bee species in the United States, where honey bees are an imported species. Furthermore, to our knowledge, no such direct comparisons of bee responses to land use have been made at the colony level. To better understand how two different social bees respond to variation in land use, we monitored the weights of A. mellifera and B. impatiens colonies placed in 12 apiaries across a range of land use in Michigan, United States in 2017. Bombus impatiens colonies gained more weight and produced more drones when surrounded by diverse agricultural land (i.e., non-corn/soybean cropland such as tree fruits and grapes), while honey bee colonies gained more weight when surrounded by more grassland/pasture land. These findings add to our understanding of how different bee species respond to agricultural landscapes, highlighting the need for further species-specific land use studies to inform tailored land management.

Land conversion and pesticide use degrade forage areas for honey bees in America's beekeeping epicenter.

A diverse range of threats have been associated with managed-bee declines globally. Recent increases of two known threats, land-use change and pesticide use, have resulted from agricultural expansion and intensification notably in the top honey-producing state in the United States: North Dakota. This study investigated the dual threat from land conversion and pesticide use surrounding ~14,000 registered apiaries in North Dakota from 2001 to 2014. We estimated the annual total insecticide use (kg) on major crops within 1.6 km of apiary sites. Of the eight insecticides quantified, six showed significant increasing trends over the time period. Specifically, applications of the newly established neonicotinoids Chlothianidin, Imidacloprid and Thiamethoxam, increased annually by 1329 kg, 686 kg, 795 kg, respectively. Also, the use of Chlorpyrifos, which was well-established in the state by 2001 and is highly toxic to honey bees, increased by ~8,800 kg annually from 6,500 kg in 2001 to 115,000 kg in 2014 on corn, soybeans and wheat. We further evaluated the relative quality changes of natural/semi-natural land covers surrounding apiaries in 2006, 2010 and 2014, a period of significant increases in cropland area. In areas surrounding apiaries, we observed changes in multiple indices of forage quality that reflect the deteriorating landscape surrounding registered apiary sites due to land-use change and pesticide-use increases. Overall, our results suggest that the application of foliar-applied insecticides, including pyrethroids and one organophosphate, increased surrounding apiaries when the use of neonicotinoid seed treatments surged and the area for producing corn and soybeans expanded. Spatially, these threats were most pronounced in southeastern North Dakota, a region hosting a high density of apiary sites that has recently experienced corn and soybean expansion. Our results highlight the value of natural and semi-natural land covers as sources of pollinator forage and refugia for bees against pesticide exposure. Our study provides insights for targeting conservation efforts to improve forage quality benefiting managed pollinators.

An updated genetic marker for detection of Lake Sinai Virus and metagenetic applications.

BACKGROUND: Lake Sinai Viruses (LSV) are common RNA viruses of honey bees (Apis mellifera) that frequently reach high abundance but are not linked to overt disease. LSVs are genetically heterogeneous and collectively widespread, but despite frequent detection in surveys, the ecological and geographic factors structuring their distribution in A. mellifera are not understood. Even less is known about their distribution in other species. Better understanding of LSV prevalence and ecology have been hampered by high sequence diversity within the LSV clade. METHODS: Here we report a new polymerase chain reaction (PCR) assay that is compatible with currently known lineages with minimal primer degeneracy, producing an expected 365 bp amplicon suitable for end-point PCR and metagenetic sequencing. Using the Illumina MiSeq platform, we performed pilot metagenetic assessments of three sample sets, each representing a distinct variable that might structure LSV diversity (geography, tissue, and species). RESULTS: The first sample set in our pilot assessment compared cDNA pools from managed A. mellifera hives in California (n = 8) and Maryland (n = 6) that had previously been evaluated for LSV2, confirming that the primers co-amplify divergent lineages in real-world samples. The second sample set included cDNA pools derived from different tissues (thorax vs. abdomen, n = 24 paired samples), collected from managed A. mellifera hives in North Dakota. End-point detection of LSV frequently differed between the two tissue types; LSV metagenetic composition was similar in one pair of sequenced samples but divergent in a second pair. Overall, LSV1 and intermediate lineages were common in these samples whereas variants clustering with LSV2 were rare. The third sample set included cDNA from individual pollinator specimens collected from diverse landscapes in the vicinity of Lincoln, Nebraska. We detected LSV in the bee Halictus ligatus (four of 63 specimens tested, 6.3%) at a similar rate as A. mellifera (nine of 115 specimens, 7.8%), but only one H. ligatus sequencing library yielded sufficient data for compositional analysis. Sequenced samples often contained multiple divergent LSV lineages, including individual specimens. While these studies were exploratory rather than statistically powerful tests of hypotheses, they illustrate the utility of high-throughput sequencing for understanding LSV transmission within and among species.

Do the Quality and Quantity of Honey Bee-Collected Pollen Vary Across an Agricultural Land-Use Gradient?

Pollen is the source of protein for most bee species, yet the quality and quantity of pollen is variable across landscapes and growing seasons. Understanding the role of landscapes in providing nutritious forage to bees is important for pollinator health, particularly in areas undergoing significant land-use change such as in the Northern Great Plains (NGP) region of the United States where grasslands are being converted to row crops. We investigated how the quality and quantity of pollen collected by honey bees (Apis mellifera L. [Hymenoptera: Apidae]) changed with land use and across the growing season by sampling bee-collected pollen from apiaries in North Dakota, South Dakota, and Minnesota, USA, throughout the flowering season in 2015-2016. We quantified protein content and quantity of pollen to investigate how they varied temporally and across a land-use gradient of grasslands to row crops. Neither pollen weight nor crude protein content varied linearly across the land-use gradient; however, there were significant interactions between land use and sampling date across the season, particularly in grasslands. Generally, pollen protein peaked mid-July while pollen weight had two maxima in late-June and late-August. Results suggest that while land use itself may not correlate with the quality or quantity of pollen resources collected by honey bees among our study apiaries, the nutritional landscape of the NGP is seasonally dynamic, especially in certain land covers, and may impose seasonal resource limitations for both managed and native bee species. Furthermore, results indicate periods of qualitative and quantitative pollen dearth may not coincide.

Nutritional status of honey bee (Apis mellifera L.) workers across an agricultural land-use gradient.

Land use, habitat, and forage quality have emerged as critical factors influencing the health, productivity, and survival of honey bee colonies. However, characterization of the mechanistic relationship between differential land-use conditions and ultimate outcomes for honey bee colonies has been elusive. We assessed the physiological health of individual worker honey bees in colonies stationed across a gradient of agricultural land use to ask whether indicators of nutritional physiology including glycogen, total sugar, lipids, and protein were associated with land-use conditions over the growing season and colony population size the subsequent spring during almond pollination. Across the observed land-use gradient, we found that September lipid levels related to growing-season land use, with honey bees from apiaries surrounded by more favorable land covers such as grassland, pasture, conservation land, and fallow fields having greater lipid reserves. Further, we observed a significant relationship between total protein during September and population size of colonies during almond pollination the following February. We demonstrate and discuss the utility of quantifying nutritional biomarkers to infer land-use quality and predict colony population size.

Past role and future outlook of the Conservation Reserve Program for supporting honey bees in the Great Plains.

Human dependence on insect pollinators continues to grow even as pollinators face global declines. The Northern Great Plains (NGP), a region often referred to as America's last honey bee (Apis mellifera) refuge, has undergone rapid land-cover change due to cropland expansion and weakened land conservation programs. We conducted a trend analysis and estimated conversion rates of Conservation Reserve Program (CRP) enrollments around bee apiaries from 2006 to 2016 and developed models to identify areas of habitat loss. Our analysis revealed that NGP apiaries lost over 53% of lands enrolled in the CRP, and the rate of loss was highest in areas of high apiary density. We estimated over 163,000 ha of CRP lands in 2006 within 1.6 km of apiaries was converted to row crops by 2012. We also evaluated how alternative scenarios of future CRP acreage caps may affect habitat suitability for supporting honey bee colonies. Our scenario revealed that a further reduction in CRP lands to 7.7 million ha nationally would reduce the number of apiaries in the NGP that meet defined forage criteria by 28% on average. Alternatively, increasing the national cap to 15 million ha would increase the number of NGP apiaries that meet defined forage criteria by 155%. Our scenarios also show that strategic placement of CRP lands near existing apiaries increased the number of apiaries that meet forage criteria by 182%. Our research will be useful for informing the potential consequences of future US farm bill policy and land management in the epicenter of the US beekeeping industry.

Land-use change reduces habitat suitability for supporting managed honey bee colonies in the Northern Great Plains.

Human reliance on insect pollination services continues to increase even as pollinator populations exhibit global declines. Increased commodity crop prices and federal subsidies for biofuel crops, such as corn and soybeans, have contributed to rapid land-use change in the US Northern Great Plains (NGP), changes that may jeopardize habitat for honey bees in a part of the country that supports >40% of the US colony stock. We investigated changes in biofuel crop production and grassland land covers surrounding ∼18,000 registered commercial apiaries in North and South Dakota from 2006 to 2014. We then developed habitat selection models to identify remotely sensed land-cover and land-use features that influence apiary site selection by Dakota beekeepers. Our study demonstrates a continual increase in biofuel crops, totaling 1.2 Mha, around registered apiary locations in North and South Dakota. Such crops were avoided by commercial beekeepers when selecting apiary sites in this region. Furthermore, our analysis reveals how grasslands that beekeepers target when selecting commercial apiary locations are becoming less common in eastern North and South Dakota, changes that may have lasting impact on pollinator conservation efforts. Our study highlights how land-use change in the NGP is altering the landscape in ways that are seemingly less conducive to beekeeping. Our models can be used to guide future conservation efforts highlighted in the US national pollinator health strategy by identifying areas that support high densities of commercial apiaries and that have exhibited significant land-use changes.

Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci.

Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota apiaries across multiple years. We used the Illumina MiSeq platform to generate sequence scaffolds from non-overlapping 300-bp paired-end sequencing reads of the ribosomal internal transcribed spacers (ITS). Full-length sequence scaffolds represented ~530 bp of ITS sequence after adapter trimming, drawn from the 5' of ITS1 and the 3' of ITS2, while skipping the uninformative 5.8S region. Operational taxonomic units (OTUs) were picked from scaffolds clustered at 97% identity, searched by BLAST against the nt database, and given taxonomic assignments using the paired-read lowest common ancestor approach. Taxonomic assignments and quantitative patterns were consistent with known plant distributions, phenology, and observational reports of pollen foraging, but revealed an unexpected contribution from non-crop graminoids and wetland plants. The mean number of plant species assignments per sample was 23.0 (+/- 5.5) and the mean species diversity (effective number of equally abundant species) was 3.3 (+/- 1.2). Bray-Curtis similarities showed good agreement among samples from the same apiary and sampling date. Rarefaction plots indicated that fewer than 50,000 reads are typically needed to characterize pollen samples of this complexity. Our results show that a pre-compiled, curated reference database is not essential for genus-level assignments, but species-level assignments are hindered by database gaps, reference length variation, and probable errors in the taxonomic assignment, requiring post-hoc evaluation. Although the effective per-sample yield achieved using custom MiSeq amplicon primers was less than the machine maximum, primarily due to lower "read2" quality, further protocol optimization and/or a modest reduction in multiplex scale should offset this difficulty. As small quantities of pollen are sufficient for amplification, our approach might be extendable to other questions or species for which large pollen samples are not available.

Comparing population patterns to processes: abundance and survival of a forest salamander following habitat degradation.

Habitat degradation resulting from anthropogenic activities poses immediate and prolonged threats to biodiversity, particularly among declining amphibians. Many studies infer amphibian response to habitat degradation by correlating patterns in species occupancy or abundance with environmental effects, often without regard to the demographic processes underlying these patterns. We evaluated how retention of vertical green trees (CANOPY) and coarse woody debris (CWD) influenced terrestrial salamander abundance and apparent survival in recently clearcut forests. Estimated abundance of unmarked salamanders was positively related to CANOPY (ß Canopy  = 0.21 (0.02-1.19; 95% CI), but not CWD (ß CWD  = 0.11 (-0.13-0.35) within 3,600 m2 sites, whereas estimated abundance of unmarked salamanders was not related to CANOPY (ß Canopy  = -0.01 (-0.21-0.18) or CWD (ß CWD  = -0.02 (-0.23-0.19) for 9 m2 enclosures. In contrast, apparent survival of marked salamanders within our enclosures over 1 month was positively influenced by both CANOPY and CWD retention (ß Canopy  = 0.73 (0.27-1.19; 95% CI) and ß CWD  = 1.01 (0.53-1.50). Our results indicate that environmental correlates to abundance are scale dependent reflecting habitat selection processes and organism movements after a habitat disturbance event. Our study also provides a cautionary example of how scientific inference is conditional on the response variable(s), and scale(s) of measure chosen by the investigator, which can have important implications for species conservation and management. Our research highlights the need for joint evaluation of population state variables, such as abundance, and population-level process, such as survival, when assessing anthropogenic impacts on forest biodiversity.

Climatic variation and the distribution of an amphibian polyploid complex.

The establishment of polyploid populations involves the persistence and growth of the polyploid in the presence of the progenitor species. Although there have been a number of animal polyploid species documented, relatively few inquiries have been made into the large-scale mechanisms of polyploid establishment in animal groups. Herein we investigate the influence of regional climatic conditions on the distributional patterns of a diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and H. versicolor (Anura: Hylidae) in the mid-Atlantic region of eastern North America. Calling surveys at breeding sites were used to document the distribution of each species. Twelve climatic models and one elevation model were generated to predict climatic and elevation values for gray treefrog breeding sites. A canonical analysis of discriminants was used to describe relationships between climatic variables, elevation and the distribution of H. chrysoscelis and H. versicolor. There was a strong correlation between several climatic variables, elevation and the distribution of the gray treefrog complex. Specifically, the tetraploid species almost exclusively occupied areas of higher elevation, where climatic conditions were relatively severe (colder, drier, greater annual variation). In contrast, the diploid species was restricted to lower elevations, where climatic conditions were warmer, wetter and exhibited less annual variation. Clusters of syntopic sites were associated with areas of high variation in annual temperature and precipitation during the breeding season. Our data suggest that large-scale climatic conditions have played a role in the establishment of the polyploid H. versicolor in at least some portions of its range. The occurrence of the polyploid and absence of the progenitor in colder, drier and more varied environments suggests the polyploid may possess a tolerance of severe environmental conditions that is not possessed by the diploid progenitor. Our findings support the hypothesis that increased tolerance to severe environmental conditions is a plausible mechanism of polyploid establishment.