Jennifer M. Standley

Royal jelly (RJ) is a glandular secretion fed to developing honey bee larvae by adult worker bees. It is also a potential source of disease transmission in and between honey bee colonies. We endeavored to characterize the microbiome, virome, and other biota present in RJ via an integrated meta‐omics approach. Using a magnetic beads‐based extraction protocol, we identified eDNA and eRNA fragments from organisms of interest in RJ using high‐throughput metagenomics (DNA‐seq), metatranscriptomics (total RNA‐seq), and parallel sequencing. This allowed us to enhance the detection of Operational Taxonomic Units (OTUs) undetectable by standard ‘omics or amplicon protocols’. Using this integrated approach, we detected OTUs present in RJ from honey bee pests and pathogens, including Melissococcus plutonius, Paenibacillus larvae, Varroa destructor, V. jacobsoni, Aethina tumida, Galleria mellonella, Vairimorpha ceranae, Apis mellifera filamentous virus, Black queen cell virus, Acute bee paralysis virus, Sacbrood virus, Deformed wing virus, Israeli acute bee paralysis virus, Kashmir bee virus, and Slow bee paralysis virus, as well as multiple beneficial gut bacteria from the genera Lactobacillus, Actinobacteria, and Gluconobacter. The presence of DNA and RNA from these organisms does not conclusively indicate the presence of live organisms in the RJ, but it does suggest some exposure of the RJ to these organisms. The results present a comprehensive eDNA and eRNA microbial profile of RJ, demonstrating that our novel method is an effective and sensitive molecular tool for high‐resolution metagenomic and metatranscriptomic profiling, and is of value for detection of pathogens of concern for the beekeeping industry.

DNA analysis of environmental samples (eDNA) provides a non-intrusive approach to identify organisms, characterize biological communities, and assess biodiversity, including the detection and monitoring of invasive plant effects. However, the use of eDNA for specific applications, such as targeted-species detection, geographic and floral source tracing, and assessment of invasive plant ecological and environmental effects, requires the development of species-specific genetic primers. Chinese tallow (Triadica sebifera (L.) Small) is a non-native high-impact invader, capable of changing fire regimes, native biodiversity, nutrient cycling, and wildlife habitat and populations, that is expanding in range and abundance throughout the southern United States. In this study, we investigated and identified specific genetic sites, markers, in the tallow chloroplast genome and developed sets of primers for tallow eDNA detection. Two sets of tallow primers were developed, tallow-specific primers and tallow-related primers. Both sets of primers can be used for tallow eDNA detection, with higher target specificity for tallow-specific primers. Primers were subsequently validated for target specificity against closely related species, samples of tallow tissue, and honey and honey bee-collected pollen from areas with tallow. We found that tallow-specific primers differentiated tallow eDNA from closely related species, demonstrating target specificity. Furthermore, a sequence analysis of the tallow-related primers in the polymerase chain reaction accurately distinguished members of the Hippomaninae subtribe, including tallow, from other subtribe or subfamily members within the Euphorbiaceae. Ultimately, the genetic markers and the corresponding sets of primers will facilitate eDNA analysis of tallow for several applications, including detection and monitoring in water and soil, assurance of honey quality and floral source tracing, and perhaps serving as a model for determining plant use by pollinators.

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.

Objective: We examined genomic variation potentially associated with the cortisol stress response in children having a painful medical procedure. Design: Children 4–10 years old having a peripheral intravenous line inserted provided saliva samples for evaluation of the cortisol response as a biological measure of distress: two on the day of the procedure and two at home on a nonstressful day for comparison values. Children and biological parents also provided samples for genotyping of variants with known or suspected association with the cortisol stress response. Analysis included child-only association and family-based transmission disequilibrium tests (TDTs). Results: Genotype and phenotype data on the cortisol stress response were available from 326 children for child-only association analyses and 376 complete family trios for TDTs. Children were 50% female, an average of 7.5 years old, and mostly (83%) White/non-Hispanic. We identified four single-nucleotide polymorphisms (SNPs) potentially associated with the cortisol stress response: rs1176744 ( HTR3B), rs10062367 ( CRHBP), rs634479 ( OPRM1), and rs8030107 ( NTRK3). Family-based analysis identified a two-SNP haplotype in HTR1B suggestive for association with the cortisol response (rs6296, rs11568817). Allelic TDTs identified rs7897947 ( NFKB2) as potentially related to cortisol response. Conclusions: Findings provide preliminary evidence for genes potentially important in cortisol response to an acute stressor in children in the serotonin, dopamine, and brain-derived neurotrophic factor pathways, the hypothalamic–pituitary–adrenal axis, and the inflammatory response. Combined with analyses of related phenotypes and clinical data, these results could help identify patients at increased risk of adverse responses to painful medical procedures who might benefit from tailored interventions.