Honey Bee Breeding, Genetics, and Physiology Laboratory

Southern shagbark hickory (Carya carolinae-septentrionalis) is one of several deciduous trees in the family Juglandaceae and genus Carya that are native to North America. Southern shagbark hickory has a restricted distribution to the Southeast U.S.A. (USDA, 2024). During a disease survey in September, 2023, symptoms of leaf scorch were noticed on trees planted in a hickory collection at the USDA-ARS, Byron, GA. Scorch was characterized by tan to light brown, irregularly shaped necrotic lesions often starting along the leaf margins, with the necrosis spreading across the entire leaflet, resulting in curling of leaflet and in some cases affecting the whole leaf (Supplementary Fig. 1). Some defoliation of leaflets on compound leaves was noted. Symptomatic shoot terminals with compound leaves were collected, stored in a refrigerator (~4°C) and processed within three days. The epidermis was stripped from a sample of leaflet petioles and ground with a tissuelyser. DNA was extracted from the petiole sample using a Zymo Research® kit (Quick-DNA™ Fungal/Bacterial Kit, Zymo Research®, Irvine, CA) following the manufacturer's protocol. Conventional PCR was performed on the sample with positive controls (DNA of Xylella strain M12) and negative controls (water, healthy pecan) using four previously characterized Xylella fastidiosa (Xf)-specific primer sets (Francis et al., 2006; Minsavage et al. 1994; Rodrigues et al., 2003). The resulting amplicons each had the characteristic size expected for X. fastidiosa. A further two samples of DNA were extracted using a NucleoSpin Plant II kit (Machereey-Nagel, Duren, Germany) following the manufacturer's protocol. The DNA samples were tested by SYBR-green real-time PCR with primer sets Teme150fc/Teme454rg (specific to Xf subsp. fastidiosa, Xff) and Dixon454fa/Dixon1261rg (specific to Xf subsp. multiplex, Xfm) (Chen et al., 2005), yielding Ct values of 31.39 and 18.96, respectively, suggesting dominant Xfm infection. One sample (designated Cc-sR5T1) was further selected and subject to next generation sequencing (NGS) using an Illumina NovaSeq 6000 (PE150) platform. A total of 58,601,960 paired reads were generated with a mapping rate of 0.97% to Xfm M12 (NC_010513.1) and 0.95% to Xff M23 (NC010577.1) using Bowtie2 (Langmead and Salzberg, 2012), confirming the Xfm status of strain Cc-sR5T1. Read coverages on both M12 and M23 genomes were >60X. Top-5 and bottom-5 reads in the mapped read data sets were selected and used as queries for a BLAST search against the National Center for Biotechnology Information (NCBI) core-nr database. All top hits were Xf subsp. multiplex (query coverage = 97 to 100%, Percentage Identity = 99 to 100%) with one exception likely related to the region of a mobile element (Supplementary Table 1). In summary, a Xfm strain was identified in a shagbark hickory tree based on leaf scorch symptoms, PCR characteristic loci, and NGS whole genome approaches. The pathogen Xfm infects other Carya (Hilton et al. 2020), including C. illinoinensis (pecan) (Sanderlin and Heyderich-Alger, 2000), which is an economically important nut crop in the southern U.S.A. Shagbark hickory is not of high economic value, but now a possible reservoir of Xfm. Knowledge of the causal agents of the disease in Carya species is important for disease management purposes in both agricultural plantings and natural stands of trees. Furthermore, Xylella pathogens have both national and international phytosanitary ramifications.

Abstract Recent declines in the health of honey bee colonies used for crop pollination pose a considerable threat to global food security. Foraging by honey bee workers represents the primary route of exposure to a plethora of toxins and pathogens known to affect bee health, but it remains unclear how foraging preferences impact colony-level patterns of stressor exposure. Resolving this knowledge gap is crucial for enhancing the health of honey bees and the agricultural systems that rely on them for pollination. To address this, we carried out a national-scale experiment encompassing 456 Canadian honey bee colonies to first characterize pollen foraging preferences in relation to major crops, then explore how foraging behaviour influences patterns of stressor exposure. We used a metagenetic approach to quantify honey bee dietary breadth and found that bees display distinct foraging preferences that vary substantially relative to crop type and proximity, and the breadth of foraging interactions can be used to predict the abundance and diversity of stressors a colony is exposed to. Foraging on diverse plant communities was associated with increased exposure to pathogens, while the opposite was associated with increased exposure to xenobiotics. Our work provides the first large-scale empirical evidence that pollen foraging behaviour plays an influential role in determining exposure to dichotomous stressor syndromes in honey bees.Significance StatementInsect-mediated pollination is an important ecological process that is crucial for food production. Managed honey bee colonies are one of the most important insect pollinators, but their health has been under threat from a variety of stressors. Bee workers are primarily exposed to stressors while foraging and understanding how bee foraging preferences are related to exposure risk could provide pivotal information to improve management efforts. Here, we studied honey bee foraging preferences in relation to prominent Canadian crops and across a gradient of modified environments. We found that honey bees show distinct, measurable foraging preferences and that dietary diversity is a strong predictor of the stressors that colonies are exposed to.

Identifying traits for adaptation to different management and environmental regimes is key to maintaining robust honey bee populations under global climate change. We compared mite-resistant (Pol-line and Russian) and Italian honey bee stocks in variable-temperature cage experiments (200 bees per cage) with respect to food consumption, thermoregulation, gene expression, and lifespan, in 3 experiments over 2 years. The Italian stock bees consumed more syrup and pollen on average than the mite-resistant stocks, but the mite-resistant stocks maintained higher cluster temperatures and had median lifespans 8 days longer, consistent with the increased expression of vitellogenin relative to Italian stock. Model results indicated that, to maintain the same colony size as the mite-resistant stocks, Italian stock colonies would need about 13% more sealed brood to offset reduced worker lifespans. These differences among bee stocks likely influence colony-level productivity and health, and showed the importance of experimental replication.

Here, we present a retrospective study on honey bee brood diseases in the USA. The Bee Disease Diagnostic Laboratory (BDDL) in the USDA-ARS Beltsville Bee Research Laboratory received a total of 4790 brood samples between 2015 and 2022 from U.S. State Apiary Inspectors and beekeepers. Samples from 49 states were analyzed by microscopy for the presence and prevalence of two bacteria, Melissococcus plutonius and Paenibacillus larvae, causing European foulbrood (EFB) and American foulbrood (AFB) diseases, respectively. Samples that tested positive for AFB were cultured and subjected to antibiotic susceptibility tests via the agar disc diffusion method to determine their resistance to tetracycline (Terramycin®) and tylosin (Tylan®) antibiotics. A comprehensive data analysis was conducted at multiple levels, including state, month, year, nationwide, and climate region. Among the 49 states examined, EFB was identified in 44 states, while AFB was found in 31 states. Infection levels vary across states, ranging from 0% to 63.6% for EFB and from 0% to 54.8% for AFB. The national average of EFB (19.2%) was significantly (p < 0.001) higher than AFB (8.6%). Contrary to AFB, significant yearly increases (p < 0.001) were identified for EFB from 2015 to 2022. Furthermore, significant monthly variations were recorded for both brood diseases, with the highest occurrence of EFB observed from April to July. States with the lowest infection rates of both diseases were NV, ND, MS, AK, and AZ. The South U.S. climate region, which comprises six states, had the highest and lowest EFB and ABF infections, respectively. On a nationwide scale, the AFB resistance to tetracycline (38%) was significantly (p < 0.001) higher than the resistance to tylosin (27%).

Like humans, animals use plants and other materials as medication against parasites. Recent decades have shown that the study of insects can greatly advance our understanding of medication behaviors. The ease of rearing insects under laboratory conditions has enabled controlled experiments to test critical hypotheses, while their spectrum of reproductive strategies and living arrangements – ranging from solitary to eusocial communities – has revealed that medication behaviors can evolve to maximize inclusive fitness through both direct and indirect fitness benefits. Studying insects has also demonstrated in some cases that medication can act through modulation of the host’s innate immune system and microbiome. We highlight outstanding questions, focusing on costs and benefits in the context of inclusive host fitness.

We tested the expression of the multi-functional gene Vitellogenin (Vg) in four commercially available stocks of honey bees (Apis mellifera: L) under three different treatments: caged bees fed only 50% sucrose solution, caged bees fed sucrose solution and pollen paste, and free-roaming bees in a colony setting. We found that seven-day old bees fed pollen and sucrose in the lab as well as those in the colony had higher Vg expression than those fed only sucrose. There were strong differences in how the stocks responded to the treatments. This ranged from the Italian bees where expression was different between all treatment groups to the Saskatraz where there were no effects of treatment on Vg expression.

The ectoparasite Varroa destructor is the greatest threat to managed honey bee (Apis mellifera) colonies globally. Despite significant efforts, novel treatments to control the mite and its vectored pathogens have shown limited efficacy, as the host remains naïve. A prospective solution lies in the development of Varroa-resistant honey bee stocks, but a paucity of rigorous selection data restricts widespread adoption. Here, we characterise the parasite and viral dynamics of a Varroa-resistant honey bee stock, designated ‘Pol-line’, using a large-scale longitudinal study. Results demonstrate markedly reduced Varroa levels in this stock, diminished titres of three major viruses (DWV-A, DWV-B, and CBPV), and a two-fold increase in survival. Levels of a fourth virus that is not associated with Varroa—BQCV—do not differ between stocks, supporting a disruption of the transmission pathway. Further, we show that when decoupled from the influence of Varroa levels, viral titres do not constitute strong independent predictors of colony mortality risk. These findings highlight the need for a reassessment of Varroa etiology, and suggest that derived stocks represent a tractable solution to the Varroa pandemic.

Various stocks of honey bees (Apis mellifera L. (Hymenoptera: Apidae)) employ multiple mechanisms to control varroa mite (Varroa destructor Anderson & Trueman (Mesostigmata: Varroidae)) infestations. Identification of trait-associated genes and markers can improve efficiency of selective breeding. Dopamine receptors show promise in this regard in their association with numerous traits in honey bees, high plasticity, and indicated association with varroa resistance through QTL analysis. We assessed the relationship between exposure to mite-infested brood and gene expression of the honey bee dopamine receptors, Amdop1, Amdop2, and Amdop3, in bees and stocks with known levels of varroa resistance, in Spring 2016 (VSH vs Italian) and Summer 2019 (Pol-line vs Italian). Relative mRNA expression levels varied both by honey bee stock and before/after exposure to varroa-infested brood, in 7-, 10-, and 14-day-old bees. However, the trials revealed contrasting patterns in expression of the three dopamine receptors. In 2016, downregulation was evident in VSH bees, but varied by days post-emergence and by gene. The 2019 trial showed upregulation post-exposure in both stocks, and at all ages, for Amdop1, Amdop2, and Amdop3, with the exception of 14 d Italian bees for Amdop2 and Amdop3. Stock comparison in 2019 showed upregulation of all three dopamine-like receptors in post-exposure bees of all ages. Season and associated differences in mite loads may have contributed to the differences observed across trials. Differential expression of all three dopamine receptors suggests a role for the dopaminergic system in varroa resistance and suggests that further characterization of these receptors for breeding potential is warranted.

During the last decades, the beekeeping industry has seen very few changes and improvements on the widely used Langstroth wooden hive. The recent development of new insulation materials with higher proprieties compared to wood could provide better alternatives for the beekeeping industry. In this study, we tested the winter insulation of polyurethane honey bee hives and compare it with the traditional Langstroth wooden hives. Eighteen honey bee colonies, equal in size, composed of nine polyurethane and nine wooden hives were randomly selected and each hive was equipped with a sensor to document the inner hive temperature and humidity. Our results show that polyurethane hives maintained a significantly (P < 0.001) higher overall temperature (10.20 ± 0.04 °C) than wooden hives (9.73 ± 0.05 °C) with a significantly more optimal relative humidity (52.05%) compared to the wooden hives (62.50%). Inner temperature patterns of the wooden hives exhibited pronounced oscillations compared to the polyurethane hives. Both hive groups showed significant differences (P < 0.001) in temperature insulation between day and night cycles. However, the polyurethane hives seem to provide better stability in humidity between days and nights compared to Langstroth wooden hives.