A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples.

Deformed wing virus (DWV) was first detected in dead honey bees in 1982 but has been in honey bees for at least 300 years. Due to its high prevalence and virulence, they have been linked with the ongoing decline in honey bee populations worldwide. A rapid, simple, semi-automated, high-throughput, and cost-effective method of screening colonies for viruses would benefit bee research and the beekeeping industry. Here we describe a semi-automated approach that combines an RNA-grade liquid homogenizer followed by magnetic bead capture for total virus nucleic acid extraction. We compare it to the more commonly applied nucleic acid column-based purification method and use qPCR plus Oxford Nanopore Technologies sequencing to evaluate the accuracy of analytical results for both methods. Our results showed high reproducibility and accuracy for both approaches. The semi-automated method described here allows for faster screening of viral loads in units of 96 samples at a time. We developed this method to monitor viral loads in honey bee colonies, but it could be easily applied for any PCR or genomic-based screening assays.

Thinking inside the box: Restoring the propolis envelope facilitates honey bee social immunity.

When wild honey bee colonies (Apis mellifera) nest in hollow tree cavities, they coat the rough cavity walls with a continuous layer of propolis, a substance comprised primarily of plant resins. Studies have shown that the resulting "propolis envelope" leads to both individual- and colony-level health benefits. Unfortunately, the smooth wooden boxes most commonly used in beekeeping do little to stimulate propolis collection. As a result, most managed bees live in hives that are propolis-poor. In this study, we assessed different surface texture treatments (rough wood boxes, boxes outfitted with propolis traps, and standard, smooth wood boxes) in terms of their ability to stimulate propolis collection, and we examined the effect of propolis on colony health, pathogen loads, immune gene expression, bacterial gene expression, survivorship, and honey production in both stationary and migratory beekeeping contexts. We found that rough wood boxes are the most effective box type for stimulating propolis deposition. Although the use of rough wood boxes did not improve colony survivorship overall, Melissococcus plutonius detections via gene expression were significantly lower in rough wood boxes, and viral loads for multiple viruses tended to decrease as propolis deposition increased. By the end of year one, honey bee populations in migratory rough box colonies were also significantly larger than those in migratory control colonies. The use of rough wood boxes did correspond with decreased honey production in year one migratory colonies but had no effect during year two. Finally, in both stationary and migratory operations, propolis deposition was correlated with a seasonal decrease and/or stabilization in the expression of multiple immune and bacterial genes, suggesting that propolis-rich environments contribute to hive homeostasis. These findings provide support for the practical implementation of rough box hives as a means to enhance propolis collection and colony health in multiple beekeeping contexts.

The Chemical Composition of Scaptotrigona mexicana Honey and Propolis Collected in Two Locations: Similarities and Differences.

The chemical composition of stingless bee honey and propolis depends on the plant sources they are derived from, and thus reflects the flora available in the vicinity of the hives, the preferences of the bee species, and the climate (altitude and temperature). To understand the relative influence of these factors, we studied the composition of honey and propolis of the stingless bee Scaptotrigona mexicana. Samples from 24 colonies were analyzed: 12 each from two S. mexicana meliponaries located in the state of Chiapas in southern Mexico, approximately 8.5 km apart, Tuxtla Chico and Cacahoatán. The chemical composition of honey and propolis was studied using nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS), respectively. The antioxidant activity of propolis was also studied. Chemometric analyses were applied. The Tuxtla Chico honey samples contained higher concentrations of glucose and fructose, while the Cacahoatán samples displayed a rich composition of di- and trisaccharides. These differences can be attributed to the distinct nectar sources utilized by the bees at each location. Propolis compositions in the two locations also demonstrated qualitative differences, indicating a specific choice of resins by the bees. The observed substantial variations in the chemical composition of propolis and honey of S. mexicana from two locations relatively close to each other supports the assumption that bee species cannot be considered the most important factor in determining their chemistry.

Correction: Do honey bee (Apis mellifera) foragers recruit their nestmates to native forbs in reconstructed prairie habitats?

[This corrects the article DOI: 10.1371/journal.pone.0228169.].

Resin Use by Stingless Bees: A Review.

Stingless bees (Meliponini) are highly social bees that are native to tropical and sub-tropical ecosystems. Resin use is vital to many aspects of stingless bee colony function. Stingless bees use resin to build essential nest structures, repel predators, and kill would-be invaders. Furthermore, resin-derived compounds have been found to enrich the cuticular chemical profiles of many stingless bee species, and resin may play an important role in shaping the microbial communities associated with stingless bees and their nests. Despite its importance for colony function, previous reviews of resin use by stingless bees are lacking. This topic grows increasingly urgent as changes in beekeeping and land use practices occur, potentially diminishing stingless bees' ability to incorporate resin into the nest environment. In this article, we review existing literature on resin use by stingless bees and discuss potential areas of future research.

Survey-derived best management practices for backyard beekeepers improve colony health and reduce mortality.

Honey bee colony losses in the US have exceeded acceptable levels for at least a decade, leaving beekeepers in need of management practices to improve colony health and survival. Here, an empirical Best Management Practice (BMP) regimen was tested, comprised of the top four management practices associated with reduced colony mortality in backyard beekeeping operations according to Bee Informed Partnership Loss and Management survey results. Seven study locations were established across the US, and each location consisted of ten colonies treated according to empirical BMPs and ten according to average beekeeping practice. After 3 years, colonies treated according to empirical BMPs experienced reduced Varroa infestation, viral infection, and mortality compared to colonies managed with Average practices. In addition, BMP colonies produced more new colonies via splits. The colonies under Average practices were given chemical Varroa treatments only once per year, and thus spent more months above economic threshold of 3.0 mites/100 bees. Increased time spent above the economic threshold was significantly correlated to both increased viral infection and colony mortality. This study demonstrates the cumulative effects of management and colony health stressors over months and years, especially the dire importance of regular Varroa monitoring and management.

Social Fever or General Immune Response? Revisiting an Example of Social Immunity in Honey Bees.

Honey bees use several strategies to protect themselves and the colony from parasites and pathogens. In addition to individual immunity, social immunity involves the cumulative effort of some individuals to limit the spread of parasites and pathogens to uninfected nestmates. Examples of social immunity in honey bees that have received attention include hygienic behavior, or the removal of diseased brood, and the collection and deposition of antimicrobial resins (propolis) on interior nest surfaces. Advances in our understanding of another form of social immunity, social fever, are lacking. Honey bees were shown to raise the temperature of the nest in response to temperature-sensitive brood pathogen, Ascosphaera apis. The increase in nest temperature (-0.6 °C) is thought to limit the spread of A. apis infection to uninfected immatures. We established observation hives and monitored the temperature of the brood nest for 40 days. This observation period was broken into five distinct segments, corresponding to sucrose solution feedings-Pre-Feed, Feed I, Challenge, Feed II, and Post-Feed. Ascosphaera apis was administered to colonies as a 1% solution of ground sporulating chalkbrood mummies in 50% v/v sucrose solution, during the Challenge period. Like previous reports, we observed a modest increase in brood nest temperature during the Challenge period. However, all hives presented signs of chalkbrood disease, suggesting that elevation of the nest temperature was not sufficient to stop the spread of infection among immatures. We also began to explore the molecular mechanisms of temperature increase by exposing adult bees in cages to A. apis, without the presence of immatures. Compared to adult workers who were given sucrose solution only, workers exposed to A. apis showed increased expression of the antimicrobial peptides abaecin (p = 0.07) and hymenoptaecin (p = 0.04), but expression of the heat shock response protein Hsp 70Ab-like (p = 0.76) and the nutritional marker vitellogenin (p = 0.72) were unaffected. These results indicate that adult honey bee workers exposed to a brood pathogen elevate the temperature of the brood nest and initiate an immune response, but the effect of this fever on preventing disease requires further study.

Propolis Envelope Promotes Beneficial Bacteria in the Honey Bee (Apis mellifera) Mouthpart Microbiome.

Honey bees collect and apply plant resins to the interior of their nest cavity, in order to form a layer around the nest cavity called a propolis envelope. Propolis displays antimicrobial activity against honey bee pathogens, but the effect of propolis on the honey bee microbiome is unknown. Honey bees do not intentionally consume propolis, but they do manipulate propolis with their mouthparts. Because honey bee mouthparts are used for collecting and storing nectar and pollen, grooming and trophallaxis between adults, feeding larvae, and cleaning the colony, they are an important interface between the bees' external and internal environments and serve as a transmission route for core gut bacteria and pathogens alike. We hypothesized that the antimicrobial activity of an experimentally applied propolis envelope would influence the bacterial diversity and abundance of the worker mouthpart microbiome. The results revealed that the mouthparts of worker bees in colonies with a propolis envelope exhibited a significantly lower bacterial diversity and significantly higher bacterial abundance compared to the mouthparts of bees in colonies without a propolis envelope. Based on the taxonomic results, the propolis envelope appeared to reduce pathogenic or opportunistic microbes and promote the proliferation of putatively beneficial microbes on the honey bee mouthparts, thus reinforcing the core microbiome of the mouthpart niche.

The Pathogen Profile of a Honey Bee Queen Does Not Reflect That of Her Workers.

Throughout a honey bee queen's lifetime, she is tended to by her worker daughters, who feed and groom her. Such interactions provide possible horizontal transmission routes for pathogens from the workers to the queen, and as such a queen's pathogen profile may be representative of the workers within a colony. To explore this further, we investigated known honey bee pathogen co-occurrence, as well as pathogen transmission from workers to queens. Queens from 42 colonies were removed from their source hives and exchanged into a second, unrelated foster colony. Worker samples were taken from the source colony on the day of queen exchange and the queens were collected 24 days after introduction. All samples were screened for Nosema spp., Trypanosome spp., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Lake Sinai virus (LSV), and deformed wing virus master variants (DWV-A, B, and C) using RT-qPCR. The data show that LSV, Nosema, and DWV-B were the most abundant pathogens in colonies. All workers (n = 42) were LSV-positive, 88% were Nosema-positive, whilst pathogen loads were low (<1 × 106 genome equivalents per pooled worker sample). All queens (n = 39) were negative for both LSV and Nosema. We found no evidence of DWV transmission occurring from worker to queen when comparing queens to foster colonies, despite DWV being present in both queens and workers. Honey bee pathogen presence and diversity in queens cannot be revealed from screening workers, nor were pathogens successfully transmitted to the queen.

Honeybee microbiome is stabilized in the presence of propolis.

Honeybees have developed many unique mechanisms to help ensure the proper maintenance of homeostasis within the hive. One method includes the collection of chemically complex plant resins combined with wax to form propolis, which is deposited throughout the hive. Propolis is believed to play a significant role in reducing disease load in the colony due to its antimicrobial and antiseptic properties. However, little is known about how propolis may interact with bee-associated microbial symbionts, and if propolis alters microbial community structure. In this study, we found that propolis appears to maintain a stable microbial community composition and reduce the overall taxonomic diversity of the honeybee microbiome. Several key members of the gut microbiota were significantly altered in the absence of propolis, suggesting that it may play an important role in maintaining favourable abundance and composition of gut symbionts. Overall, these findings suggest that propolis may help to maintain honeybee colony microbial health by limiting changes to the overall microbial community.

Do honey bee (Apis mellifera) foragers recruit their nestmates to native forbs in reconstructed prairie habitats?

Honey bee (Apis mellifera) colonies are valued for the pollination services that they provide. However, colony mortality has increased to unsustainable levels in some countries, including the United States. Landscape conversion to monocrop agriculture likely plays a role in this increased mortality by decreasing the food sources available to honey bees. Many land owners and organizations in the Upper Midwest region of the United States would like to restore/reconstruct native prairie habitats. With increasing public awareness of high bee mortality, many landowners and beekeepers have wondered whether these restored prairies could significantly improve honey bee colony nutrition. Conveniently, honey bees have a unique communication signal called a waggle dance, which indicates the locations of the flower patches that foragers perceive as highly profitable food sources. We used these communication signals to answer two main questions: First, is there any part of the season in which the foraging force of a honey bee colony will devote a large proportion of its recruitment efforts (waggle dances) to flower patches within prairies? Second, will honey bee foragers advertise specific taxa of native prairie flowers as profitable pollen sources? We decoded 1528 waggle dances in colonies located near two large, reconstructed prairies. We also collected pollen loads from a subset of waggle-dancing bees, which we then analyzed to determine the flower taxon advertised. Most dances advertised flower patches outside of reconstructed prairies, but the proportion of dances advertising nectar sources within prairies increased significantly in the late summer/fall at one site. Honey bees advertised seven native prairie taxa as profitable pollen sources, although the three most commonly advertised pollen taxa were non-native. Our results suggest that including certain native prairie flower taxa in reconstructed prairies may increase the chances that colonies will use those prairies as major food sources during the period of greatest colony growth and honey production.

Social-medication in bees: the line between individual and social regulation.

We use the term social-medication to describe the deliberate consumption or use of plant compounds by social insects that are detrimental to a pathogen or parasite at the colony level, result in increased inclusive fitness to the colony, and have potential costs either at the individual or colony level in the absence of parasite infection. These criteria for social-medication differ from those for self-medication in that inclusive fitness costs and benefits are distinguished from individual costs and benefits. The consumption of pollen and nectar may be considered a form of social immunity if they help fight infection, resulting in a demonstrated increase in colony health and survival. However, the dietary use of pollen and nectar per se is likely not a form of social-medication unless there is a detriment or cost to their consumption in the absence of parasite infection, such as when they contain phytochemicals that are toxic at certain doses. We provide examples among social bees (bumblebees, stingless bees and honey bees) in which the consumption or use of plant compounds have a demonstrated role in parasite defense and health of the colony. We indicate where more work is needed to distinguish between prophylactic and therapeutic effects of these compounds, and whether the effects are observed at the individual or colony level.

Honey Bee Exposure to Pesticides: A Four-Year Nationwide Study.

Pollinators, including honey bees, are responsible for the successful reproduction of more than 87% of flowering plant species: they are thus vital to ecosystem health and agricultural services world-wide. To investigate honey bee exposure to pesticides, 168 pollen samples and 142 wax comb samples were collected from colonies within six stationary apiaries in six U.S. states. These samples were analyzed for evidence of pesticides. Samples were taken bi-weekly when each colony was active. Each apiary included thirty colonies, of which five randomly chosen colonies in each apiary were sampled for pollen. The pollen samples were separately pooled by apiary. There were a total of 714 detections in the collected pollen and 1008 detections in collected wax. A total of 91 different compounds were detected: of these, 79 different pesticides and metabolites were observed in the pollen and 56 were observed in the wax. In all years, insecticides were detected more frequently than were fungicides or herbicides: one third of the detected pesticides were found only in pollen. The mean (standard deviation (SD)) number of detections per pooled pollen sample varied by location from 1.1 (1.1) to 8.7 (2.1). Ten different modes of action were found across all four years and nine additional modes of action occurred in only one year. If synergy in toxicological response is a function of simultaneous occurrence of multiple distinct modes of action, then a high frequency of potential synergies was found in pollen and wax-comb samples. Because only pooled pollen samples were obtained from each apiary, and these from only five colonies per apiary per year, more data are needed to adequately evaluate the differences in pesticide exposure risk to honey bees among colonies in the same apiary and by year and location.

Is the Brood Pattern within a Honey Bee Colony a Reliable Indicator of Queen Quality?

Failure of the queen is often identified as a leading cause of honey bee colony mortality. However, the factors that can contribute to "queen failure" are poorly defined and often misunderstood. We studied one specific sign attributed to queen failure: poor brood pattern. In 2016 and 2017, we identified pairs of colonies with "good" and "poor" brood patterns in commercial beekeeping operations and used standard metrics to assess queen and colony health. We found no queen quality measures reliably associated with poor-brood colonies. In the second year (2017), we exchanged queens between colony pairs (n = 21): a queen from a poor-brood colony was introduced into a good-brood colony and vice versa. We observed that brood patterns of queens originally from poor-brood colonies significantly improved after placement into a good-brood colony after 21 days, suggesting factors other than the queen contributed to brood pattern. Our study challenges the notion that brood pattern alone is sufficient to judge queen quality. Our results emphasize the challenges in determining the root source for problems related to the queen when assessing honey bee colony health.

Brood Affects Hygienic Behavior in the Honey Bee (Hymenoptera: Apidae).

Despite receiving much attention, the ectoparasitic mite Varroa destructor (Anderson and Trueman) and the pathogens it vectors remain critical threats to the health of the honey bee Apis mellifera (Linnaeus) (Hymenoptera: Apidae). One promising intervention approach is the breeding of hygienic honey bees, which have an improved ability to detect and remove unhealthy brood from the colony, and are thus more resistant to Varroa. While much hygienic behavior-related research has focused on enhanced adult honey bee olfaction, less attention has been paid to the olfactory signals that originate inside the brood cell, triggering hygienic removal. Here, we hypothesized that selection for hygienic behavior in honey bees has influenced brood signaling, predicting that: 1) in a common social environment, removal rates differ among brood with different selective breeding histories, and 2) the removal rates of brood positively correlate to the hygiene level of the brood's colony of origin. To test these predictions, we cross-fostered brood subjected to control, wound, or Varroa treatment in unselected (UNS), Minnesota Hygienic (HYG), and Varroa-Sensitive Hygienic (VSH) colonies, and monitored individual brood cells for hygienic removal. Results confirmed both predictions, as brood from hygienic colonies was more likely to be removed than brood from UNS colonies, regardless of where the brood was fostered. These findings suggest that hygiene-related brood signals complement previously identified characteristics of hygienic adults, constituting an important mechanism of social immunity in honey bees. Thus, selective breeding for honey bee hygienic behavior may be improved through the utilization of field assays containing compounds related to larval signaling.

Effects of neonicotinoid imidacloprid exposure on bumble bee (Hymenoptera: Apidae) queen survival and nest initiation.

Neonicotinoids are highly toxic to insects and may systemically translocate to nectar and pollen of plants where foraging bees may become exposed. Exposure to neonicotinoids can induce detrimental sublethal effects on individual and colonies of bees and may have long-term impacts, such as impaired foraging, reduced longevity, and reduced brood care or production. Less well-studied are the potential effects on queen bumble bees that may become exposed while foraging in the spring during colony initiation. This study assessed queen survival and nest founding in caged bumble bees [Bombus impatiens (Cresson) (Hymenoptera: Apidae)] after chronic (18-d) dietary exposure of imidacloprid in syrup (1, 5, 10, and 25 ppb) and pollen (0.3, 1.7, 3.3, and 8.3 ppb), paired respectively. Here we show some mortality in queens exposed at all doses even as low as 1 ppb, and, compared with untreated queens, significantly reduced survival of treated queens at the two highest doses. Queens that survived initial imidacloprid exposure commenced nest initiation; however, they exhibited dose-dependent delay in egg-laying and emergence of worker brood. Furthermore, imidacloprid treatment affected other parameters such as nest and queen weight. This study is the first to show direct impacts of imidacloprid at field-relevant levels on individual B. impatiens queen survival and nest founding, indicating that bumble bee queens are particularly sensitive to neonicotinoids when directly exposed. This study also helps focus pesticide risk mitigation efforts and highlights the importance of reducing exposure rates in the early spring when bumble bee queens, and other wild bees are foraging and initiating nests.

Propolis envelope in Apis mellifera colonies supports honey bees against the pathogen, Paenibacillus larvae.

Honey bees have immune defenses both as individuals and as a colony (e.g., individual and social immunity). One form of honey bee social immunity is the collection of antimicrobial plant resins and the deposition of the resins as a propolis envelope within the nest. In this study, we tested the effects of the propolis envelope as a natural defense against Paenibacillus larvae, the causative agent of American foulbrood (AFB) disease. Using colonies with and without a propolis envelope, we quantified: 1) the antimicrobial activity of larval food fed to 1-2 day old larvae; and 2) clinical signs of AFB. Our results show that the antimicrobial activity of larval food was significantly higher when challenged colonies had a propolis envelope compared to colonies without the envelope. In addition, colonies with a propolis envelope had significantly reduced levels of AFB clinical signs two months following challenge. Our results indicate that the propolis envelope serves as an antimicrobial layer around the colony that helps protect the brood from bacterial pathogen infection, resulting in a lower colony-level infection load.

Propolis Counteracts Some Threats to Honey Bee Health.

Honey bees (Apis mellifera) are constantly dealing with threats from pathogens, pests, pesticides and poor nutrition. It is critically important to understand how honey bees' natural immune responses (individual immunity) and collective behavioral defenses (social immunity) can improve bee health and productivity. One form of social immunity in honey bee colonies is the collection of antimicrobial plant resins and their use in the nest architecture as propolis. We review research on the constitutive benefits of propolis on the honey bee immune system, and its known therapeutic, colony-level effects against the pathogens Paenibacillus larvae and Ascosphaera apis. We also review the limited research on the effects of propolis against other pathogens, parasites and pests (Nosema, viruses, Varroa destructor, and hive beetles) and how propolis may enhance bee products such as royal jelly and honey. Although propolis may be a source of pesticide contamination, it also has the potential to be a detoxifying agent or primer of detoxification pathways, as well as increasing bee longevity via antioxidant-related pathways. Throughout this paper, we discuss opportunities for future research goals and present ways in which the beekeeping community can promote propolis use in standard colonies, as one way to improve and maintain colony health and resiliency.

3-Acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis.

Honey bees, Apis mellifera, collect antimicrobial plant resins from the environment and deposit them in their nests as propolis. This behavior is of practical concern to beekeepers since the presence of propolis in the hive has a variety of benefits, including the suppression of disease symptoms. To connect the benefits that bees derive from propolis with particular resinous plants, we determined the identity and botanical origin of propolis compounds active against bee pathogens using bioassay-guided fractionation against the bacterium Paenibacillus larvae, the causative agent of American foulbrood. Eleven dihydroflavonols were isolated from propolis collected in Fallon, NV, including pinobanksin-3-octanoate. This hitherto unknown derivative and five other 3-acyl-dihydroflavonols showed inhibitory activity against both P. larvae (IC50 = 17-68 µM) and Ascosphaera apis (IC50 = 8-23 µM), the fungal agent of chalkbrood. A structure-activity relationship between acyl group size and antimicrobial activity was found, with longer acyl groups increasing activity against P. larvae and shorter acyl groups increasing activity against A. apis. Finally, it was determined that the isolated 3-acyl-dihydroflavonols originated from Populus fremontii, and further analysis showed these compounds can also be found in other North American Populus spp.