Properties of essential oils absorbed on the surface of cardboard pieces after using atmospheric-pressure plasma treatments to develop long-lasting Varroa miticides in honeybees (Apis mellifera).

The ectoparasitic mite, Varroa destructor is the most serious widespread pest of managed honeybees (Apis mellifera). Several acaricide products, which include essential oils, have been proposed for mite control. In this study, we aimed to apply atmospheric-pressure plasma to modify a cardboard piece surface in order to prolong the delivery of essential oils for controlling Varroa in honeybee colonies. Absorption capacity, release rates and evaporation rates of essential oils were determined. Cardboard piece showed a higher absorption capacity of cinnamon compared to citronella and clove. Surface modification of cardboard pieces using argon plasma at different gas flow rates and treatment durations, significantly affected the absorption of clove oil. Additionally, the release rate of cinnamon, citronella and clove was significantly enhanced after argon plasma treatments. Evaporation of cinnamon was dramatically increased by plasma treatment at 6-h of incubation. The highest evaporation rate was obtained by plasma-treated cardboard piece at a gas flow rate of 0.5 Lpm for 60 s (0.2175 ± 0.0148 µl/g•h). Efficiency of plasma-treated cardboard piece, impregnated with essential oils, was also investigated for Varroa control in honeybee colonies. In the first experiment, formic acid 65% (v/v) showed the highest efficiency of 90.60% and 81.59% with the percent of mite infestation was 0.23 ± 0.13% and 0.47 ± 0.19% at 21 and 35 days, respectively after treatment. The efficacy of cardamon oil (5% (v/v)) delivered using plasma-treated cardboard pieces was 57.71% (0.70 ± 0.16% of mite infestation) at day 21 of experiment. However, the delivery of cardamon oil at the concentration of 1% and 5% (v/v) by untreated cardboard piece had 16.93% and 24.05% of efficacy to control mites. In the 2nd experiment, the application of plasma-treated cardboard pieces impregnated with 5% (v/v) clove oil induced a 38.10% reduction in the population of Varroa mites followed by 5% (v/v) of cardamon with 30% efficiency. Although, the infestation rate of Varroa in colonies was not significant different between treatments, essential oils delivered using plasma-treated cardboard pieces tended to decrease Varroa population in the treated colonies. Hence, atmospheric-pressure plasma for the modification of other materials, should be further investigated to provide alternative control treatment applications against honeybee mites.

Atmospheric non-thermal plasma inactivation of Ascosphaera apis, the causative agent of chalkbrood disease in honeybee.

Ascosphaera apis is a worldwide pathogenic fungi of honeybees that can cause a decline in bee populations. In this study, we investigated the antifungal activity of non-thermal plasma on fungal growth. Spore inactivation after exposure to gas plasma by liquid phase and plasma activated water (PAW) and pathogenicity of A. apis in vivo were also examined. The results demonstrated that the mycelial growth of fungi was completely inhibited after argon plasma treatment. Both gas plasma and PAW exposures resulted in a significant decrease of A. apis spore numbers, maximum reduction of 1.71 and 3.18-fold, respectively. Germinated fungal spores on potato dextrose agar were also reduced after plasma treatment. SEM analysis revealed a disruption in the morphological structure of the fungal spores. The pathogenicity of A. apis on honeybee larvae was decreased after spores treated by gas plasma and PAW with a disease inhibition of 63.61 ± 7.28% and 58.27 ± 5.87%, respectively after 7 days of cultivation. Chalkbrood in honey bees have limited control options and our findings are encouraging. Here, we demonstrate a possible alternative control method using non-thermal plasma for chalkbrood disease in honeybees.

Preliminary Survey of Pathogens in the Asian Honey Bee (Apis cerana) in Thailand.

Widespread parasites, along with emerging threats, globalization, and climate change, have greatly affected honey bees' health, leading to colony losses worldwide. In this study, we investigated the detection of biotic stressors (i.e., viruses, microsporidian, bacteria, and fungi) in Apis cerana by surveying the colonies across different regions of Thailand (Chiang Mai in the north, Nong Khai and Khon Kaen in the northeast, and Chumphon and Surat Thani in the south, in addition to the Samui and Pha-ngan islands). In this study, we detected ABPV, BQCV, LSV, and Nosema ceranae in A. cerana samples through RT-PCR. ABPV was only detected from the samples of Chiang Mai, whereas we found BQCV only in those from Chumphon. LSV was detected only in the samples from the Samui and Pha-ngan islands, where historically no managed bees are known. Nosema ceranae was found in all of the regions except for Nong Khai and Khon Kaen in northeastern Thailand. Paenibacillus larvae and Ascosphaera apis were not detected in any of the A. cerana samples in this survey. The phylogenetic tree analysis of the pathogens provided insights into the pathogens' movements and their distribution ranges across different landscapes, indicating the flow of pathogens among the honey bees. Here, we describe the presence of emerging pathogens in the Asian honey bee as a valuable step in our understanding of these pathogens in terms of the decline in eastern honey bee populations.

Natural extracts as potential control agents for Nosema ceranae infection in honeybees, Apis mellifera.

Nosema disease is one factor that can cause colony decline in honeybees (Apis mellifera L.) worldwide. Nosema ceranae has outcompeted Nosema apis in the Western honeybee (A. mellifera) which is its original host. Fumagilin is an effective antibiotic treatment to control Nosema infection but currently it is forbidden in many countries. In this study, 12 plant extracts were evaluated for their toxicity to adult bees and antimicrosporidian activity under laboratory and field conditions. N. ceranae-infected adult bees were fed ad libitum with 50% sucrose solution containing 1% and 5% (w/v) of each plant extract. Bee mortality in N. ceranae-infected groups fed with plant extracts was higher than that in the control group treated with fumagilin. The results demonstrated that 9 of 12 extracts had high antimicrosporidian activity against N. ceranae and their efficacies were comparable to fumagilin. Spore reduction in infected bees was 4-6 fold less after extract treatment. Following laboratory screening, Annona squamosa, Ocimum basilicum, Psidium guajava and Syzygium jambos were tested in honeybee colonies. Plant extracts of 2% concentration (w/v) inhibited the development of Nosema spores after 30 days of treatment. At the end of experiment (90 days), spores in the plant extract treated groups were lower than in group treated with fumagilin but there was no significant difference. Although, extracts tested in this study showed high toxicity to bee in laboratory cages, they did not show negative affects on bees under whole colony conditions. Therefore, the effectiveness of plant extracts tested in this study was notable and warrants further study as potential Nosema control agents in honey bees. Plant extracts would offer a non-antibiotic alternative for Nosema control and help reduce the overuse of antibiotics in livestock.

Microbial communities associated with honey bees in Brazil and in the United States.

Honey bee colony losses worldwide call for a more in-depth understanding of the pathogenic and mutualistic components of the honey bee microbiota and their relation with the environment. In this descriptive study, we characterized the yeast and bacterial communities that arise from six substrates associated with honey bees: corbicular pollen, beebread, hive debris, intestinal contents, body surface of nurses and forager bees, comparing two different landscapes, Minas Gerais, Brazil and Maryland, United States. The sampling of five hives in Brazil and four in the USA yielded 217 yeast and 284 bacterial isolates. Whereas the yeast community, accounted for 47 species from 29 genera, was dominated in Brazil by Aureobasidium sp. and Candida orthopsilosis, the major yeast recovered from the USA was Debaryomyces hansenii. The bacterial community was more diverse, encompassing 65 species distributed across 31 genera. Overall, most isolates belonged to Firmicutes, genus Bacillus. Among LAB, species from Lactobacillus were the most prevalent. Cluster analysis evidenced high structuration of the microbial communities, with two distinguished microbial groups between Brazil and the United States. In general, the higher difference among sites and substrates were dependents on the turnover effect (~ 93% of the beta diversity), with a more pronounced effect of nestedness (~ 28%) observed from Brazil microbiota change. The relative abundance of yeasts and bacteria also showed the dissimilarity of the microbial communities between both environments. These results provide a comprehensive view of microorganisms associated with A. mellifera, highlighting the importance of the environment in the establishment of the microbiota associated with honey bees.

Pesticides in honey bee colonies: Establishing a baseline for real world exposure over seven years in the USA.

Honey bees Apis mellifera forage in a wide radius around their colony, bringing back contaminated food resources that can function as terrestrial bioindicators of environmental pesticide exposure. Evaluating pesticide exposure risk to pollinators is an ongoing problem. Here we apply five metrics for pesticide exposure risk (prevalence, diversity, concentration, significant pesticide prevalence, and hazard quotient (HQ)) to a nation-wide field study of honey bees, Apis mellifera in the United States. We examined samples from 1055 apiaries over seven years for 218 different pesticide residues and metabolites, determining that bees were exposed to 120 different pesticide products with a mean of 2.78 per sample. Pesticides in pollen were highly prevalent and variable across states. While pesticide diversity increased over time, most detections occurred at levels predicted to be of low risk to colonies. Varroacides contributed most to concentration, followed by fungicides, while insecticides contributed most to diversity above a toxicity threshold. High risk samples contained one of 12 different insecticides or varroacides. Exposures predicted to be low-risk were nevertheless associated with colony morbidity, and low-level fungicide exposures were tied to queen loss, Nosema infection, and brood diseases.

Trade-offs between sperm viability and immune protein expression in honey bee queens (Apis mellifera).

Queens of many social hymenoptera keep sperm alive within their specialized storage organ, the spermatheca, for years, defying the typical trade-off between lifespan and reproduction. However, whether honey bee (Apis mellifera) queens experience a trade-off between reproduction and immunity is unknown, and the biochemical processes underlying sperm viability are poorly understood. Here, we survey quality metrics and viral loads of honey bee queens from nine genetic sources. Queens rated as 'failed' by beekeepers had lower sperm viability, fewer sperm, and higher levels of sacbrood virus and black queen cell virus. Quantitative proteomics on N = 123 spermathecal fluid samples shows, after accounting for sperm count, health status, and apiary effects, five spermathecal fluid proteins significantly correlating with sperm viability: odorant binding protein (OBP)14, lysozyme, serpin 88Ea, artichoke, and heat-shock protein (HSP)10. The significant negative correlation of lysozyme-a conserved immune effector-with sperm viability is consistent with a reproduction vs. immunity trade-off in honey bee queens.

Candidate stress biomarkers for queen failure diagnostics.

BACKGROUND: Queen failure is a persistent problem in beekeeping operations, but in the absence of overt symptoms it is often difficult, if not impossible, to ascertain the root cause. Stressors like heat-shock, cold-shock, and sublethal pesticide exposure can reduce stored sperm viability and lead to cryptic queen failure. Previously, we suggested candidate protein markers indicating heat-shock in queens. Here, we further investigate these heat-shock markers and test new stressors to identify additional candidate protein markers. RESULTS: We found that heat-shocking queens for upwards of 1 h at 40 °C was necessary to induce significant changes in the two strongest candidate heat-shock markers, and that relative humidity significantly influenced the degree of activation. In blind heat-shock experiments, we tested the efficiency of these markers at assigning queens to their respective treatment groups and found that one marker was sufficient to correctly assign queens 75% of the time. Finally, we compared cold-shocked queens at 4 °C and pesticide-exposed queens to controls to identify candidate markers for these additional stressors, and compared relative abundances of all markers to queens designated as 'healthy' and 'failing' by beekeepers. Queens that failed in the field had higher expression of both heat-shock and pesticide protein markers, but not cold-shock markers. CONCLUSIONS: This work offers some of the first steps towards developing molecular diagnostic tools to aid in determining cryptic causes of queen failure. Further work will be necessary to determine how long after the stress event a marker's expression remains elevated, and how accurate these markers will be for field diagnoses.

Impact of Sacbrood Virus on Larval Microbiome of Apis mellifera and Apis cerana.

In this study, we examined the impact of Sacbrood virus (SBV), the cause of larval honeybee (Apis mellifera) death, producing a liquefied a larva sac, on the gut bacterial communities on two larval honeybee species, Apis mellifera and Apis cerana. SBV was added into a worker jelly food mixture and bee larvae were grafted into each of the treatment groups for 24 h before DNA/RNA extraction. Confirmation of SBV infection was achieved using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and visual symptomology. The 16S rDNA was sequenced by Illumina sequencing. The results showed the larvae were infected with SBV. The gut communities of infected A. cerana larvae exhibited a dramatic change compared with A. mellifera. In A. mellifera larvae, the Illumina sequencing revealed the proportion of Gilliamella, Snodgrassella and Fructobacillus was not significantly different, whereas in A. cerana, Gilliamella was significantly decreased (from 35.54% to 2.96%), however, with significant increase in Snodgrassella and Fructobacillus. The possibility of cross-infection should be further investigated.

Feminizer and doublesex knock-outs cause honey bees to switch sexes.

Honey bees are experts at refuting societal norms. Their matriarchal hives are headed by queens, backed by an all-female workforce, and males die soon after copulation. But the biochemical basis of how these distinct castes and sexes (queens, workers, and drones) arise is poorly understood, partly due to a lack of efficient tools for genetic manipulation. Now, Roth and colleagues have used clustered regularly interspaced short palindromic repeats (CRISPR) to knock out two key genes (feminizer and doublesex) that guide sexual development. Their technique yielded remarkably low rates of genetic mosaicism and offers a promising tool for engineering and phenotyping bees for diverse applications.

Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), is the primary bacterial pathogen affecting honeybees and beekeeping. The main methods for controlling AFB are incineration of diseased colonies or prophylactic antibiotic treatment (e.g., with tylosin), neither of which is fully satisfactory. The search for superior means for controlling AFB has led to an increased interest in the natural relationships between the honeybee-pathogenic and mutualistic microorganisms and, in particular, the antagonistic effects of honeybee-specific lactic acid bacteria (hbs-LAB) against P. larvae These effects have been demonstrated only on individual larvae in controlled laboratory bioassays. Here we investigated whether supplemental administration of hbs-LAB had a similar beneficial effect on P. larvae infection at colony level. We compared experimentally AFB-infected colonies treated with hbs-LAB supplements to untreated and tylosin-treated colonies and recorded AFB symptoms, bacterial spore levels, and two measures of colony health. To account for the complexity of a bee colony, we focused on (Bayesian) probabilities and magnitudes of effect sizes. Tylosin reduced AFB disease symptoms but also had a negative effect on colony strength. The tylosin treatment did not, however, affect P. larvae spore levels and might therefore "mask" the potential for disease. hbs-LAB tended to reduce brood size in the short term but was unlikely to affect AFB symptoms or spores. These results do not contradict demonstrated antagonistic effects of hbs-LAB against P. larvae at the individual bee level but rather suggest that supplementary administration of hbs-LAB may not be the most effective way to harness these beneficial effects at the colony level.IMPORTANCE The previously demonstrated antagonistic effects of honeybee-derived bacterial microbiota on the infectivity and pathogenicity of P. larvae in laboratory bioassays have identified a possible new approach to AFB control. However, honeybee colonies are complex superorganisms where social immune defenses play a major role in resistance against disease at the colony level. Few studies have investigated the effect of beneficial microorganisms on bee diseases at the colony level. Effects observed at the individual bee level do not necessarily translate into similar effects at the colony level. This study partially fills this gap by showing that, unlike at the individual level, hbs-LAB supplements did not affect AFB symptoms at the colony level. The inference is that the mechanisms regulating the honeybee microbial dynamics within a colony are too strong to manipulate positively through supplemental feeding of live hbs-LAB and that new potential remedies identified through laboratory research have to be tested thoroughly in situ, in colonies.

Effects of Temperature During Package Transportation on Queen Establishment and Survival in Honey Bees (Hymenoptera: Apidae).

Honey bee (Apis mellifera) (Linnaeus) (Hymenoptera: Apidae) queens, the reproductive female caste, are crucial for colony success, and many management problems that beekeepers face are related to their diminished reproductive quality and premature failure. Previous research has suggested that temperature extremes may affect the viability of stored sperm in queens' spermathecae, thus the abiotic conditions of queens during transport may be germane to these problems. We recorded the temperatures experienced by queens during 2 yr of package transportation and tracked the newly installed colonies through establishment and buildup. During this critical 6-8 wk period, we observed typically high rates of queen failure (~25%) but found no indication that these postinstallation queen events were driven by temperature-related damage to stored sperm (an essential component of queen quality) incurred during transportation. We also found no indication of significant hot or cold zones across the truckloads of packages that would suggest a problem in how packages are insulated during transportation. However, we did observe significantly higher temperatures (31.2 vs. 29.9°C) and lower temperature variance (8.8 vs. 12.2) in queens that ultimately failed during the observation period, indicating that workers may respond differently to these queens in a way that manifests as more insulating clusters around queen cages. If so, then the collective process by which workers accept or reject a foreign queen may already be detectable even if it does not ultimately conclude until some weeks later. Nevertheless, it remains unclear why large numbers of otherwise high-quality queens are failing in newly installed packages.

New Viruses from the Ectoparasite Mite Varroa destructor Infesting Apis mellifera and Apis cerana.

Varroa destructor is an ectoparasitic mite of Asian or Eastern honeybees Apis cerana(A. cerana) which has become a serious threat to European subspecies of Western honeybees Apis mellifera (A. mellifera) within the last century. V.destructor and its vectored honeybee viruses became serious threats for colony survival. This is a short period for pathogen- and host-populations to adapt. To look for possible variation in the composition of viral populations we performed RNA metagenomic analysis of the Western honeybee subspecies A. m. ligustica, A. m.syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites. The analysis revealed two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor and a Hubei like-virga virus-14 homolog in V. destructor. VOV-1 was more prevalent in V. destructor than in A. mellifera and we found evidence for viral replication in both hosts. Interestingly, we found differences in viral loads of A. cerana and their V. destructor, A. m. intermissa, and its V. destructor showed partial similarity, while A. m.ligustica and A. m.syriaca and their varroa where very similar. Deformed wing virus exhibited 82.20%, 99.20%, 97.90%, and 0.76% of total viral reads in A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana, respectively. This is the first report of a complete segmented-single-stranded negative-sense RNA virus genome in honeybees and V. destructor mites.

Chemical and cultural control of Tropilaelaps mercedesae mites in honeybee (Apis mellifera) colonies in Northern Thailand.

At least two parasitic mites have moved from Asian species of honeybees to infest Apis mellifera. Of these two, Varroa destructor is more widespread globally while Tropilaelaps mercedesae has remained largely in Asia. Tropilaelaps mites are most problematic when A. mellifera is managed outside its native range in contact with Asian species of Apis. In areas where this occurs, beekeepers of A. mellifera treat aggressively for Tropilaelaps and Varroa is either outcompeted or is controlled as a result of the aggressive treatment regime used against Tropilaelaps. Many mite control products used worldwide may in fact control both mites but environmental conditions differ globally and thus a control product that works well in one area may be less or ineffective in other areas. This is especially true of volatile compounds. In the current research we tested several commercial products known to control Varroa and powdered sulfur for efficacy against Tropilaelaps. Additionally, we tested the cultural control method of making a hive division to reduce Tropilaelaps growth in both the parent and offspring colony. Making a split or nucleus colony significantly reduced mite population in both the parent and nucleus colony when compared to un-manipulated control colonies. The formic acid product, Mite-Away Quick Strips®, was the only commercial product that significantly reduced mite population 8 weeks after initiation of treatment without side effects. Sulfur also reduced mite populations but both sulfur and Hopguard® significantly impacted colony growth by reducing adult bee populations. Apivar® (amitraz) strips had no effect on mite or adult bee populations under the conditions tested.

Correction: Assessment of Chronic Sublethal Effects of Imidacloprid on Honey Bee Colony Health.

[This corrects the article DOI: 10.1371/journal.pone.0118748.].

Multilocus sequence typing, biochemical and antibiotic resistance characterizations reveal diversity of North American strains of the honey bee pathogen Paenibacillus larvae.

Paenibacillus larvae is a Gram positive bacterium and the causative agent of the most widespread fatal brood disease of honey bees, American foulbrood (AFB). A total of thirty-three independent Paenibacillus larvae isolates from various geographical origins in North America and five reference strains were investigated for genetic diversity using multilocus sequence typing (MLST). This technique is regarded to be a powerful tool for epidemiological studies of pathogenic bacteria and is widely used in genotyping assays. For MLST, seven housekeeping gene loci, ilvD (dihydroxy-acid dyhydrogenase), tri (triosephosphate isomerase), purH (phospharibosyl-aminoimidazolecarboxamide), recF (DNA replication and repair protein), pyrE (orotate phosphoribosyltransferase), sucC (succinyl coenzyme A synthetase ß subunit) and glpF (glycerol uptake facilitator protein) were studied and applied for primer designs. Previously, ERIC type DNA fingerprinting was applied to these same isolates and the data showed that almost all represented the ERIC I type, whereas using BOX-PCR gave an indication of more diversity. All isolates were screened for resistance to four antibiotics used by U.S. beekeepers, showing extensive resistance to tetracycline and the first records of resistance to tylosin and lincomycin. Our data highlight the intraspecies relationships of P. larvae and the potential application of MLST methods in enhancing our understanding of epidemiological relationships among bacterial isolates of different origins.

In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States.

This study measured part of the in-hive pesticide exposome by analyzing residues from live in-hive bees, stored pollen, and wax in migratory colonies over time and compared exposure to colony health. We summarized the pesticide burden using three different additive methods: (1) the hazard quotient (HQ), an estimate of pesticide exposure risk, (2) the total number of pesticide residues, and (3) the number of relevant residues. Despite being simplistic, these models attempt to summarize potential risk from multiple contaminations in real-world contexts. Colonies performing pollination services were subject to increased pesticide exposure compared to honey-production and holding yards. We found clear links between an increase in the total number of products in wax and colony mortality. In particular, we found that fungicides with particular modes of action increased disproportionally in wax within colonies that died. The occurrence of queen events, a significant risk factor for colony health and productivity, was positively associated with all three proxies of pesticide exposure. While our exposome summation models do not fully capture the complexities of pesticide exposure, they nonetheless help elucidate their risks to colony health. Implementing and improving such models can help identify potential pesticide risks, permitting preventative actions to improve pollinator health.

Correction: Colony Failure Linked to Low Sperm Viability in Honey Bee (Apis mellifera) Queens and an Exploration of Potential Causative Factors.

[This corrects the article DOI: 10.1371/journal.pone.0147220.].

Rising atmospheric CO2 is reducing the protein concentration of a floral pollen source essential for North American bees.

At present, there is substantive evidence that the nutritional content of agriculturally important food crops will decrease in response to rising levels of atmospheric carbon dioxide, Ca However, whether Ca-induced declines in nutritional quality are also occurring for pollinator food sources is unknown. Flowering late in the season, goldenrod (Solidago spp.) pollen is a widely available autumnal food source commonly acknowledged by apiarists to be essential to native bee (e.g. Bombus spp.) and honeybee (Apis mellifera) health and winter survival. Using floral collections obtained from the Smithsonian Natural History Museum, we quantified Ca-induced temporal changes in pollen protein concentration of Canada goldenrod (Solidago canadensis), the most wide spread Solidago taxon, from hundreds of samples collected throughout the USA and southern Canada over the period 1842-2014 (i.e. a Ca from approx. 280 to 398 ppm). In addition, we conducted a 2 year in situtrial of S. Canadensis populations grown along a continuous Ca gradient from approximately 280 to 500 ppm. The historical data indicated a strong significant correlation between recent increases in Ca and reductions in pollen protein concentration (r(2)= 0.81). Experimental data confirmed this decrease in pollen protein concentration, and indicated that it would be ongoing as Ca continues to rise in the near term, i.e. to 500 ppm (r(2)= 0.88). While additional data are needed to quantify the subsequent effects of reduced protein concentration for Canada goldenrod on bee health and population stability, these results are the first to indicate that increasing Ca can reduce protein content of a floral pollen source widely used by North American bees.