Honey Bee Brood Research Articles

Unique brood ester profile in a Varroa destructor resistant population of European honey bee (Apis mellifera)

Varroa destructor is one of the greatest threats to Apis mellifera worldwide and if left untreated will kill a colony in less than three years. A Varroa-resistant population from Gotland, Sweden, has managed to survive for 25 years with little to no Varroa treatment by reducing the mite’s reproductive success. The underlying mechanisms of this trait is currently not known, though previous research indicates that it is the honey bee brood, and not adult bee influence, that contributes to this phenotype. As the mite’s own reproduction is synchronized with the brood’s development though the interception of brood pheromones, it is possible that a change in pheromone profile would disrupt the mite’s reproductive timing. To investigate this, we characterized the brood ester pheromone (BEP) profile of our resistant Gotland population compared to a non-resistant control. This was done by extracting and analyzing key cuticular compounds of the BEP using gas chromatography. A significant difference was found immediately after brood capping, indicating a divergence in their pheromonal production at this time point. This is an important step to understanding the mechanisms of the Gotland population’s Varroa-resistance and contributes to our global understanding of Varroa destructor infestation and survival.

Evaluation of Prebiotic and Health-Promoting Functions of Honeybee Brood Biopeptides and Their Maillard Reaction Conjugates.

This study addresses the growing interest in natural functional ingredients by evaluating the prebiotic and health-promoting functions of honeybee brood biopeptides (HBb-Bps) and their conjugates. The purpose was to investigate their antioxidant activities, enzyme inhibition properties, and effects on probiotic growth and short-chain fatty acid (SCFA) production. The HBb-Bps were conjugated with honey, glucose, and fructose via the Maillard reaction. Antioxidant activities were assessed using DPPH and ABTS assays. The inhibitory effects on amylase, pancreatic lipase, and the angiotensin-converting enzyme (ACE) were measured. Probiotic growth and SCFA production were evaluated using L. plantarum TISTR846, and L. lactis TISTR1464. The HBb-Bps and their conjugates exhibited enhanced antioxidant activities post-Maillard reaction. They showed moderate enzyme inhibition, which decreased after conjugation. However, ACE inhibition increased with conjugation. The HBb-Bps significantly promoted probiotic growth and SCFA production, with further enhancement by the Maillard reaction. Overall, the HBb-Bps and their conjugates demonstrate significant prebiotic and health-promoting functions, suggesting their potential as natural ingredients in functional foods and nutraceuticals. Further research should focus on the in vivo effects and, given their solubility and stability these biopeptides could be incorporated into functional food formulations, such as health beverages, protein bars, and other fortified foods designed to deliver specific health benefits.

Honey bee larval toxicity study designs: Applicability of the current study protocols and endpoints asa predictor of pesticide hazard for pollinators.

The assessment of pesticide risks to bees in North America currently relies in part on Tier 1 honey bee laboratory toxicity studies to support the registration and registration review processes for crop protection chemicals. For immature stages, the studies follow two standardized test designs recommended by the Organization for Economic Cooperation (OECD), evaluating acute (seven-day single-dose, TG OECD 237) and chronic (22-day repeated-dose, GD OECD 239) toxicity in bee larvae. In this article, we aim to evaluate the current approach for generating and interpreting honey bee larval toxicity data, enhancing pesticide risk assessment for pollinators. First, by considering that the repeated-dose larval study covers all stages of honey bee brood development up to adult emergence, we compared endpoints (larval LD/ED50 and LC/EC50 values) from seven-day acute exposure studies with the 22-day chronic exposure studies. Our goal was to identify the study design offering greater sensitivity in assessing pesticide toxicity to immature bees. Our second objective involved analyzing available weight data from emerged adults and comparing it to survival endpoints (e.g., NOEL and LD50) to determine if the weight after adult emergence would accurately represent a sensitive indicator of pesticide effects on developing honey bees. Our analysis determined that the use of a single 22-day chronic exposure study adequately covers all immature stages and that the toxicity values based on cumulative dose are more accurate and representative measures of exposure for immature bees than using endpoints based on estimated daily doses. Furthermore, our analysis suggests that measuring the weight of emerged adults was a more sensitive indicator than mortality of treatment-related effects in 22% of the compounds included in our analysis. Here we also discuss the importance of standardized protocols for proper collection of weight after emergence and the need for further discussion on the relevance of this parameter at risk assessment scheme. Integr Environ Assess Manag 2024;20:2283-2293. © 2024 Pollinator Research Task Force. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Application of Carboxymethyl Cellulose and Glycerol Monostearate as Binder Agents for Protein Powder Production from Honey Bee Brood Using Foam-Mat Drying Technique.

This study investigates the development of protein powder from honey bee drone broods using foam-mat drying, a scalable method suitable for community enterprises, as well as the preservation of bee broods as a food ingredient. Initially, honey bee broods were pre-treated by boiling and steaming, with steamed bee brood (S_BB) showing the highest protein content (44.71 g/100 g dry basis). A factorial design optimized the powder formulation through the foam-mat drying process, incorporating varying concentrations of S_BB, glycerol monostearate (GMS), and carboxymethyl cellulose (CMC). The physicochemical properties of the resulting powder, including yield, color spaces, water activity, solubility, protein content, and total amino acids, were evaluated. The results showed that foam-mat drying produced a stable protein powder. The binders (CMC and GMS) increased the powder's yield and lightness but negatively affected the hue angle (yellow-brown), protein content, and amino acid content. The optimal quantities of the three variables (S_BB, GMS, and CMC) were determined to be 30 g, 6 g, and 1.5 g, or 80%, 16%, and 4%, respectively. Under this formulation, the protein powder exhibited a protein content of 19.89 g/100 g. This research highlights the potential of bee brood protein powder as a sustainable and nutritious alternative protein source, enhancing food diversification and security.

Sacbrood viruses and select Lake Sinai virus variants dominated Apis mellifera colonies symptomatic for European foulbrood.

European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (-, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB - colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB.IMPORTANCEThis study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

A field realistic model to assess the effects of pesticides residues and adulterants on honey bee gene expression.

While studies on the sublethal effects of chemical residues in beeswax on adult honey bees are increasing, the study protocols assessing the impacts on honey bee brood in realistic conditions still need to be investigated. Moreover, little is known about the residue's effect on gene expression in honey bee brood. This study reports the effects of chlorpyriphos-ethyl, acrinathrin and stearin worker pupae exposure through contaminated or adulterated beeswax on the gene expression of some key health indicators, using a novel in vivo realistic model. Larvae were reared in acrinathrin (12.5, 25, 10 and 100 ppb) and chlorpyriphos-ethyl (5, 10, 500 and 5000 ppb) contaminated or stearin adulterated beeswax (3, 4, 5, 6 and 9%) in newly formed colonies to reduce the influence of external factors. On day 11, mortality rates were assessed. Honey bee pupae were extracted from the comb after 19 days of rearing and were analysed for the gene expression profile of four genes involved in the immune response to pathogens and environmental stress factors (Imd, dorsal, domeless and defensin), and two genes involved in detoxifications mechanisms (CYP6AS14 and CYP9Q3). We found no linear relation between the increase in the pesticide concentrations and the brood mortality rates, unlike stearin where an increase in stearin percentage led to an exponential increase in brood mortality. The immune system of pupae raised in acrinathrin contaminated wax was triggered and the expression of CYP6AS14 was significantly upregulated (exposure to 12.5 and 25 ppb). Almost all expression levels of the tested immune and detoxification genes were down-regulated when pupae were exposed to chlorpyrifos-contaminated wax. The exposure to stearin triggered the immune system and detoxification system of the pupae. The identification of substance-specific response factors might ultimately serve to identify molecules that are safer for bees and the ecosystem's health.

Antioxidant Capacity, Phytochemicals, Minerals, and Chemical Pollutants in Worker Honey Bee (Apis mellifera L.) Broods from Northern Thailand: A Safe and Sustainable Food Source.

Honey bee brood (HBB) (Apis mellifera L.), a traditional protein source, has been studied for its nutritional value, but bio-functional properties and safety concerns have not been verified. This study examined the Antioxidant capacity, phytochemicals, minerals, and chemical pollutants in worker broods from several apiaries in Northern Thailand. HBB samples were lyophilized to evaluate antioxidant capacity using ABTS, DPPH, and FRAP assays, tests with water, and 70% ethanol extracts. Phytochemicals were identified using LC-QTOF-MS; pollutants were analyzed chromatographically, and minerals were determined using ICP-OES. The results showed that the evaluated antioxidant capacity of the ethanol extracts included DPPH 2.04-3.37 mg/mL, ABTS 21.22-33.91 mg/mL, and FRAP 50.07-104.15 mg AAE/100 g dry weight. Water extracts had outstanding antioxidant activities except for ABTS, with DPPH 10.67-84.97 mg/mL, ABTS 9.25-13.54 mg/mL, and FRAP 57.66-177.32 mgAAE/100 g dry weight. Total phenolics and flavonoids in ethanol extracts ranged from 488.95-508.87 GAE/100 g to 4.7-12.98 mg QE/g dry weight, respectively. Thirteen phytochemicals were detected and contained adequate mineral contents in the HBBs from different locations found, which were K, Ca, Mg, and Na, and no heavy metals or pollutants exceeded safe levels. These results imply that HBB from different apiaries in Northern Thailand is a nutritious food source with considerable antioxidants and a safe and sustainable food source.

Comprehensive Approach to Phenotype Varroa destructor Reproduction in Honey Bee Drone Brood and Its Correlation with Decreased Mite Reproduction (DMR).

The mechanisms of action behind decreased mite reproduction (DMR) are still unknown, but current hypotheses state that DMR is the result of brood-intrinsic and/or external disturbances in the V. destructor-honey bee pupa signal interactions. For accurate and precise DMR phenotyping, sufficient single infested honey bee brood cells are required (e.g., 35), which requires extensive labor and time and may exclude many samples not reaching the threshold. We defined a new comprehensive trait called the 'mean V. destructor reproduction rate' (mVR), which describes the mean number of offspring mites per infested cell in the sample while compensating for the reduced number of offspring with increasing multiple infested cells. We found a significant correlation between mVR and DMR, allowing for an estimation of DMR based on the mVR only. When the mVR was calculated with 10 infested cells, we found an average variation in mVR of 16.8%. For the same variation in DMR determination, 40 single infested cells are required. This broader look at V. destructor resistance phenotyping can improve the applicability and effectiveness of traits related to V. destructor reproduction in honey bee breeding programs.

Core proteome mediated subtractive approach for the identification of potential therapeutic drug target against the honeybee pathogen Paenibacillus larvae.

American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium Paenibacillus larvae, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against P. larvae, a causative agent of American foulbrood disease in honey bee. We investigated effective novel drug targets through a comprehensive in silico pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of P.larvae genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (n>36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand. Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against P. larvae. Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry.

Micronutrient Deficiency May Be Associated with the Onset of Chalkbrood Disease in Honey Bees.

Chalkbrood is a disease of honey bee brood caused by the fungal parasite Ascosphaera apis. Many factors such as genetics, temperature, humidity and nutrition influence the appearance of clinical symptoms. Poor nutrition impairs the immune system, which favors the manifestation of symptoms of many honey bee diseases. However, a direct link between dietary ingredients and the symptoms of chalkbrood disease has not yet been established. We show here that the elemental composition of chalkbrood mummies and healthy larvae from the same infected hives differ, as well as that mummies differ from larvae from healthy hives. Chalkbrood mummies had the highest concentration of macroelements such as Na, Mg, P, S, K and Ca and some microelements such as Rb and Sn, and at the same time the lowest concentration of B, As, Sr, Ag, Cd, Sb, Ba and Pb. Larvae from infected hives contained less Pb, Ba, Cs, Sb, Cd, Sr, As, Zn, Cu, Ni, Co, Mn, Cr, V and Al in contrast to healthy larvae from a disease-free apiary. This is the first study to demonstrate such differences, suggesting that an infection alters the larval nutrition or that nutrition is a predisposition for the outbreak of a chalkbrood infection. Though, based on results obtained from a case study, rather than from a controlled experiment, our findings stress the differences in elements of healthy versus diseased honey bee larvae.

Effects of Disinfectants on Bacterium Paenibacillus larvae in Laboratory Conditions.

American foulbrood is an infectious disease of the honeybee brood that causes multiple types of damage to beekeeping. The causative agent of the disease is the bacterium Paenibacillus larvae, which forms resistant infective spores and is viable for decades. After the eradication measures have been implemented, in cases of clinically visible disease, it is necessary to conduct effective final disinfections of equipment and tools. This study aimed to determine the effect of ten commercially available and commonly used disinfectants on certified strains of P. larvae under laboratory conditions, as well as to compare the obtained results among individual genotypes of P. larvae. Selected products were tested by determining the zone of inhibition using an agar diffusion test, a suspension test for viable bacteria, a surface disinfectant test, and a sporicidal effect in the suspension test. Incidin OxyFoam S and Sekusept Aktiv are both effective against all examined genotypes of P. larvae. Despadac and Despadac Secure have a bactericidal effect, but their sporocidal effect is not as satisfactory as that of Genox. Genoll does not exhibit a sporicidal effect, and Ecocide S at 1%, Bee protect H forte, and Bee protect F did not exhibit a satisfactory sporocidal effect. Additionally, EM® PROBIOTIC FOR BEES did not exhibit any bactericidal effect. The effective application of control measures and proper application of final disinfection can reduce the reoccurrence of visible clinical signs of disease, whereas methods of early diagnosis can significantly reduce the incidence of the disease.

Protective covers impact honey bee colony performance and access to outside resources

Protective crop covers are used extensively to protect high-value crops from bird and hail damage, however, these structures may present challenges for honey bees (Apis mellifera L.), the main pollinators in these systems. Some studies have indicated that protective covers result in declines in colony size and resource storage, but few replicated field trials exist. To determine the impact of protective cropping structures on honey bee brood production and resource storage, 14 honey bee colonies were placed inside and outside of protective covers on four blueberry (Vaccinium corymbosum and V. virgatum) farms in northern New South Wales, Australia. We measured changes in brood production, pollen and honey storage, hive weight and ambient hive temperature fortnightly over a 12-week period. Brood production, pollen and honey storage, and hive weight all displayed greater reductions within four weeks of placement under net cover, compared to hives outside of net cover. Although brood and honey metrics gradually increased in all hives during the remainder of the 12-week monitoring period, hives located under protective netting had relatively smaller gains in brood production and pollen storage when compared to hives located outside the protective covering. Hives under protective netting that were in the shade and/or whose entrances were facing south were most impacted by the end of the monitoring period. Our findings highlight the need for protected crop management strategies to consider pollinator health as well as crop pollination needs so that both bee health and pollination services are maintained in these systems.

An improved technique for direct visualization of plasmid patterns in strains of Paenibacillus larvae

American Foulbrood disease (AFB) caused by Paenibacillus larvae is the most devastating bacterial disease affecting honey bee brood worldwide. Beekeepers have been using oxytetracycline for decades to prevent and control AFB. In some countries with high disease incidence, antibiotics are still employed with the consequent risks of the appearance of tetracycline-resistant phenotypes associated with small plasmids carrying a tetracycline-resistance gene. Techniques for plasmid visualization in Gram-positive bacteria, particularly spore-forming, are not always successful. The present study aims to improve a method for direct visualization of plasmid patterns in P. larvae and other spore-forming bacteria isolated from apiarian sources. We tested 51 P. larvae strains from different geographical areas, including tetracycline-sensitive and resistant ones. The results revealed six different plasmid profiles, named I, II, III, IV, and V. Within tetracycline-resistant strains, four strains showed pattern IV with two plasmids of 5,000 and 8,000 bp, and two strains exhibited pattern V, with bands of about 5,000, 7,000, and 8,000 bp. The plasmid band of 5,000 bp present in the six resistant strains corresponds to the tetracycline-resistance mobilizable plasmid reported for P. larvae. Within the tetracycline-sensitive phenotypes, 33 strains showed no plasmids (pattern I); while two additional profiles were detected in the rest, i.e., pattern III with three plasmids in one strain and pattern VI showing two plasmids in six strains. Patterns II, III, and VI are probably related to the presence of cryptic plasmids. In conclusion, the improved technique allows us to identify plasmid patterns in P. larvae, ensuring their reproducibility and feasibility.

Quantifying Abdominal Coloration of Worker Honey Bees.

The main drawback in using coloration to identify honey bee subspecies is the lack of knowledge regarding genetic background, subjectivity of coloration grading, and the effect of the environment. The aim of our study was to evaluate the effect of environmental temperature on the abdominal coloration of honey bee workers and to develop a tool for quantifying abdominal coloration. We obtained four frames of honey bee brood from two colonies and incubated them at two different temperatures (30 and 34 °C). One colony had workers exhibiting yellow marks on the abdomen, while the other did not. We collected hatched workers and photographed abdomens. Images were analyzed using custom-written R script to obtain vectors that summarize the coloration over the abdomen length in a single value-coloration index. We used UMAP to reduce the dimensions of the vectors and to develop a classification procedure with the support vector machine method. We tested the effect of brood origin and temperature on coloration index with ANOVA. UMAP did not distinguish individual abdomens according to experimental group. The trained classifier sufficiently separated abdomens incubated at different temperatures. We improved the performance by preprocessing data with UMAP. The differences among the mean coloration index values were not significant between the gray groups incubated at different temperatures nor between the yellow groups. However, the differences between the gray and yellow groups were significant, permitting options for application of our tool and the newly developed coloration index. Our results indicate that the environmental temperature in the selected range during development does not seem to impact honey bee coloration significantly. The developed color-recording protocol and statistical analysis provide useful tools for quantifying abdominal coloration in honey bees.

Occurrence and distribution of two bacterial brood diseases (American and European foulbrood) in US honey bee colonies and resistance to antibiotics from 2015 to 2022

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%).

The effect of Israeli acute paralysis virus infection on honey bee brood care behavior

To protect themselves from communicable diseases, social insects utilize social immunity—behavioral, physiological, and organizational means to combat disease transmission and severity. Within a honey bee colony, larvae are visited thousands of times by nurse bees, representing a prime environment for pathogen transmission. We investigated a potential social immune response to Israeli acute paralysis virus (IAPV) infection in brood care, testing the hypotheses that bees will respond with behaviors that result in reduced brood care, or that infection results in elevated brood care as a virus-driven mechanism to increase transmission. We tested for group-level effects by comparing three different social environments in which 0%, 50%, or 100% of nurse bees were experimentally infected with IAPV. We investigated individual-level effects by comparing exposed bees to unexposed bees within the mixed-exposure treatment group. We found no evidence for a social immune response at the group level; however, individually, exposed bees interacted with the larva more frequently than their unexposed nestmates. While this could increase virus transmission from adults to larvae, it could also represent a hygienic response to increase grooming when an infection is detected. Together, our findings underline the complexity of disease dynamics in complex social animal systems.

Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

Efficacy and trade-offs of an innovative hyperthermia device to control Varroa destructor in honeybee colonies

For decades, the honeybee, Apis mellifera, has suffered from severe colony losses due to the ectoparasitic mite Varroa destructor. Various strategies based on chemicals fail to adequately control varroa mite populations, and often comprise side-effects on the host, parasite resistance and residues in hive products. Reduced temperature tolerance of V. destructor compared to its host has long been recognised and accordingly, the potential of hyperthermia to disrupt mite reproduction within honeybee brood cells or even kill adult parasites. Yet, earlier studies on hyperthermia remain largely anecdotal, and readily implementable solutions have so far been lacking. This study investigates autonomously controlled interval heating from within brood combs throughout the season compared to control colonies maintained according to good apicultural practice. We documented treatment-dependent colony growth dynamics and honey production at three apiaries, complemented by regular monitoring of varroa mite levels and comprehensive digital assessments of brood development over time. Our one-year field trial suggests the evaluated hyperthermia device efficiently suppresses mite populations below critical thresholds until autumn. Whilst a general winter reference-curing revealed similar mite infestations of colonies previously treated with hyperthermia versus formic acid (control), only the latter imposed substantial overwintering burdens indirectly through frequent late-season queen supersedure. However, relative to targeted pupae, increased mortality particularly of heat-treated non-target brood stages (eggs and larvae) appeared to trigger compensatory colony-level responses, translating into temporarily decreased numbers of adult workers coupled with increased pollen foraging and overall lower honey harvests. Valuable insights into previously unrecognised side-effects of hyperthermia and mitigation thereof may ultimately permit successful routine applications of this chemical-free approach to combat the major threat to honeybees worldwide.

Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box-Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7-10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 μmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.

Oxytetracycline-resistant Paenibacillus larvae identified in commercial beekeeping operations in Saskatchewan using pooled honey sampling.

American foulbrood (AFB) is an infectious disease of honey bee brood caused by the endospore-forming bacterium Paenibacillus larvae. P. larvae spores are resilient in the environment, thus colonies with clinical signs of AFB are often destroyed by burning to eradicate the causative agent. To prevent outbreaks of AFB, oxytetracycline metaphylaxis is widely used in North America, resulting in sustained selective pressure for oxytetracycline resistance in P. larvae. To determine if antimicrobial resistance (AMR) is present among P. larvae isolates from commercial beekeeping operations in Saskatchewan, Canada, we performed antimicrobial susceptibility testing of 718 P. larvae samples cultured from pooled, extracted honey collected from 52 beekeepers over a 2-y period, 2019 and 2020. We found that 65 of 718 (9%) P. larvae samples collected from 8 beekeepers were resistant to oxytetracycline with minimum inhibitory concentration (MIC) values of 64-256 µg/mL. Eight of 718 (1%) samples from 4 beekeepers had intermediate resistance to oxytetracycline (MIC: 4-8 µg/mL). Susceptibility testing for tylosin and lincomycin indicated that P. larvae in Saskatchewan continue to be susceptible to these antimicrobials (tylosin MIC: <1 µg/mL, lincomycin MIC: ≤2 µg/mL). Most oxytetracycline-resistant P. larvae samples were identified in northeastern Saskatchewan. Whole-genome sequence analysis identified the P. larvae-specific plasmid pMA67 with tetracycline-resistance gene tet(L) in 9 of 11 oxytetracycline-resistant P. larvae isolates sequenced. Our results highlight the advantage of using pooled, extracted honey as a surveillance tool for monitoring AMR in P. larvae.