Comparing the efficacy of synthetic Varroacides and Varroa destructor phenotypic resistance using Apiarium and Mason jar bioassay techniques.

BACKGROUND: Varroa mite, Varroa destructor, is a major threat for honey bee, Apis mellifera, colonies. Beekeepers have used synthetic Varroacides against Varroa mite for decades, but resistance to organophosphates, pyrethroids and formamidine has been reported in many locations worldwide. The goals of this study were to develop a reliable bioassay to assess efficacy and phenotypic resistance to commercial Varroacides. In this study, efficacy and Varroa resistance was evaluated using the Apiarium technique in comparison to the Mason jar method. RESULTS: Among tested Varroacides, a high efficacy (89%) for Apivar was identified when compared to Bayvarol (58%), Apistan (44%) and CheckMite (6%), in a 24 h assessment. We also found that CheckMite was toxic to bees in the Mason jar method. In addition, the Apiarium technique revealed a case of phenotypic resistance to Bayvarol, Apistan and CheckMite in the mite population evaluated. CONCLUSION: A laboratory protocol was developed using the Apiarium method to evaluate Apivar efficacy. Collectively, the findings indicated that the Apiarium methodology provided a reliable technique to measure Varroacide efficacy and determine the presence of phenotypic resistance in V. destructor. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In Vivo Inhibitory Assessment of Potential Antifungal Agents on Nosema ceranae Proliferation in Honey Bees.

Nosema ceranae Fries, 1996, causes contagious fungal nosemosis disease in managed honey bees, Apis mellifera L. It is associated around the world with winter losses and colony collapse disorder. We used a laboratory in vivo screening assay to test curcumin, fenbendazole, nitrofurazone and ornidazole against N. ceranae in honey bees to identify novel compounds with anti-nosemosis activity compared to the commercially available medication Fumagilin-B®. Over a 20-day period, Nosema-inoculated bees in Plexiglas cages were orally treated with subsequent dilutions of candidate compounds, or Fumagilin-B® at the recommended dose, with three replicates per treatment. Outcomes indicated that fenbendazole suppressed Nosema spore proliferation, resulting in lower spore abundance in live bees (0.36 ± 1.18 million spores per bee) and dead bees (0.03 ± 0.25 million spores per bee), in comparison to Fumagilin-B®-treated live bees (3.21 ± 2.19 million spores per bee) and dead bees (3.5 ± 0.6 million spores per bee). Our findings suggest that Fumagilin-B® at the recommended dose suppressed Nosema. However, it was also likely responsible for killing Nosema-infected bees (24% mortality). Bees treated with fenbendazole experienced a greater survival probability (71%), followed by ornidazole (69%), compared to Nosema-infected non-treated control bees (20%). This research revealed that among screened compounds, fenbendazole, along with ornidazole, has potential effective antifungal activities against N. ceranae in a controlled laboratory environment.

Miticidal activity of fenazaquin and fenpyroximate against Varroa destructor, an ectoparasite of Apis mellifera.

BACKGROUND: The Varroa mite (Varroa destructor) is an ectoparasite that can affect the health of honey bees (Apis mellifera) and contributes to the loss of colony productivity. The limited availability of Varroacides with different modes of action in Canada has resulted in the development of chemical resistance in mite populations. Therefore, an urgent need to evaluate new potential miticides that are safe for bees and exhibit high efficacy against Varroa exists. In this study, the acute contact toxicity of 26 active ingredients (19 chemical classes), already available on the market, was evaluated on V. destructor and A. mellifera under laboratory conditions using an apiarium bioassay. In this assay, groups of Varroa-infested worker bees were exposed to different dilutions of candidate compounds. In semi-field trials, Varroa-infested honey bees were randomly treated with four vetted candidate compounds from the apiarium assay in mini-colonies. RESULTS: Among tested compounds, fenazaquin (quinazoline class) and fenpyroximate (pyrazole class) had higher mite mortality and lower bee mortality over a 24 h exposure period in apiariums. These two compounds, plus spirotetramat and spirodiclofen, were selected for semi-field evaluation based on the findings of the apiarium bioassay trials and previous laboratory studies. Consistent with the apiarium bioassay, semi-field results showed fenazaquin and fenpyroximate had high efficacy (>80%), reducing Varroa abundance by 80% and 68%, respectively. CONCLUSION: These findings suggest that fenazaquin would be an effective Varroacide, along with fenpyroximate, which was previously registered for in-hive use as Hivastan. Both compounds have the potential to provide beekeepers with an alternative option for managing Varroa mites in honey bee colonies. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

New bioassay cage methodology for in vitro studies on Varroa destructor and Apis mellifera.

Varroa destructor Anderson and Trueman, is an ectoparasitic mite of honey bees, Apis mellifera L., that has been considered a major cause of colony losses. Synthetic miticides have been developed and registered to manage this ectoparasite, however, resistance to registered pyrethroid and organophosphate Varroacides have already been reported in Canada. To test toxicity of miticides, current contact-based bioassay methods are designed to evaluate mites and bees separately, however, these methods are unlikely to give an accurate depiction of how miticides interact at the colony level. Therefore, the objective of this study was to develop a bioassay cage for testing the toxicity of miticides on honey bees and Varroa mites simultaneously using amitraz as a reference chemical. A 800 mL polypropylene plastic cage holding 100-150 bees was designed and officially named "Apiarium". A comparison of the effects of three subsequent dilutions of amitraz was conducted on: Varroa mites placed in glass vials, honey bees in glass Mason jars, and Varroa-infested bees in Apiariums. Our results indicated cumulative Varroa mortality was dose-dependent in the Apiarium after 4 h and 24 h assessments. Apiarium and glass vial treatments at 24 h also had high mite mortality and a positive polynomial regression between Varroa mortality and amitraz dose rates. Moreover, chemical application in the Apiarium was less toxic for bees compared to the Mason jar method. Considering these results, the Apiarium bioassay provides a simple, cheap and reliable method for simultaneous chemical screening on V. destructor and A. mellifera. Furthermore, as mites and bees are tested together, the Apiarium simulates a colony-like environment that provides a necessary bridge between laboratory bioassay testing and full field experimentation. The versatility of the Apiarium allows researchers to test a multitude of different honey bee bioassay experiments including miticide screening, delivery methods for chemical products, or development of new mite resistance-testing methodology.

Evaluation of potential miticide toxicity to Varroa destructor and honey bees, Apis mellifera, under laboratory conditions.

The honey bee, Apis mellifera L., is the world's most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

Influence of Honey Bee Genotype and Wintering Method on Wintering Performance of Varroa destructor (Parasitiformes: Varroidae)-Infected Honey Bee (Hymenoptera: Apidae) Colonies in a Northern Climate.

The objective of this study was to assess the effectiveness of a cooperative breeding program designed to enhance winter survival of honey bees (Apis mellifera L.) when exposed to high levels of varroa (Varroa destructor Anderson and Trueman) in outdoor-wintered and indoor-wintered colonies. Half of the colonies from selected and unselected stocks were randomly assigned to be treated with late autumn oxalic acid treatment or to be left untreated. Colonies were then randomly assigned to be wintered either indoors (n = 37) or outdoors (n = 40). Late autumn treatment with oxalic acid did not improve wintering performance. However, genotype of bees affected colony survival and the proportion of commercially viable colonies in spring, as indicated by greater rates of colony survival and commercially viable colonies for selected stock (43% survived and 33% were viable) in comparison to unselected stock (19% survived and 9% were viable) across all treatment groups. Indoor wintering improved spring bee population score, proportion of colonies surviving, and proportion of commercially viable colonies relative to outdoor wintering (73% of selected stock and 41% of unselected stock survived during indoor wintering). Selected stock showed better "tolerance" to varroa as the selected stock also maintained higher bee populations relative to unselected stock. However, there was no evidence of "resistance" in selected colonies (reduced mite densities). Collectively, this experiment showed that breeding can improve tolerance to varroa and this can help minimize colony loss through winter and improve colony wintering performance. Overall, colony wintering success of both genotypes of bees was better when colonies were wintered indoors than when colonies were wintered outdoors.

The Potential of Bee-Generated Carbon Dioxide for Control of Varroa Mite (Mesostigmata: Varroidae) in Indoor Overwintering Honey bee (Hymenoptera: Apidae) Colonies.

The objective of this study was to manipulate ventilation rate to characterize interactions between stocks of honey bees (Apis mellifera L.) and ventilation setting on varroa mite (Varroa destructor Anderson and Trueman) mortality in honey bee colonies kept indoors over winter. The first experiment used colonies established from stock selected locally for wintering performance under exposure to varroa (n = 6) and unselected bees (n = 6) to assess mite and bee mortality and levels of carbon dioxide (CO2) and oxygen (O2) in the bee cluster when kept under a simulated winter condition at 5°C. The second experiment, used colonies from selected bees (n = 10) and unselected bees (n = 12) that were exposed to either standard ventilation (14.4 liter/min per hive) or restricted ventilation (0.24 liter/min per hive, in a Plexiglas ventilation chamber) during a 16-d treatment period to assess the influence of restricted air flow on winter mortality rates of varroa mites and honey bees. Experiment 2 was repeated in early, mid-, and late winter. The first experiment showed that under unrestricted ventilation with CO2 concentrations averaging <2% there was no correlation between CO2 and varroa mite mortality when colonies were placed under low temperature. CO2 was negatively correlated with O2 in the bee cluster in both experiments. When ventilation was restricted, mean CO2 level (3.82 ± 0.31%, range 0.43-8.44%) increased by 200% relative to standard ventilation (1.29 ± 0.31%; range 0.09-5.26%) within the 16-d treatment period. The overall mite mortality rates and the reduction in mean abundance of varroa mite over time was greater under restricted ventilation (37 ± 4.2%) than under standard ventilation (23 ± 4.2%) but not affected by stock of bees during the treatment period. Selected bees showed overall greater mite mortality relative to unselected bees in both experiments. Restricting ventilation increased mite mortality, but did not affect worker bee mortality relative to that for colonies under standard ventilation. Restricted ventilation did not affect the overall level of Nosema compared with the control. However, there was an interaction between stock, season, and time of the trial. Unselected stock showed an increase in Nosema over time in the late winter trial that did not occur in the selected stock. In conclusion, these findings suggested that restricted ventilation has potential to suppress varroa mite in overwintering honey bee colonies via a low-cost and environmentally friendly measure.

The influence of Nosema (Microspora: Nosematidae) infection on honey bee (Hymenoptera: Apidae) defense against Varroa destructor (Mesostigmata: Varroidae).

The objectives of this study were to quantify the costs and benefits of co-parasitism with Varroa (Varroa destructor Anderson and Trueman) and Nosema (Nosema ceranae Fries and Nosema apis Zander) on honey bees (Apis mellifera L.) with different defense levels. Newly-emerged worker bees from either high-mite-mortality-rate (high-MMR) bees or low-mite-mortality-rate (low-MMR) bees were confined in forty bioassay cages which were either inoculated with Nosema spores [Nosema (+) group] or were left un-inoculated [Nosema (-) group]. Caged-bees were then inoculated with Varroa mites [Varroa (+) group] or were left untreated [Varroa (-) group]. This established four treatment combinations within each Nosema treatment group: (1) low-MMR Varroa (-), (2) high-MMR Varroa (-), (3) low-MMR Varroa (+) and (4) high-MMR Varroa (+), each with five replicates. Overall mite mortality in high-MMR bees (0.12±0.02 mites per day) was significantly greater than in the low-MMR bees (0.06±0.02 mites per day). In the Nosema (-) groups bee mortality was greater in high-MMR bees than low-MMR bees but only when bees had a higher mite burden. Overall, high-MMR bees in the Nosema (-) group showed greater reductions in mean abundance of mites over time compared with low-MMR bees, when inoculated with additional mites. However, high-MMR bees could not reduce mite load as well as in the Nosema (-) group when fed with Nosema spores. Mean abundance of Nosema spores in live bees and dead bees of both strains of bees was significantly greater in the Nosema (+) group. Molecular analyses confirmed the presence of both Nosema species in inoculated bees but N. ceranae was more abundant than N. apis and unlike N. apis increased over the course of the experiment. Collectively, this study showed differential mite mortality rates among different genotypes of bees, however, Nosema infection restrained Varroa removal success in high-MMR bees.

The effect of queen pheromone status on Varroa mite removal from honey bee colonies with different grooming ability.

The objective of this study was to assess the effects of honey bees (Apis mellifera L.) with different grooming ability and queen pheromone status on mortality rates of Varroa mites (Varroa destructor Anderson and Trueman), mite damage, and mortality rates of honey bees. Twenty-four small queenless colonies containing either stock selected for high rates of mite removal (n = 12) or unselected stock (n = 12) were maintained under constant darkness at 5 °C. Colonies were randomly assigned to be treated with one of three queen pheromone status treatments: (1) caged, mated queen, (2) a synthetic queen mandibular pheromone lure (QMP), or (3) queenless with no queen substitute. The results showed overall mite mortality rate was greater in stock selected for grooming than in unselected stock. There was a short term transitory increase in bee mortality rates in selected stock when compared to unselected stock. The presence of queen pheromone from either caged, mated queens or QMP enhanced mite removal from clusters of bees relative to queenless colonies over short periods of time and increased the variation in mite mortality over time relative to colonies without queen pheromone, but did not affect the proportion of damaged mites. The effects of source of bees on mite damage varied with time but damage to mites was not reliably related to mite mortality. In conclusion, this study showed differential mite removal of different stocks was possible under low temperature. Queen status should be considered when designing experiments using bioassays for grooming response.