Fetal Alcohol Spectrum Disorder: The Honey Bee as a Social Animal Model.

Animal models have been essential for advancing research of fetal alcohol spectrum disorder (FASD) in humans, but few animal species effectively replicate the behavioural and clinical signs of FASD. The honey bee (Apis mellifera) is a previously unexplored research model for FASD that offers the distinct benefit of highly social behaviour. In this study, we chronically exposed honey bee larvae to incremental concentrations of 0, 3, 6, and 10% ethanol in the larval diet using an in vitro rearing protocol and measured developmental time and survival to adult eclosion, as well as body weight and motor activity of newly emerged adult bees. Larvae reared on 6 and 10% dietary ethanol demonstrated significant, dose-responsive delays to pupation and decreased survival and adult body weight. All ethanol-reared adults showed significantly decreased motor activity. These results suggest that honey bees may be a suitable social animal model for future FASD research.

Fall Treatment with Fumagillin Contributes to an Overwinter Shift in Vairimorpha Species Prevalence in Honey Bee Colonies in Western Canada.

(1) Background: Microsporidiosis (nosemosis) is an intestinal disorder of adult honey bees caused by the microsporidian pathogens Vairimorpha apis and Vairimorpha ceranae. In Canada, fumagillin is an approved antibiotic used to treat this disease. However, the recommended dosage is based on efficacy studies for V. apis, the native pathogen in European honey bees. Since the detection of V. ceranae in Apis mellifera, V. ceranae became more prevalent in managed European honey bees and seems to have replaced V. apis due to yet unknown reasons. (2) Methods: This colony study investigated the efficacy of fumagillin administered in the fall to colonies infected with both V. apis and V. ceranae and its effects on the Vairimorpha species' prevalence overwinter. Spore loads in control and fumagillin-treated colonies were analysed by microscopy; Vairimorpha species prevalence was determined molecularly and infection and treatment effects on colony productivity were assessed. (3) Results: Fall fumagillin treatment was associated with a temporary reduction in spore load, but there was no difference in spore loads between treated and control colonies the following spring. Interestingly, fumagillin-treated colonies had a significantly greater prevalence of V. ceranae relative to V. apis the following spring, suggesting fumagillin is less effective in controlling V. ceranae.

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.

Comparison of individual and pooled sampling methods for estimation of Vairimorpha (Nosema) spp. levels in experimentally infected honey bee colonies.

The microsporidian pathogens Vairimorpha apis and V. ceranae are known to cause intestinal infection in honey bees and are associated with decreased colony productivity and colony loss. The widely accepted method for determining Vairimorpha colony infection level for risk assessment and antibiotic treatment is based on spore counts of 60 pooled worker bees using light microscopy. Given that honey bee colonies consist of as many as 1,000 times more individuals, the number of bees collected for Vairimorpha detection may significantly impact the estimated colony infection level, especially in the case of uneven distribution of high- and low-infected individuals within a hive. Hence, we compared the frequency and severity of Vairimorpha infection in individual bees to pooled samples of 60, 120, and 180 bees, as well as compared the Vairimorpha spp. prevalence in pooled samples of 60 and 180 bees. Overall, we did not find significant differences in spore counts in pooled samples containing incremental numbers of bees, although we observed that, in less-infected colonies, a low frequency of highly infected individuals influenced the estimated colony infection level. Moreover, Vairimorpha spp. prevalence did not differ significantly among the pooled bee samples tested. Increasing the number of pooled bees from the recommended 60 bees to 180 bees did not yield a more accurate representation of colony infection level for highly infected colonies, but the clinical importance of a low frequency of highly infected individuals in less-infected colonies needs to be addressed in future studies.

Investigation of Melissococcus plutonius isolates from 3 outbreaks of European foulbrood disease in commercial beekeeping operations in western Canada.

European foulbrood (EFB) disease is an economically important bacterial disease of honey bee larvae caused by enteric infection with Melissococcus plutonius. In this study, we investigated 3 clinical outbreaks of EFB disease in commercial beekeeping operations in western Canada in the summer of 2020 and characterized the Melissococcus plutonius isolates cultured from these outbreaks according to genetic multi-locus sequence type and i n vitro larval pathogenicity. We isolated M. plutonius sequence type 19 from EFB outbreaks in British Columbia and Alberta, and a novel M. plutonius sequence type 36 from an EFB outbreak in Saskatchewan. In vitro larval infection with each M. plutonius isolate was associated with decreased larval survival in vitro by 58.3 to 70.8% (P < 0.001) compared to non-infected controls. Further elucidation of mechanisms of virulence of M. plutonius, paired with epidemiologic investigation, is imperative to improve EFB management strategies and mitigate risks of EFB outbreaks in western Canada.

Enquête sur des isolats de Melissococcus plutonius provenant de trois éclosions de loque e uropéenne dans des exploitations apicoles commerciales de l'Ouest canadien. La loque européenne (EFB) est une maladie bactérienne économiquement importante des larves d'abeilles mellifères causée par une infection entérique par Melissococcus plutonius. Dans cette étude, nous avons enquêté sur trois éclosions cliniques de la maladie EFB dans des exploitations apicoles commerciales dans l'ouest du Canada à l'été 2020 et caractérisé les isolats de Melissococcus plutonius cultivés à partir de ces éclosions selon le typage génomique multilocus et la pathogénicité larvaire in vitro. Nous avons isolé le type de séquence 19 de M. plutonius des éclosions d'EFB en Colombie-Britannique et en Alberta, et une nouvelle séquence de type 36 de M. plutonius d'une éclosion d'EFB en Saskatchewan. L'infection larvaire in vitro avec chaque isolat de M. plutonius était associée à une diminution de la survie larvaire in vitro de 58,3 à 70,8 % (P < 0,001) par rapport aux témoins non infectés. Une élucidation plus poussée des mécanismes de virulence de M. plutonius, associée à une enquête épidémiologique, est impérative pour améliorer les stratégies de gestion de l'EFB et atténuer les risques d'épidémies d'EFB dans l'Ouest canadien.(Traduit par Dr Serge Messier).

Establishment of apiary-level risk of American foulbrood through the detection of Paenibacillus larvae spores in pooled, extracted honey in Saskatchewan.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), produces spores that may be detectable within honey. We analyzed the spore content of pooled, extracted honey from 52 large-scale (L) and 64 small-scale (S) Saskatchewan beekeepers over a two-year period (2019-2020). Our objectives were: (i) establish reliable prognostic reference ranges for spore concentrations in extracted honey to determine future AFB risk at the apiary level; (ii) identify management practices as targets for mitigation of risk. P. larvae spores were detected in 753 of 1476 samples (51%). Beekeepers were stratified into low (< 2 spores/gram), moderate (2- < 100 spores/gram), and high (≥ 100 spores/gram) risk categories. Of forty-nine L beekeepers sampled in 2019, those that reported AFB in 2020 included 0/26 low, 3/18 moderate, and 3/5 high risk. Of twenty-seven L beekeepers sampled in 2020, those that reported AFB in 2021 included 0/11 low, 2/14 moderate, and 1/2 high risk. Predictive modelling included indoor overwintering of hives, purchase of used equipment, movement of honey-producing colonies between apiaries, beekeeper demographic, and antimicrobial use as risk category predictors. Saskatchewan beekeepers with fewer than 2 spores/gram in extracted honey that avoid high risk activities may be considered at low risk of AFB the following year.

Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees.

European foulbrood (EFB) is a disease of honey bee larvae caused by Melissococcus plutonius. In North America, oxytetracycline (OTC) is approved to combat EFB disease though tylosin (TYL) and lincomycin (LMC) are also registered for use against American foulbrood disease. Herein, we report and characterize an OTC-resistant M. plutonius isolate from British Columbia, Canada, providing an antimicrobial sensitivity to the three approved antibiotics and studying their abilities to alter larval survival in an in vitro infection model. Specifically, we investigated OTC, TYL, and LMC as potential treatment options for EFB disease using laboratory-reared larvae infected with M. plutonius. The utility of the three antibiotics were compared through an experimental design that either mimicked metaphylaxis or antimicrobial intervention. At varying concentrations, all three antibiotics prevented clinical signs of EFB disease following infection with M. plutonius 2019BC1 in vitro. This included treatment with 100 µg/mL of OTC, a concentration that was ~ 3× the minimum inhibitory concentration measured to inhibit the strain in nutrient broth. Additionally, we noted high larval mortality in groups treated with doses of OTC corresponding to ~ 30× the dose required to eliminate bacterial growth in vitro. In contrast, TYL and LMC were not toxic to larvae at concentrations that exceed field use. As we continue to investigate antimicrobial resistance (AMR) profiles of M. plutonius from known EFB outbreaks, we expect a range of AMR phenotypes, reiterating the importance of expanding current therapeutic options along with alternative management practices to suppress this disease.

Comparison of individual hive and apiary-level sample types for spores of Paenibacillus larvae in Saskatchewan honey bee operations.

Three commercial honey bee operations in Saskatchewan, Canada, with outbreaks of American foulbrood (AFB) and recent or ongoing metaphylactic antibiotic use were intensively sampled to detect spores of Paenibacillus larvae during the summer of 2019. Here, we compared spore concentrations in different sample types within individual hives, assessed the surrogacy potential of honey collected from honey supers in place of brood chamber honey or adult bees within hives, and evaluated the ability of pooled, extracted honey to predict the degree of spore contamination identified through individual hive testing. Samples of honey and bees from hives within apiaries with a recent, confirmed case of AFB in a single hive (index apiaries) and apiaries without clinical evidence of AFB (unaffected apiaries), as well as pooled, apiary-level honey samples from end-of-season extraction, were collected and cultured to detect and enumerate spores. Only a few hives were heavily contaminated by spores in any given apiary. All operations were different from one another with regard to both the overall degree of spore contamination across apiaries and the distribution of spores between index apiaries and unaffected apiaries. Within operations, individual hive spore concentrations in unaffected apiaries were significantly different from index apiaries in the brood chamber (BC) honey, honey super (HS) honey, and BC bees of one of three operations. Across all operations, BC honey was best for discriminating index apiaries from unaffected apiaries (p = 0.001), followed by HS honey (p = 0.06), and BC bees (p = 0.398). HS honey positively correlated with both BC honey (rs = 0.76, p < 0.0001) and bees (rs = 0.50, p < 0.0001) and may be useful as a surrogate for either. Spore concentrations in pooled, extracted honey seem to have predictive potential for overall spore contamination within each operation and may have prognostic value in assessing the risk of future AFB outbreaks at the apiary (or operation) level.

Testicular Changes of Honey Bee Drones, Apis mellifera (Hymenoptera: Apidae), During Sexual Maturation.

The normal developmental anatomy and histology of the reproductive tract of the honey bee drone, Apis mellifera (Linnaeus, 1758), has been well documented. The post-emergence maturation changes of the accessory glands are likewise well understood, but the normal histological changes of the testicle undergoing physiologic atrophy are not well characterized. To address this knowledge gap, herein we describe the anatomy and sequential histological stages of normal testicular atrophy of drones sampled daily from emergence to sexual maturity in the spring (June) and early summer (July). Testicular histological changes during maturation are characterized by the following stages: I) conclusion of spermiogenesis; II) evacuation of spermatodesms from tubular lumens; III) progressive follicular cell atrophy, and IV) complete atrophy and collapse of testicular parenchyma. Tubular changes occur in a basilar to apical direction where segments closer to the vas deferens are histologically more mature than corresponding apical segments. In addition, the rate of testicular maturation was found to change with seasonal progression. This description of physiologic testicular atrophy should be useful for future studies investigating potential pathological effects of stressors on drone testes during sexual maturation.

Reproductive fitness of honey bee queens exposed to thiamethoxam during development.

The productivity and survival of honey bee (Apis mellifera) colonies depend on queen bee health. Colony-level neonicotinoid exposure has negative effects on reproductive fitness of honey bee queens. However, it is unclear if the observed effects are a direct outcome of neonicotinoid toxicity or result from suboptimal care of developing queens by exposed workers. The aim of this study was to evaluate larval survival, reproductive fitness, and histopathology of honey bee queens exposed to incremental doses (0, 5, 50 ng) of the neonicotinoid thiamethoxam (THI) applied directly to individual late larvae (7 days post-oviposition) of queens. The 5 ng dose represents a calculated high environmental level of exposure for honey bee queen larvae. Morphometric evaluation revealed that the total area of mandibular gland epithelium in queens exposed to 5 and 50 ng THI was reduced by 14% (P = .12) and 25% (P = .001), respectively. Decreased mandibular gland size may alter pheromone production, which could in part explain previously observed negative effects of THI on the reproductive fitness of queens. We also found that late larval exposure to THI reduced larval and pupal survival and decreased sperm viability in mated queens. These changes may interfere with queen development and reproductive longevity.

Investigation of clinical outbreaks of American foulbrood in honey-bee operations in Saskatchewan.

Four outbreaks of American foulbrood were investigated in honey-bee operations in Saskatchewan during the summer of 2019. Clinical signs were confirmed by the Saskatchewan Provincial Specialist in Apiculture and the causative agent was cultured and identified through matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Evaluation of management practices revealed off-label metaphylactic use of oxytetracycline in 3 of 4 operations and a discontinuation of antibiotic use in the fourth. Recent regulatory changes regarding access to medically important antimicrobials has provided an opportunity for veterinarians to promote evidence-based use of antimicrobials in apiculture while safe-guarding the health of commercial honeybee populations and the economic viability of their producers.

Enquête sur des poussées de cas cliniques de loque américaine dans des opérations d'abeilles mellifères en Saskatchewan. Quatre poussées de cas de loque américaine furent investiguées dans des opérations d'abeilles mellifères en Saskatchewan durant l'été 2019. Les signes cliniques furent confirmés par le Spécialiste provincial en apiculture de la Saskatchewan et l'agent causal fut cultivé et identifié par spectroscopie de masse par ionisation laser assistée par une matrice et analyse à temps de vol (MALDI-TOF MS). Une évaluation des pratiques de gestion a révélé l'utilisation métaphylactique en dérogation d'oxytétracycline dans trois des quatre opérations et un arrêt de l'utilisation d'antibiotique dans la quatrième. Des changements réglementaires récents concernant l'accès à des antimicrobiens importants médicalement ont fourni une opportunité aux vétérinaires de faire la promotion de l'utilisation factuelle des antimicrobiens en apiculture tout en conservant la santé des populations d'abeilles mellifères et la viabilité économique des apiculteurs.(Traduit par Dr Serge Messier).

In Vitro Effects of Pesticides on European Foulbrood in Honeybee Larvae.

Neonicotinoid and fungicide exposure has been linked to immunosuppression and increased susceptibility to disease in honeybees (Apis mellifera). European foulbrood, caused by the bacterium Melissococcus plutonius, is a disease of honeybee larvae which causes economic hardship for commercial beekeepers, in particular those whose colonies pollinate blueberries. We report for the first time in Canada, an atypical variant of M. plutonius isolated from a blueberry-pollinating colony. With this isolate, we used an in vitro larval infection system to study the effects of pesticide exposure on the development of European foulbrood disease. Pesticide doses tested were excessive (thiamethoxam and pyrimethanil) or maximal field-relevant (propiconazole and boscalid). We found that chronic exposure to the combination of thiamethoxam and propiconazole significantly decreased the survival of larvae infected with M. plutonius, while larvae chronically exposed to thiamethoxam and/or boscalid or pyrimethanil did not experience significant increases in mortality from M. plutonius infection in vitro. Based on these results, individual, calculated field-realistic residues of thiamethoxam and/or boscalid or pyrimethanil are unlikely to increase mortality from European foulbrood disease in honeybee worker brood, while the effects of field-relevant exposure to thiamethoxam and propiconazole on larval mortality from European foulbrood warrant further study.

Effects of chronic dietary thiamethoxam and prothioconazole exposure on Apis mellifera worker adults and brood.

BACKGROUND: Chronic exposure of honey bees (Apis mellifera Linnaeus) to the neonicotinoid thiamethoxam and the fungicide prothioconazole is common during foraging in agricultural landscapes. We evaluated the survival and hypopharyngeal gland development of adult worker honey bees, and the survival of the worker brood when chronically exposed to thiamethoxam or thiamethoxam and prothioconazole in combination. RESULTS: We found that 30 days of exposure to 40 µg kg-1 of thiamethoxam significantly (P < 0.001) increased the frequency of death in worker adults by four times relative to solvent control. The worker brood required 23 times higher doses of thiamethoxam (1 mg L-1 or 909 µg kg-1 ) before a significant (P = 0.04), 3.9 times increase in frequency of death was observed relative to solvent control. No additive effects of simultaneous exposure of worker adults or brood to thiamethoxam and prothioconazole were observed. At day 8 and day 12, the hypopharyngeal gland acinar diameter was not significantly different (P > 0.05) between controls and adult workers exposed to thiamethoxam and/or prothioconazole. CONCLUSION: These results indicate that chronic exposure to field-realistic doses of thiamethoxam and/or prothioconazole are unlikely to affect the survival of adult workers and brood. © 2019 Society of Chemical Industry.

Deformed Wing Virus Infection in Honey Bees (Apis mellifera L.).

Deformed wing virus (DWV) is a single-stranded RNA virus of honey bees (Apis mellifera L.) transmitted by the parasitic mite Varroa destructor. Although DWV represents a major threat to honey bee health worldwide, the pathological basis of DWV infection is not well documented. The objective of this study was to investigate clinicopathological and histological aspects of natural DWV infection in honey bee workers. Emergence of worker honey bees was observed in 5 colonies that were clinically affected with DWV and the newly emerged bees were collected for histopathology. DWV-affected bees were 2 times slower to emerge and had 30% higher mortality compared to clinically normal bees. Hypopharyngeal glands in bees with DWV were hypoplastic, with fewer intracytoplasmic secretory vesicles; cells affected by apoptosis were observed more frequently. Mandibular glands were hypoplastic and were lined by cuboidal epithelium in severely affected bees compared to tall columnar epithelium in nonaffected bees. The DWV load was on average 1.7 × 106 times higher (P < .001) in the severely affected workers compared to aged-matched sister honey bee workers that were not affected by deformed wing disease based on gross examination. Thus, DWV infection is associated with prolonged emergence, increased mortality during emergence, and hypoplasia of hypopharyngeal and mandibular glands in newly emerged worker honey bees in addition to previously reported deformed wing abnormalities.

Chronic High-Dose Neonicotinoid Exposure Decreases Overwinter Survival of Apis mellifera L.

Overwinter colony mortality is an ongoing challenge for North American beekeepers. During winter, honey bee colonies rely on stored honey and beebread, which is frequently contaminated with the neonicotinoid insecticides clothianidin and thiamethoxam. To determine whether neonicotinoid exposure affects overwinter survival of Apis mellifera L., we chronically exposed overwintering field colonies and winter workers in the laboratory to thiamethoxam or clothianidin at different concentrations and monitored survival and feed consumption. We also investigated the sublethal effects of chronic thiamethoxam exposure on colony pathogen load, queen quality, and colony temperature regulation. Under field conditions, high doses of thiamethoxam significantly increased overwinter mortality compared to controls, with field-realistic doses of thiamethoxam showing no significant effect on colony overwinter survival. Under laboratory conditions, chronic neonicotinoid exposure significantly decreased survival of winter workers relative to negative control at all doses tested. Chronic high-dose thiamethoxam exposure was not shown to impact pathogen load or queen quality, and field-realistic concentrations of thiamethoxam did not affect colony temperature homeostasis. Taken together, these results demonstrate that chronic environmental neonicotinoid exposure significantly decreases survival of winter workers in the laboratory, but only chronic high-dose thiamethoxam significantly decreases overwinter survival of colonies in the field.

Comparative chronic toxicity of three neonicotinoids on New Zealand packaged honey bees.

BACKGROUND: Thiamethoxam, clothianidin, and imidacloprid are the most commonly used neonicotinoid insecticides on the Canadian prairies. There is widespread contamination of nectar and pollen with neonicotinoids, at concentrations which are sublethal for honey bees (Apis mellifera Linnaeus). OBJECTIVE: We compared the effects of chronic, sublethal exposure to the three most commonly used neonicotinoids on honey bee colonies established from New Zealand packaged bees using colony weight gain, brood area, and population size as measures of colony performance. METHODS: From May 7 to July 29, 2016 (12 weeks), sixty-eight colonies received weekly feedings of sugar syrup and pollen patties containing 0 nM, 20 nM (median environmental dose), or 80 nM (high environmental dose) of one of three neonicotinoids (thiamethoxam, clothianidin, and imidacloprid). Colonies were weighed at three-week intervals. Brood area and population size were determined from digital images of colonies at week 12. Statistical analyses were performed by ANOVA and mixed models. RESULTS: There was a significant negative effect (-30%, p<0.01) on colony weight gain (honey production) after 9 and 12 weeks of exposure to 80 nM of thiamethoxam, clothianidin, or imidacloprid and on bee cluster size (-21%, p<0.05) after 12 weeks. Analysis of brood area and number of adult bees lacked adequate (>80%) statistical power to detect an effect. CONCLUSIONS: Chronic exposure of honey bees to high environmental doses of neonicotinoids has negative effects on honey production. Brood area appears to be less sensitive to detect sublethal effects of neonicotinoids.