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.

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.

Infection effects of the new microsporidian species Tubulinosema suzukii on its host Drosophila suzukii.

Microsporidian infections of insects are important natural constraints of population growth, often reducing lifespan, fecundity and fertility of the infected host. The recently discovered Tubulinosema suzukii infects Drosophila suzukii (spotted wing drosophila, SWD), an invasive pest of many fruit crops in North America and Europe. In laboratory tests, fitness effects on larval and adult stages were explored. High level infection after larval treatment caused up to 70% pupal mortality, a decreased lifespan and a 70% reduced oviposition of emerging adults in biparental infection clusters. A shift to higher proportion of female offspring compared to controls suggested a potential parthenogenetic effect after microsporidian infection. A clear sex-linkage of effects was noted; females were specifically impaired, as concluded from fecundity tests with only infected female parents. Additive effects were noted when both parental sexes were infected, whereas least effects were found with only infected male parents, though survival of males was most negatively affected if they were fed with T. suzukii spores in the adult stage. Although most negative effects on fitness parameters were revealed after larval treatment, infection of offspring was never higher than 4%, suggesting limited vertical transmission. For that reason, a self-reliant spread in natural SWD populations would probably only occur by spore release from cadavers or frass.

Molecular and morphological characterisation of a novel microsporidian species, Tubulinosema suzukii, infecting Drosophila suzukii (Diptera: Drosophilidae).

A microsporidium showing morphological characteristics typical of a Tubulinosema species was discovered in Drosophila suzukii. All developmental stages were diplokaryotic and grew in direct contact with the host cell cytoplasm. Spores from fresh preparations were ovoid to slightly pyriform and measured 4.29 × 2.47 µm in wet mount preparations. The spore wall consisted of a 125 nm thick endospore covered by a double layered exospore of 39 nm and 18 nm. The polar filament measured 67 µm in length, was slightly anisofilar and was arranged in ten coils in one or rarely two rows. The two posterior coils were 95 nm in diameter while the anterior coils were 115 nm in diameter. Early developmental stages were surrounded by electron-dense, 35.3 nm diameter, surface ornaments scattered over the membrane. Tubular elements with diameters of approximately 75 nm were seen attaching to the periphery of meronts and sporonts. Tissues infected included fat body, midgut and muscle. A 1915 bp rDNA fragment, covering the small subunit (SSU), the internal transcribed spacer (ITS) and the 5' end of the large subunit ribosomal DNA, was amplified by PCR and sequenced. Phylogenetic analyses of the SSU rDNA fragment revealed closest relationship to Tubulinosema pampeana (Host: Bombus atratus, South America) and Tubulinosema loxostegi (Host: Loxostege sticticalis, ubiquitous), but using the complete dataset of SSU-ITS-LSU rDNA genes revealed T. hippodamiae (Host: Hippodamiae convergens) as the most closely related species. Based on the morphological and genetic features a new species, Tubulinosema suzukii sp. nov., is proposed for this microsporidium isolated from D. suzukii.