Developing an in vivo toxicity assay for RNAi risk assessment in honey bees, Apis mellifera L.

Maize plants expressing dsRNA for the management of Diabrotica virgifera virgifera are likely to be commercially available by the end of this decade. Honey bees, Apis mellifera, can potentially be exposed to pollen from transformed maize expressing dsRNA. Consequently, evaluation of the biological impacts of RNAi in honey bees is a fundamental component for ecological risk assessment. The insecticidal activity of a known lethal dsRNA target for D. v. virgifera, the vATPase subunit A, was evaluated in larval and adult honey bees. Activity of both D. v. virgifera (Dvv)- and A. mellifera (Am)-specific dsRNA was tested by dietary exposure to dsRNA. Larval development, survival, adult eclosion, adult life span and relative gene expression were evaluated. The results of these tests indicated that Dvv vATPase-A dsRNA has limited effects on larval and adult honey bee survival. Importantly, no effects were observed upon exposure of Am vATPase-A dsRNA suggesting that the lack of response involves factors other than sequence specificity. The results from this study provide guidance for future RNAi risk analyses and for the development of a risk assessment framework that incorporates similar hazard assessments.

Evaluating the impact of a community developed collaborative project for the prevention of early childhood caries: the Healthy Smile Happy Child project.

INTRODUCTION: To determine the effectiveness of the Healthy Smile Happy Child (HSHC) project, a community-developed initiative promoting early childhood oral health in Manitoba, Canada. Specific aims were to assess improvements in caregiver knowledge, attitudes, and behaviours relating to early childhood oral health, and the burden of early childhood caries (ECC) and severe ECC (S-ECC). METHODS: A serial cross-sectional study design was selected to contrast findings following the Healthy Smile Happy Child (HSHC) campaign in four communities with the previous baseline data. One community was a remote First Nation in northern Manitoba and another was a rural First Nation in southern Manitoba. The other two communities were urban centres, one of which was located in northern Manitoba. A community-development approach was adopted for the project to foster community solutions to address ECC. Goals of the HSHC program were to promote the project in each community, use existing community-based programs and services to deliver the oral health promotion and ECC prevention activities, and recruit and train natural leaders to assist in program development and to deliver the ECC prevention program. The HSHC coordinator worked with communities to develop a comprehensive list of potential strategies to address ECC. Numerous activities occurred in each community to engage members and increase their knowledge of early childhood oral health and ultimately lead them to adopt preventive oral health practices for their young children. Children under 71 months of age and their primary caregivers participated in this follow-up study. A -value ≤0.05 was statistically significant. RESULTS: 319 children (mean age 38.2±18.6 months) and their primary caregivers participated. Significant improvements in caregiver knowledge and attitudes were observed following the HSHC campaign, including that baby teeth are important (98.8%), that decay involving primary teeth can impact on health (94.3%), and the importance of a dental visit by the first birthday (82.4%). Significantly more respondents indicated that their child had visited the dentist (50.2%) and had started brushing their child's teeth (86.7%) when compared to baseline. Overall, 52.0% had ECC, 38.6% had S-ECC. The mean deft score was 3.85±4.97 (range 0-20). There was no significant change is ECC prevalence between the follow-up and baseline investigations. However, age-adjusted logistic regression for S-ECC in this follow-up study revealed a significant reduction in prevalence compared with the baseline study (=0.021). Similarly, age-adjusted Poisson regression revealed that there were significant reductions in both the decayed teeth and decayed, extracted and filled teeth scores between follow-up and baseline study periods (0.016 and .0001, respectively). CONCLUSIONS: Follow-up study results suggest that the HSHC initiative may have contributed to improvements in caregiver knowledge, attitudes, and behaviours towards early childhood oral health and subsequently modest yet statistically significant reductions in caries scores and the prevalence of S-ECC.

Evaluating sub-lethal effects of orchard-applied pyrethroids using video-tracking software to quantify honey bee behaviors.

Managed honey bee, Apis mellifera L., colonies are contracted to pollinate fruit and nut orchards improving crop quality and yield. Colonies placed in orchards are potentially exposed to pyrethroid insecticides used for broad-spectrum pest control. Pyrethroids have been reported to pose minimal risk to bees due to their low application rates in the field and putative repellent properties. This repellency is believed to alter foraging behavior with the benefit of preventing bees from encountering a lethal dose in the field. However, sub-lethal exposure to pyrethroids may adversely impact bee behavior potentially resulting in social dysfunction or disruption of foraging. This study quantified behaviors associated with sub-lethal exposure to orchard-applied pyrethroids including, lambda-cyhalothrin, esfenvalerate, and permethrin, using video tracking software, Ethovision XT (Noldus Information Technologies). Bee locomotion, social interaction, and time spent near a food source were measured over a 24-h period. Bees treated with a pyrethroid traveled 30-71% less than control bees. Social interaction time decreased by 43% for bees treated with a high sub-lethal dose of esfenvalerate. Bees exposed to a high sub-lethal dose of permethrin spent 67% less time in social interaction and spent more than 5 times as long in the food zone compared to control bees.

Acaricide, fungicide and drug interactions in honey bees (Apis mellifera).

BACKGROUND: Chemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects. METHODOLOGY/PRINCIPAL FINDINGS: Laboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested. CONCLUSIONS/SIGNIFICANCE: Interactions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an important role. Evidence of non-transivity, year-to-year variation and induction of detoxication enzymes indicates that pesticide interactions in bees may be as complex as drug interactions in mammals.

Dentist's views on a province-wide campaign promoting early dental visits for young children.

INTRODUCTION: The Canadian Dental Association recommends that children have their first visit to a dental professional no later than 12 months of age. In 2010, the Manitoba Dental Association launched the Free First Visit (FFV) program to increase access to early visits in the province. The purpose of the study reported here was to survey dentists about their views on the FFV program and to gain an understanding of their attitudes and practice patterns relating to the oral health of infants and toddlers and first dental visits. METHODS: A survey was mailed to registered general and pediatric dentists in Manitoba according to a modified Dillman methodology. Dentists were asked about their views on the FFV, their knowledge of early childhood oral health and the timing of first dental visits. Descriptive statistics, bivariate analyses and logistic regression analyses were performed. A p value of 0.05 or less was considered significant. RESULTS: The overall response rate was 63.2% (375 eligible responses out of 593 surveys mailed). The majority of respondents were men (255/373 [68.4%]), and most respondents were general dentists (355/372 [95.4%]). A total of 63.5% (231/364) felt that the FFV program improved access to care, 64.6% (223/345) believed that public awareness of young children's oral health has increased, and 76.2% (266/349) thought that the FFV initiative should continue past the planned end date of March 31, 2013. On average (± standard deviation), respondents thought that the first dental visit should occur at 18.1 ± 10.0 months, but in their practices, they actually recommended a slightly older age (18.9 ± 10.4 months). Compared with results from a previous survey, conducted in 2005, dentists who responded to this survey recommended that children have their first visit at a significantly younger age. A greater proportion of dentists reported seeing children 12-23 months of age in their practices than in the past (81.9% vs. 73.7%). CONCLUSIONS: A majority of dentists who responded to the survey approved of the FFV program and thought it should continue. Although these dentists recommended early first dental visits, the average age recommended by respondents was 6 months later than the target age of 12 months. It appears that, over time, dentists are becoming more aware of prevention and management techniques relating to infants and toddlers.

Using video-tracking to assess sublethal effects of pesticides on honey bees (Apis mellifera L.).

Concern about the role of pesticides in honey bee decline has highlighted the need to examine the effects of sublethal exposure on bee behaviors. The video-tracking system EthoVisionXT (Noldus Information Technologies) was used to measure the effects of sublethal exposure to tau-fluvalinate and imidacloprid on honey bee locomotion, interactions, and time spent near a food source over a 24-h observation period. Bees were either treated topically with 0.3, 1.5, and 3 µg tau-fluvalinate or exposed to 0.05, 0.5, 5.0, 50, and 500 ppb imidacloprid in a sugar agar cube. Tau-fluvalinate caused a significant reduction in distance moved at all dose levels (p < 0.05), as did 50 and 500 ppb imidacloprid (p < 0.001). Bees exposed to 50 and 500 ppb spent significantly more time near the food source than control bees (p < 0.05). Interaction time decreased as time in the food zone increased for both chemicals. This study documents that video-tracking of bee behavior can enhance current protocols for measuring the effects of pesticides on honey bees at sublethal levels. It may provide a means of identifying problematic compounds for further testing.

Comparative toxicity of acaricides to honey bee (Hymenoptera: Apidae) workers and queens.

Acaricides are used to treat honey bee (Apis mellifera L.) colonies to control the varroa mite (Varroa destructor Anderson & Trueman), a worldwide threat to honey bee health. Although acaricides control a serious honey bee parasite and mitigate bee loss, they may cause harm to bees as well. We topically applied five acaricides, each with a different mode of action, to young adult queen and worker bees to generate dose-response curves and LD50. Twenty-four hours after treatment, queens were found to be three-times more tolerant of tau-fluvalinate and six-times more tolerant of thymol than workers when adjusted for body weight differences between workers (108 mg) and queens (180 mg). Queens survived the highest administered doses of fenpyroximate (1620 microg/g) and coumaphos (2700 microg/g) indicating that queens are at least 11-fold more tolerant of coumaphos and at least 54-fold more tolerant of fenpyroximate than workers. However, queens treated with as little as 54 microg/g of fenpyroximate exhibited reduced survival over 6 wk after treatment. Amitraz was the only acaricide tested for which queens were not more tolerant than workers. The striking difference in acaricide tolerance of queen and worker honey bees suggests physiological differences in how the two castes are affected by xenobiotics.

Oxalic acid: a prospective tool for reducing Varroa mite populations in package bees.

Numerous studies have investigated using oxalic acid (OA) to control Varroa mites in honey bee colonies. In contrast, techniques for treating package bees with OA have not been investigated. The goal of this study was to develop a protocol for using OA to reduce mite infestation in package bees. We made 97 mini packages of Varroa-infested adult bees. Each package contained 1,613 +/- 18 bees and 92 +/- 3 mites, and represented an experimental unit. We prepared a 2.8% solution of OA by mixing 35 g OA with 1 l of sugar water (sugar:water = 1:1; w:w). Eight treatments were assigned to the packages based on previous laboratory bioassays that characterized the acute contact toxicity of OA to mites and bees. We administered the treatments by spraying the OA solution directly on the bees through the mesh screen cage using a pressurized air brush and quantified mite and bee mortality over a 10-day period. Our results support applying an optimum volume of 3.0 ml of a 2.8% OA solution per 1,000 bees to packages for effective mite control with minimal adult bee mortality. The outcome of our research provides beekeepers and package bee shippers guidance for using OA to reduce mite populations in package bees.

Acute contact toxicity of oxalic acid to Varroa destructor (Acari: Varroidae) and their Apis mellifera (Hymenoptera: Apidae) hosts in laboratory bioassays.

Laboratory bioassays were performed to characterize the acute contact toxicity of oxalic acid (OA) to Varroa destructor (Anderson and Trueman) and their honey bee hosts (Apis mellifera L.). Specifically, glass-vial residual bioassays were conducted to determine the lethal concentration of OA for V. destructor, and topical applications of OA in acetone were conducted to determine the lethal dose for honey bees. The results indicate that OA has a low acute toxicity to honey bees and a high acute toxicity to mites. The toxicity data will help guide scientists in delivering optimum dosages of OA to the parasite and its host, and will be useful in making treatment recommendations. The data will also facilitate future comparisons of toxicity if mite resistance to OA becomes evident.