NEONICOTINOID TOXICITY IN REPRODUCTIVE HONEY BEE CASTES

High pollinator losses of recent decades have been attributed to several causes including increased exposure to pesticides, particularly neonicotinoid insecticides. This has spiked re-evaluation of neonicotinoid use in agriculture and has heightened the need to enhance pesticide risk assessment. Currently, pesticide risk assessment for pollinators is based on assays focused predominantly on worker bee mortality in response to pesticide exposure. This approach has been criticized for overlooking sublethal toxic effects and its lack of proper evaluation of the reproductively active honey bee drones and queens. Accordingly, the overarching goal of this thesis was to incorporate the evaluation of honey bee reproductive castes in toxicologic assays through the use of “gold standard” research techniques routinely used in vertebrate toxicology studies. First, we reviewed, documented, and summarized the normal anatomical structures of the mated queen reproductive tract in order to establish reference material of the normal morphology expected in healthy queen bees. Next, we investigated the effect of thiamethoxam (THI), a commonly used neonicotinoid, on developing queens. We found that direct THI toxicity in queens can result in hypoplasia of the pheromone producing mandibular glands. Such morphologic organ changes precede compromised organ function. Therefore, it is reasonable to hypothesize that dysregulation and disruption of pheromone production in intoxicated queens may partially explain the increased queen failure rates reported by beekeepers in association with high colony losses. Importantly, this study highlights that THI can have a direct negative effect on queen bees, indicating that it may be prudent to include all castes in pesticide risk assessment. This finding is further supported by our final studies, where we describe that THI toxicity is highly caste and age specific. Namely, we found that developing (i.e., larval) queens are highly sensitive to THI toxicity, but become more resilient following emergence. The opposite was found to be true for drones. Furthermore, detoxification enzyme activity in bees is similarly caste and age specific, although the enzymes tested in our studies did not change in response to THI treatment. Overall, these finding indicate that using worker bees alone in pesticide risk assessment may be suboptimal since toxicity in workers may not fully reflect toxicity observed in other honey bee castes. In addition, applying histologic and biochemical assays in pesticide risk assessment can potentially enhance our understanding of bee toxicity, improve the detection of sublethal toxic changes, and assist in the establishment of safe dose ranges of pesticides to protect pollinators while ensuring proper protection of agricultural crops

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

<div><p>Background</p><p>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 (<i>Apis mellifera</i> Linnaeus).</p><p>Objective</p><p>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.</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

Weekly and cumulative feed consumption per colony over 12 weeks.

<p>Over twelve weeks, cumulative consumption of syrup (B) and pollen patty (D) was comparable for all experimental groups with the exception of colonies exposed to 20 nM thiamethoxam consuming 18.2% (2.98 kg) less syrup compared to controls. Shaded area indicates significant differences (P<0.01) in weekly pollen patty (C) consumption between control colonies and colonies exposed to 80 nM neonicotinoids. Treatment colonies were exposed to clothianidin (CLO), imidacloprid (IMD), or thiamethoxam (THI) at 20 or 80 nanomolar concentrations. Mean weekly (A, C) or cumulative (B, D) consumption per colony ± SD is indicated for each group. * significantly different from control, P<0.01. The timing of the canola and alfalfa bloom surrounding the study site is indicated (A, C).</p

Cumulative weight gain of colonies exposed to sublethal doses of individual neonicotinoids for twelve weeks.

<p>Treatment colonies were exposed to clothianidin (CLO) (A), imidacloprid (IMD) (B), or thiamethoxam (THI) (C) at 20 or 80 nanomolar concentrations. Colonies exposed to 80 nM CLO (A) and 80 nM IMD (B) demonstrated significant decreases in weight gain from controls at weeks 9 and 12 and week 9, respectively. The bars show mean cumulative colony weight gain ± SD for each group (left y-axis). The curves show mean cumulative consumption of neonicotinoid per colony ± SD in micromoles for the treatment groups (right y-axis). * significantly different from control, P<0.01. The timing of the canola and alfalfa bloom surrounding the study site is indicated (A).</p

Capped brood area of colonies exposed to sublethal doses of neonicotinoid for twelve weeks.

<p>Treatment colonies were exposed for twelve weeks to clothianidin (CLO), imidacloprid (IMD), or thiamethoxam (THI) at 20 or 80 nanomolar concentrations. Brood area was quantified by analysis of digital images of brood frames with brood recognition software. There was no statistical difference among experimental groups but analyses lacked adequate (>80%) statistical power due to high variability. Mean ± SD are indicated for each group.</p

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

Table_1_Are fungicides a driver of European foulbrood disease in honey bee colonies pollinating blueberries?.XLSX

IntroductionBlueberry producers in Canada depend heavily on pollination services provided by honey bees (Apis mellifera L.). Anecdotal reports indicate an increased incidence of European foulbrood (EFB), a bacterial disease caused by Melissococcus plutonius, is compromising pollination services and colony health. Fungicidal products are commonly used in blueberry production to prevent fungal diseases such as anthracnose and botrytis fruit rot. Pesticide exposure has been implicated in honey bee immunosuppression; however, the effects of commercial fungicidal products, commonly used during blueberry pollination, on honey bee larval susceptibility to EFB have not been investigated.MethodsUsing an in vitro infection model of EFB, we infected first instar honey bee larvae with M. plutonius 2019 BC1, a strain isolated from an EFB outbreak in British Columbia, Canada, and chronically exposed larvae to environmentally relevant concentrations of fungicide products over 6 days. Survival was monitored until pupation or eclosion.ResultsWe found that larvae chronically exposed to one, two, or three fungicidal products [Supra® Captan 80WDG (Captan), low concentration of Kenja™ 400SC (Kenja), Luna® Tranquility (Luna), and/or Switch® 62.5 WG (Switch)], did not significantly reduce survival from EFB relative to infected controls. When larvae were exposed to four fungicide products concurrently, we observed a significant 24.2% decrease in survival from M. plutonius infection (p = 0.0038). Similarly, higher concentrations of Kenja significantly reduced larval survival by 24.7–33.0% from EFB (p DiscussionThese in vitro results suggest that fungicides may contribute to larval susceptibility and response to M. plutonius infections. Further testing of other pesticide combinations is warranted as well as continued surveillance of pesticide residues in blueberry-pollinating colonies.</p

Image_1_Are fungicides a driver of European foulbrood disease in honey bee colonies pollinating blueberries?.JPEG

IntroductionBlueberry producers in Canada depend heavily on pollination services provided by honey bees (Apis mellifera L.). Anecdotal reports indicate an increased incidence of European foulbrood (EFB), a bacterial disease caused by Melissococcus plutonius, is compromising pollination services and colony health. Fungicidal products are commonly used in blueberry production to prevent fungal diseases such as anthracnose and botrytis fruit rot. Pesticide exposure has been implicated in honey bee immunosuppression; however, the effects of commercial fungicidal products, commonly used during blueberry pollination, on honey bee larval susceptibility to EFB have not been investigated.MethodsUsing an in vitro infection model of EFB, we infected first instar honey bee larvae with M. plutonius 2019 BC1, a strain isolated from an EFB outbreak in British Columbia, Canada, and chronically exposed larvae to environmentally relevant concentrations of fungicide products over 6 days. Survival was monitored until pupation or eclosion.ResultsWe found that larvae chronically exposed to one, two, or three fungicidal products [Supra® Captan 80WDG (Captan), low concentration of Kenja™ 400SC (Kenja), Luna® Tranquility (Luna), and/or Switch® 62.5 WG (Switch)], did not significantly reduce survival from EFB relative to infected controls. When larvae were exposed to four fungicide products concurrently, we observed a significant 24.2% decrease in survival from M. plutonius infection (p = 0.0038). Similarly, higher concentrations of Kenja significantly reduced larval survival by 24.7–33.0% from EFB (p DiscussionThese in vitro results suggest that fungicides may contribute to larval susceptibility and response to M. plutonius infections. Further testing of other pesticide combinations is warranted as well as continued surveillance of pesticide residues in blueberry-pollinating colonies.</p

Image_2_Are fungicides a driver of European foulbrood disease in honey bee colonies pollinating blueberries?.TIF

IntroductionBlueberry producers in Canada depend heavily on pollination services provided by honey bees (Apis mellifera L.). Anecdotal reports indicate an increased incidence of European foulbrood (EFB), a bacterial disease caused by Melissococcus plutonius, is compromising pollination services and colony health. Fungicidal products are commonly used in blueberry production to prevent fungal diseases such as anthracnose and botrytis fruit rot. Pesticide exposure has been implicated in honey bee immunosuppression; however, the effects of commercial fungicidal products, commonly used during blueberry pollination, on honey bee larval susceptibility to EFB have not been investigated.MethodsUsing an in vitro infection model of EFB, we infected first instar honey bee larvae with M. plutonius 2019 BC1, a strain isolated from an EFB outbreak in British Columbia, Canada, and chronically exposed larvae to environmentally relevant concentrations of fungicide products over 6 days. Survival was monitored until pupation or eclosion.ResultsWe found that larvae chronically exposed to one, two, or three fungicidal products [Supra® Captan 80WDG (Captan), low concentration of Kenja™ 400SC (Kenja), Luna® Tranquility (Luna), and/or Switch® 62.5 WG (Switch)], did not significantly reduce survival from EFB relative to infected controls. When larvae were exposed to four fungicide products concurrently, we observed a significant 24.2% decrease in survival from M. plutonius infection (p = 0.0038). Similarly, higher concentrations of Kenja significantly reduced larval survival by 24.7–33.0% from EFB (p DiscussionThese in vitro results suggest that fungicides may contribute to larval susceptibility and response to M. plutonius infections. Further testing of other pesticide combinations is warranted as well as continued surveillance of pesticide residues in blueberry-pollinating colonies.</p