Acute toxicity of the fungicide captan to honey bees and mixed evidence for synergism with the insecticide thiamethoxam

Acute toxicity of the fungicide captan to honey bees and mixed evidence for synergism with the insecticide thiamethoxam | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Acute toxicity of the fungicide captan to honey bees and mixed evidence for synergism with the insecticide thiamethoxam Daiana De Souza, Christine M. Urbanowicz, Wee Hao Ng, Nicolas Baert, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3944102/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Honey bees are commonly co-exposed to pesticides during crop pollination, including the fungicide captan and neonicotinoid insecticide thiamethoxam. We assessed the impact of exposure to these two pesticides individually and in combination, at a range of field-realistic doses. In laboratory assays, mortality of larvae/pupae treated with captan was 80–90% greater than controls, dose-independent, and similar to mortality from the lowest dose of thiamethoxam. There was evidence of synergism (i.e., a non-additive response) from captan-thiamethoxam co-exposure at the highest dose of thiamethoxam, but not at lower doses. In the field, we exposed whole colonies to the lowest doses used in the laboratory. Exposure to captan and thiamethoxam individually and in combination resulted in minimal impacts on population growth or colony mortality, and there was no evidence of synergism or antagonism. These results suggest captan and thiamethoxam are each acutely toxic to immature honey bees, but whole colonies can potentially compensate for detrimental effects, at least at the low doses used in our field trial. Further work is needed to assess how compensation occurs, potentially via increased queen egg laying, and whether short-term compensation leads to long-term costs. Other crop pollinators that lack the social detoxification capabilities of honey bees may also be less resilient. Apis mellifera ecotoxicology synergism antagonism in vitro rearing Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The western honey bee, Apis mellifera , is the most important managed pollinator in the world, contributing approximately half of crop pollination services worldwide [ 1 ]. Unfortunately, in recent years high rates of honey bee colony losses have been observed [ 2 – 6 ]. Lack of floral resources, exposure to pesticides, and pathogens/parasites are among the major stressors contributing to high colony loss rates and weakened pollinator populations [ 7 ]. Furthermore, pesticide residues in pollen and nectar collected by bees are often found at levels known to influence honey bee susceptibility to parasites and pathogens [ 8 – 10 ] foraging behaviors [ 11 , 12 ], and growth and survival of bees [ 11 , 13 – 15 ]. During crop pollination, honey bees are commonly co-exposed to multiple pesticides [ 16 – 19 ]. Fungicide residues are often the most abundant pesticides found in bee-collected pollen, bee bread, and other hive products [ 17 – 24 ]. This should not be surprising since fungicide applications are recommended during crop bloom, when honey bees and other pollinators are actively foraging at flowers. But honey bees are also exposed to insecticides and other pesticides during crop pollination. For example, honey bees conducting blueberry pollination in Michigan were simultaneously exposed to an average of 35 pesticides in bee-collected pollen [ 16 ] with the majority of risk coming from the organophosphate insecticide chlorpyrifos (max concentration in pollen = 214 parts per billion; ppb), and the neonicotinoid insecticides clothianidin (max concentration = 35 ppb), imidacloprid (max concentration = 9 ppb), and thiamethoxam (max concentration = 15 ppb; [ 25 ]. Honey bees conducting apple pollination in New York were also commonly exposed to insecticides; freshly collected beebread from hives at 24 of 30 orchards contained insecticides, with the majority of risk attributed to the oxadiazine insecticide indoxacarb (max concentration in pollen = 918 ppb) and the neonicotinoid insecticide thiamethoxam (max concentration in pollen = 48 ppb [ 19 ]. Neonicotinoids contribute to honey bee risk during crop pollination, and a worldwide review of pesticide risk to bees found that the neonicotinoid thiamethoxam was one of the three highest-risk pesticides to honey bees [ 22 , 26 ]. Thiamethoxam is a systemic and environmentally persistent insecticide, which increases the likelihood of exposure via pollen, nectar, plant guttation fluids, soils, and other environmental matrices for days, months, or even years [ 27 – 29 ]. Thiamethoxam’s action as a neurotoxin can lead to paralysis and death of adult bees by binding to nicotinic acetylcholine receptors, and at sublethal exposure levels, can affect the ability of bees to perform important tasks inside and outside of the hive [ 28 , 30 – 32 ]. Exposure of larvae to thiamethoxam is also known to affect survival and physiology of the honey bee postembryonic stages [ 33 – 35 ]. Although fungicide exposure is generally considered safe for bees, concern has recently been raised about the risk posed from some fungicides, including captan [ 36 – 38 ]. Captan is one of the most widely used fungicides in the United States and the most-sprayed fungicide during apple bloom in New York State [ 19 ]. Officially, captan is considered relatively non-toxic to adult honey bees [ 39 , 40 ]. However, little is known regarding interactions between captan and any insecticides, including thiamethoxam. Such information is important since some fungicide-insecticide combinations lead to synergisms and some do not [ 41 ], which can be useful in guiding recommendations regarding which fungicides are safest to apply during crop pollination. Here, we investigate the individual and interactive effects of two commonly used pesticides, the neonicotinoid insecticide thiamethoxam and the fungicide captan, on honey bee larval development and full-colony growth and survival. In the lab, we exposed larvae to three field-realistic doses of each pesticide individually and in combination, monitoring survival and development. In the field, we exposed full colonies to each pesticide individually and in combination at the lowest dose used in the laboratory assays, monitoring colony performance and survival over a full calendar year. This study addresses three main questions: 1) What are the direct effects of captan and thiamethoxam on development and survival of individual larvae and full colonies? 2) Does captan-thiamethoxam co-exposure synergistically affect development and survival of individual larvae and full colonies? and 3) Do in vitro assays with individual larvae scale up to predict colony-level outcomes? Results In vitro laboratory assays We assessed the impact of treatment on survival of immature bees in two ways: first using a Cox proportional hazards model for survivorship (Fig. 1 ), and second using a logistic model for the percentage mortality at the end of the trial (Fig. 2 ). Survival of immature bees in the positive and negative control groups (with and without acetone used as a solvent) were 74% and 69%, respectively, and were not significantly different in either model (Cox model p Tukey = 0.95; logistic model p Tukey = 0.97). All pesticide treatments resulted in significantly greater mortality than the positive control (Tables S2 and S3). Treatment of larvae with the fungicide captan resulted in an 80–90% increase in mortality compared to the positive controls, with no significant difference between dosages (Fig. 2 , Tables S2 and S3). Treatment of larvae with the insecticide thiamethoxam at the low, medium and high dosages increased the mortality rates by 90%, 120% and 150%, respectively, compared to the positive control, although again the differences between dosages were not significant (Fig. 2 , Tables S2 and S3). Finally, the combination of captan (highest dosage) with thiamethoxam at the low, medium and high dosages demonstrated 130%, 140% and 210% increase in mortality rates, respectively, compared to the positive control (Fig. 2 ). Mortality from the combination with the high dose of thiamethoxam was significantly greater than mortality from the combination with the medium or low doses via the Cox model (Table S2), but these differences were not observed via the logistic model (Table S3). We developed an improved method to assess interactions between pesticides that identifies non-additive responses (synergism or antagonism) using a log-binomial generalized linear model (GLM) for survivorship. We did not detect any significant interactions on the overall survivorship (see Table S4). However, when survivorship within each developmental stage was analyzed separately, we detected statistically significant synergistic interactions (leading to higher-than-expected mortality) at the larval stage in the bioassay when captan was crossed with thiamethoxam at highest dose (LR = 7.932, p Holm = 0.044). For other developmental stages and chemical dosages, no significant interactions were observed (see Table S4). Field trials with whole colonies We assessed the impact of treatment using nine performance metrics in all colonies over a full year – number of adult bees, nectar cells, pollen cells, worker brood cells, drone brood cells, varroa levels, chalkbrood presence, queen supersedure events, and colony survival. No significant difference was observed among treatment groups and there was no treatment × month interaction for: number of adult bees, nectar cells, pollen cells, worker brood cells, or drone brood cells, but each of these performance metrics varied significantly among months (Table 1 , Fig. 3 ; Supplemental material Fig. S1 ). Table 1 Likelihood ratio test results for separate models to test the effect of pesticide treatment on five performance metrics - area of adult bees, nectar cells, pollen cells, worker brood cells, and drone brood cells. Response Source Degrees of freedom χ 2 p Adult bees Treatment 3 0.92 0.82 Month 4 39.99 < 0.0001 Bee yard 2 0.47 0.79 Treatment × month 12 10.61 0.56 Nectar cells Treatment 3 0.60 0.90 Month 4 97.57 < 0.0001 Area of adult bees 1 3.43 0.064 Bee yard 2 1.99 0.37 Treatment × month 12 17.15 0.14 Pollen cells Treatment 3 3.05 0.38 Month 4 26.22 < 0.0001 Area of adult bees 1 1.40 0.24 Bee yard 2 2.61 0.27 Treatment × month 12 13.31 0.35 Brood cells Treatment 3 1.81 0.61 Month 4 159.27 < 0.0001 Area of adult bees 1 18.33 < 0.0001 Bee yard 2 0.40 0.82 Treatment × month 12 6.52 0.89 Drone brood cells Treatment 3 3.66 0.30 Month 4 62.77 < 0.0001 Area of adult bees 1 31.97 < 0.0001 Bee yard 2 7.21 0.027 Treatment × month 12 5.47 0.94 Consumption of pollen patties containing the treatments (captan, thiamethoxam, captan + thiamethoxam, or control) did not vary by treatment, bee yard, or the treatment × month interaction (Supplemental material Table S5), but consumption varied significantly among months and was positively correlated with the area of adult bees in a colony (β = 4.35 ± 0.06; Table S5, Fig. S2). Queen supersedure occurred in eleven of the 46 colonies, but supersedure was not related to treatment (χ 2 3 = 6.43, p = 0.093). Varroa levels varied significantly among months, with levels higher in September 2016 than in July 2016 or April 2017 (Supplemental material Table S6; Fig. S3), but did not vary by treatment. Chalkbrood was present in 25 of the 46 hives. The presence of chalkbrood at any point in the study was not related to treatment (χ 2 3 = 2.15, p = 0.54) or bee yard (χ 2 3 = 2.26, p = 0.32). Finally, no significant difference in colony survival was observed (χ 2 11 = 10.4, p = 0.51, Fig. 4 ). Discussion Our data indicate that exposure of honey bee larvae to field-realistic levels of the fungicide captan, neonicotinoid insecticide thiamethoxam, and the combination of captan + thiamethoxam significantly increased mortality. Perhaps most importantly, we observed that the 80–90% increase in mortality of immature bees exposed to captan was dose-independent and similar in magnitude to the lowest dose of thiamethoxam. Furthermore, synergism between captan and thiamethoxam was observed to non-additively increase larval mortality at the highest dose of thiamethoxam, but there was no evidence of synergism or antagonism for other developmental stages or chemical dosages. At the full-colony level in the field, the effects of pesticide exposure at the lowest doses used in the laboratory assays did not translate to changes in colony performance as measured by bee population numbers, resistance to parasites/pathogens, or colony survivorship. Thus, we find that captan and thiamethoxam are each acutely toxic to immature honey bees, but whole colonies can potentially compensate for detrimental effects, at least at the low doses and duration used for our field trial. Fungicides are not intended to control insects, and their acute toxicity to honey bees is generally low[ 42 ]. That said, a recent review shows that risk from some fungicide exposures can be high for bees [ 38 ]. Impacts of fungicides on honey bee brood have been observed [ 39 , 43 , 44 ], which is in agreement with our findings regarding captan’s acute toxicity to developing larvae/pupae. Captan inhibits respiratory pathways in fungal cells, but this mode of action may also be detrimental to animals such as honey bees since they use similar metabolic pathways [ 45 , 46 ]. The larval stages of honey bees may be particularly susceptible to captan due to the high rate of mitochondrial biogenesis and respiratory metabolism in response to the large ATP demand at this stage of development [ 47 ]. In addition to the direct acute toxicity of captan by itself, we also addressed the potential interaction of this fungicide with the neonicotinoid insecticide thiamethoxam. The combination of captan with thiamethoxam at the highest concentration tested did synergistically increase mortality of larvae. However, synergisms at other concentrations and at other developmental stages were not observed. Thiamethoxam is an insecticide distinguished for its lethality to bees in both immature and adult stages [ 31 , 33 , 48 , 49 ]. Despite this neonicotinoid impacting neurologic [ 28 , 30 , 32 , 50 ] and immune systems [ 51 , 52 ], the honey bee detoxification system does have some mechanisms to metabolize nicotine-like compounds [ 42 , 53 ]. Sterol biosynthesis-inhibiting (SBI) fungicides are known to interfere with these detoxification mechanisms [ 54 ], but captan does not have the same mode of action as SBI fungicides and therefore the mechanism underlying synergism is unclear. Due to their limited number of detoxification genes compared to other insects, honey bees may have difficulty processing multiple xenobiotics simultaneously [ 53 , 55 ]. Despite the findings of the in vitro assays, our field trial with full colonies did not observe impacts of pesticide exposure over one year at the low concentrations used in the study. The incongruity among lab and field toxicology studies of bees is important to consider. Three main factors that can lead to incongruence between laboratory-based and field-based toxicology studies: pesticide concentration, duration of exposure, and the capacity of foragers to choose between foraging sites in the field. Our choice of pesticide concentrations for the in vitro lab assays (100, 500, and 2,000 ppb for captan, and 10, 70, and 1,440 ppb for thiamethoxam) were based on a careful search of pesticide residues found in field surveys of honey bee-collected pollen and beebread (see Tables S7 & S8) and the concentrations chosen provide a range of field-realistic exposure possibilities. In addition, in the in vitro lab assay, larvae were exposed to pesticide treatments via diet from the fourth day of development until reaching adulthood. This protocol is what is advised by the Organization for Economic Cooperation and Development guidelines for toxicological studies (Test No. 239 (OECD, 2016)[ 56 ], indicating our laboratory bioassays were appropriate. Thus, our lab-based study was sound, and the field-based study shows that full colonies likely have mechanisms to compensate for increased larval mortality. For example, the queen may compensate for increased larval mortality by laying more eggs. Short-term colony resilience has been described among colonies exposed to sub-lethal dosages of neonicotinoids, whereby colonies increase brood initiation rate to compensate for increased brood mortality [ 57 , 58 ]. While effective in the short term, this could lead to longer-term costs for the queen and an increased incidence of queen failure [ 59 , 60 ]. The degree to which nurse bees in honey bee colonies can detoxify xenobiotic substances in pollen before secreting jelly to the brood is poorly known, but the limited evidence to date suggests this can be part of an effective “social detoxification system” [ 53 , 61 , 62 ]. For example, Ricke et al. (2021) found that less than 2% of pesticides in pollen fed to nurse bees made it into jelly excreted by the workers[ 63 ]. Moreover, royal jelly from colonies exposed to pesticide-contaminated pollen contained negligible residues, although queens reared with this jelly had reduced reproductive quality [ 64 ]. At immature stages, exposure occurs transdermally and orally at the same time. This may be especially important given that early life stages of bees are generally more sensitive to contaminants relative to adult stages [ 13 ]. Overall, our laboratory and field results show that field-realistic exposures to the fungicide captan greatly increase immature honey bee mortality, and synergism with the neonicotinoid thiamethoxam occurs, but synergism is dose-dependent and full colonies may be able to compensate for losses. Therefore, we suggest that further effort be placed in understanding how exposure to captan and co-exposure with other pesticides affects honey bee colony dynamics and mortality of solitary and sub-social wild bees, which lack the social detoxification capabilities of honey bees and can therefore be more susceptible to pesticides in the field. Notably, captan affected honey bee larval survivorship regardless of the concentration and at field-realistic levels. Most studies investigating captan describe effects after chronic exposure [ 39 , 46 , 65 , 66 ], while our study found an impact via a single exposure event. Since captan is actively applied to crops during bloom while honey bees and wild pollinators are carrying out critical pollination services, further investigations of this fungicide are warranted to ensure the sustainability of crop pollination and agricultural production. Methods Chemicals The agrochemicals used for toxicity assays were the fungicide captan (CAS number 133-06-2, ≥ 98% purity); and the insecticide thiamethoxam (CAS number 153719-23-4, ≥ 98% purity), both from Sigma-Aldrich. The fungicide captan is one of the most-used fungicides in the United States and is the most-sprayed fungicide during apple bloom in New York [ 19 ]. Thiamethoxam is a widely used neonicotinoid insecticide and has been identified as one of the highest-risk pesticides to honey bees worldwide [ 22 ].The agrochemicals used for toxicity assays were the fungicide captan (CAS number 133-06-2, ≥ 98% purity); and the insecticide thiamethoxam (CAS number 153719-23-4, ≥ 98% purity), both from Sigma-Aldrich. The fungicide captan is one of the most-used fungicides in the United States and is the most-sprayed fungicide during apple bloom in NY [ 19 ]. Thiamethoxam is a widely used neonicotinoid insecticide [ 27 ] and contributes to worldwide risk to honey bees [ 22 ]. In vitro laboratory assays We used an in vitro rearing method to assess the impact of pesticide exposure on individual honey bees during development. Worker larvae were sampled from six honey bee colonies (unselected stock) located at the Dyce Lab for Honey Bee Studies at Cornell University in Ithaca, New York. Worker larvae at the first instar were grafted individually in commercial plastic queen cups, which were placed inside 96-well microcentrifuge tube racks containing equal numbers of wells filled with a saturated K 2 SO 4 , and using a feeding protocol previously described for honey bee worker rearing in an artificial environment [ 56 , 67 ] with slight modifications on the volume of liquid diet provided (Table 2 ). Table 2 Larval feeding regimen adopted to the in vitro rearing system. The diet provided were A (44.25% of royal jelly, 5.3% of D-Glucose, 5.3% of D-Fructose, 0.9% of Yeast extract and 44.25 distilled water), B (42.94% of royal jelly, 6.4% of D-Glucose, 6.4% of D-Fructose, 1.3% of Yeast extract and 42.95 distilled water) or C (50% of royal jelly, 9% of D-Glucose, 9% of D-Fructose, 2% of Yeast extract and 30% distilled water). Larval progress Diet Amount of diet (µl) Day 0 (at grafting) A 30 Day 1 n/a 0 Day 2 B 30 Day 3 C 40 Day 4 C 50 Day 5 C 60 Glucose (CAS Number: 50-99-7) and Fructose (CAS Number: 57-48-7) were obtained from Sigma-Aldrich (St. Louis, MO, USA), yeast extract (#288620) was obtained from Life Technologies Corp. (Sparks, MD, USA) and organic royal jelly was obtained from Glorybee (Eugene, OR, USA). Diets were prepared and provided in different amounts according to each larval stage. The larvae were kept in an incubator at 34.5 ˚C and 80% RH until the defecation stage, then at 70% RH during the pupal stage until emergence of the adults [ 68 ]. Under normal development, worker larvae switch from an exclusive royal jelly diet to a mix of the same secretions with pollen and nectar between the fourth to the fifth larval instar [ 69 ]. Thus, we applied the pesticide treatments at this phase, following the OECD 239 study design [ 70 ]. Cautious attention was taken to select only larvae that had achieved the fourth larval instar [ 71 ], thus ensuring the next stages of developmental monitoring were accurate. Treatment solutions were mixed into the diet and supplied individually to larvae according to each treatment group. The concentrations used were based on sublethal concentrations previously found in the literature (see Tables S7 and S8). For the insecticide thiamethoxam, the concentrations were: low (10 ng/ml = 10 parts per billion), medium (70 ng/ul) and high (1,440 ng/ml). For the fungicide captan, the concentrations were: low (100 ng/ml), medium (500 ng/ml) and high (2,000) ng/ml. We tested for potential interactions between these pesticides by using a blend of captan at high concentration (2,000 ng/ml) and thiamethoxam at low (10 ng/ml = 10 parts per billion), medium (70 ng/ul) and high (1,440 ng/ml) concentrations. Both chemicals were solubilized in acetone before combining with the jelly diet. Positive (+ acetone) and negative (no acetone) control groups were tested to measure possible effects of the solvent. Three experimental trials were conducted at different times; see Table S1 for the number of treated larvae in each treatment group and trial. The cups containing the diet with different treatments were randomly distributed in the racks to avoid site effects. All larvae were monitored twice a day and larvae that died (movement stopped, discoloring, drowned in the food) or if there was any evidence of contamination, were immediately recorded and removed. Survivorship and developmental advances were recorded during all ontogenic stages until each larva reached adulthood. Adults were considered viable if they successfully crossed the pupation stages and reached adulthood healthy, morphologically well-shaped, and actively walking around. Field trials with whole colonies Forty-eight nucleus honey bee colonies were acquired from a commercial beekeeper (Chuck Kutik, Norwich, NY). All nucleus colonies were transferred into a new 10-frame box with plastic foundation and allowed to draw comb for two weeks in a common location. Before starting the experiments, all colonies were checked for queen status, number of bees, and frame composition. Frames were redistributed among colonies such that all colonies had a similar composition of brood, bees, pollen and nectar immediately prior to enrollment in the field experiment. In addition, each colony was fitted with a pollen trap, placed at the entrance of the hive, to restrict pollen flow into the hive and therefore ensure consumption of the treated pollen patties [ 58 ]. Twelve colonies were assigned to each of four treatments: control (no pesticides added), thiamethoxam (10 ppb), captan (1,000 ppb), and thiamethoxam + captan (10 ppb thiamethoxam + 1,000 ppb captan). We used the lowest dose of thiamethoxam from our laboratory assays (10 ppb) and a medium/high dose of captan (1,000 ppb) because these contamination levels are commonly observed from our own studies during crop pollination [ 16 , 19 ]; and the broader literature (see Tables S7 and S8). Pollen patty treatments were prepared the day before they were fed to the bees using captan (CAS number 133-06-2, ≥ 98% purity) and thiamethoxam (CAS number 153719-23-4, ≥ 98% purity), both from Sigma-Aldrich (St. Louis, MO, USA), and Bee Pro from Mann Lake (Hackensack, MN, USA). The pollen patties were provided to each colony weekly from the first week of June to the first week in October 2016. Patties were kept on for 3–7 days, with an average of 4 days, until they were almost entirely consumed. Two hives had patties left on for 14 days in one month. Because the colonies accumulated minimal honey during the summer, we supplementally fed each colony three liters of sugar syrup (30% by volume) from the third week of August until the first week first week of October. Sugar syrup was prepared weekly according to treatments: thiamethoxam (10 ppb), captan (1,000 ppb), and thiamethoxam + captan (10 ppb thiamethoxam + 1,000 ppb captan). The colonies were equally distributed among three bee yards: Varna (42.463730, -76.440748), Turkey Hill (42.437293, -76.427063), and Sarkaria (42.443964, -76.452238); which are ~ 2 km from each other. The colonies were not treated for varroa mites during the experiment, and overwintering preparation consisted of placing a moisture board and foam-insulated inner cover under the telescoping outer cover. Performance and survival of colonies were assessed via monthly inspections in June, July, August, and October 2016, and April 2017. During each visit, we estimated the number of adult bees, brood cells, nectar cells, and pollen cells of each surviving colony. We followed the Liebefelder method [ 72 ], visually estimating the proportion of each frame covered by adult bees or each type of cell and summing these proportions across all frames in a hive. Additionally, each colony had one drone comb frame, and the proportion of the frame covered by drone brood was recorded once per month in July, August, September, October 2016 and April 2017. The inspections also accounted for queen supersedure events, varroa levels (measured as mites per 300 bees using the sugar roll method) and chalkbrood presence. Statistical analyses In vitro laboratory assays We assessed the impact of treatment on the survival of immature bees using a Cox proportional hazards model for the survivorship, as well as a logistic model for the percentage mortality at the end of the trial (Fig. 2 ). In each model, both the pesticide treatment and trial number were included as additive fixed effects, the latter to account for variation in natural mortality between trials. Post-hoc tests for pairwise differences between treatments, marginalized across trials, were performed for both models, adjusting for multiple comparisons using Tukey's HSD. We used Bliss' definition of independence to define synergism or antagonism between different pesticides [ 73 ]. Although Bliss independence is traditionally defined using mortality rates, it can also be defined using survival rates. Let s C be the control survival rate, s C · s A the survival rate when exposed to pesticide A, and s C · s B the survival rate when exposed to pesticide B; here s A and s B can be interpreted as Abbott-corrected survival rates [ 74 ]. If pesticides A and B are Bliss-independent, then the survival rate when simultaneously exposed to both pesticides is given by s C · s A · s B ; if the actual survival rate is lower, then the two pesticides interact synergistically, whereas if the reverse is true, then they interact antagonistically. Biologically, Bliss independence requires that the two pesticides have independent biological action, and also that individual susceptibilities to A and to B are uncorrelated. The second requirement is important but often overlooked: as an extreme example, if the susceptibilities were perfectly correlated, then all bees that survive pesticide A will also survive any additional exposure to pesticide B, so the survival rate remains at s C · s A < s C · s A · s B , leading to an apparent antagonism. We developed a new approach to assess Bliss independence between pesticides A and B, based on the well-established framework of the generalized linear model (GLM). The idea is as follows. We fitted survivorship using a binomial GLM with a log link function (as opposed to typical binomial GLMs for mortality with logit link functions), with binary (0/1) predictors x A and x B , representing exposure to pesticide A and to B, as well as the interaction x A · x B . The log link function means that log(p surv ) = β 0 + β A · x A + β B · x B + β AB · x A · x B , so the survival rates are exp( β 0 ) for control, exp( β 0 )·exp( β A ) for pesticide A only, exp( β 0 )·exp( β B ) for pesticide B only, and exp( β 0 )·exp( β A )·exp( β B )·exp( β AB ) for simultaneous exposure. If pesticides A and B are Bliss-independent, then the survival rate for the latter should simply be exp( β 0 )·exp( β A )·exp( β B ), so we require that exp( β AB ) = 1, or equivalently β AB = 0. Therefore, Bliss independence is equivalent to the null hypothesis that the interaction x A x B has no effect, which can be evaluated using a likelihood ratio test. A significant negative β AB would imply synergism, and a significant positive β AB would imply antagonism. The use of a log-binomial GLM also allows us to include the trial number as an additional fixed effect to account for variations in natural mortality between trials. Hence our method improves upon the method in (Sgolastra et al., 2017) [ 75 ], not only by accounting for natural mortality, but also by allowing for variations between experimental replicates. We tested for interactions between the fungicide and the insecticide at three different chemical dosages on the overall survivorship, as well as the survivorship within each of three benchmarks of ontogenic development (5th larval instar, Pre-pupae and pupation). Holm correction was used to account for multiple testing. All analyses and graphical presentation were conducted in R version 3.5.1 [ 76 ], using the packages "survival" [ 77 ] and “survminer” [ 78 ] for the analysis of survival curves, "emmeans" [ 79 ] for post-hoc pairwise tests between treatments, and “lbreg” [ 80 ] for fitting log-binomial models in the Bliss independence analysis. Field trials with whole colonies First, we tested whether pollen patty consumption (grams consumed per day) varied across treatments. Treatment, month, the treatment × month interaction, and bee yard were fixed effects, and colony identity was a random effect. We also included the estimated number of adult bees during performance assessments, as a covariate. Month was treated as a categorical predictor because the number of adult bees was not measured in September, we estimated this value by averaging the number of adult bees in October and August. Significance of model terms here and below was assessed with likelihood ratio tests. We used post-hoc Tukey pairwise comparisons to test for differences in pollen patty consumption between months. Regarding the survival analysis, we performed a Kaplan-Meier survival curves for each treatment; surv function , ‘survival’ package [ 77 ]. We assessed statistical differences in survival among treatments with a log-rank test, using treatment as the main effect and bee yard as a frailty term ( survdiff function ). To examine the performance ratios, we used separate linear mixed models to test how treatment influenced the frame area of adult bees, nectar cells, pollen cells, and worker brood cells. Treatment, month, the treatment × month interaction, and bee yard were fixed effects, and colony identity was a random effect. We also included the frame area of adult bees as a covariate when modeling the frame area of brood cells, nectar cells, and pollen cells. The proportion of the drone brood frame covered by drone brood was modeled with the same fixed and random effects using a generalized linear mixed model and a Tweedie error distribution; glmmTMB function, ‘TMB’ package [ 81 ]. When month was significant in any of the above performance metrics, we used post-hoc Tukey pairwise comparisons to test for differences between months. The possible event of supersedure of the original queen (yes/no) at any point in the study was modeled as a function of treatment and bee yard using penalized logistic regression due to small number of positive cases (logistf function, ‘logistf’ package, [ 82 ]. For parasites and pathogens, we assessed Varroa levels (mites per 300 bees, log(x + 1) transformed) with a linear mixed model. Treatment, month, the treatment × month interaction, and site (bee yards) were included as fixed effects, and colony identity was included as a random effect. We modeled the presence of chalkbrood (yes/no) at any point in the study as a function of treatment and bee yard using logistic regression. Declarations Acknowledgements We thank Alina Xiao, Trebor Hall & David Lewis for help with the field experiment, and Connor Hinsley and Hailey Scofield for help with lab experiments. Author Contributions DDS, MLS, and SHM conceived and designed the experiments. DDS, MLS, AAF, and NB collected the data. DDS, CMU, WHN and SHM analyzed and/or interpreted the data. DDS and CMU drafted the manuscript. All authors revised the manuscript. Funding This work was supported by USDA-NIFA AFRI grant 2018-08603 and the New York State Environmental Protection Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. Competing Interests The authors declare no competing interests. References Kleijn, D., et al., Delivery of crop pollination services is an insufficient argument for wild pollinator conservation. Nature Communications, 2015. 6 (1): p. 7414. Van der Zee, R., et al., Title: Managed honey bee colony losses in Canada, China, Europe, Israel and Turkey, for the winters of 2008-9 and 2009-10. Journal of Apicultural Research, 2012. 51 : p. 91-114. Traynor, K., et al., Multiyear survey targeting disease incidence in US honey bees. Apidologie, 2016. 47 . Traynor, K.S., et al., In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States. Scientific Reports, 2016. 6 : p. 33207. Seitz, N., et al., A national survey of managed honey bee 2014–2015 annual colony losses in the USA. Journal of Apicultural Research, 2015. 54 (4): p. 292-304. Kulhanek, K., et al., A national survey of managed honey bee 2015–2016 annual colony losses in the USA. Journal of Apicultural Research, 2017. 56 (4): p. 328-340. Goulson, D., et al., Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science, 2015. 347 (6229): p. 1255957. Alaux, C., et al., Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera). Environmental Microbiology, 2010. 12 (3): p. 774-782. Pettis, J.S., et al., Pesticide exposure in honey bees results in increased levels of the gut pathogen Nosema. Naturwissenschaften, 2012. 99 (2): p. 153-158. Wu, J.Y., et al., Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection. Journal of Invertebrate Pathology, 2012. 109 (3): p. 326-329. Henry, M., et al., A common pesticide decreases foraging success and survival in honey bees. Science, 2012. 336 (6079): p. 348-350. Stanley, D.A., et al., Neonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees. Nature, 2015. 528 (7583): p. 548-550. Desneux, N., A. Decourtye, and J.-M. Delpuech, The sublethal effects of pesticides on beneficial arthropods. Annual Review of Entomology, 2006. 52 (1): p. 81-106. Whitehorn, P.R., et al., Neonicotinoid pesticide reduces bumble bee colony growth and queen production. Science, 2012. 336 (6079): p. 351-352. Vidau, C., et al., Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae. PLoS ONE, 2011. 6 (6): p. e21550. Graham, K.K., et al., Identities, concentrations, and sources of pesticide exposure in pollen collected by managed bees during blueberry pollination. Scientific Reports, 2021. 11 (1): p. 16857. Pettis, J., et al., Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae. PloS one, 2013. 8 : p. e70182. Frazier, M., et al., Assessing Honey Bee (Hymenoptera: Apidae) Foraging Populations and the Potential Impact of Pesticides on Eight U.S. Crops. Journal of Economic Entomology, 2015. 108 : p. tov195. McArt, S.H., et al., High pesticide risk to honey bees despite low focal crop pollen collection during pollination of a mass blooming crop. Scientific Reports, 2017. 7 : p. 46554. Chauzat, M.-P., et al., A Survey of Pesticide Residues in Pollen Loads Collected by Honey Bees in France. Journal of economic entomology, 2006. 99 : p. 253-62. Mullin, C.A., et al., High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health. PLOS ONE, 2010. 5 (3): p. e9754. Sanchez-Bayo, F. and K. Goka, Pesticide Residues and Bees – A Risk Assessment. PLOS ONE, 2014. 9 (4): p. e94482. Mullin, C., et al., The formulation makes the honey bee poison. Pesticide Biochemistry and Physiology, 2015. 120 . Milano, N., et al., Comparative survival and fitness of bumble bee colonies in natural, suburban, and agricultural landscapes. Agriculture, Ecosystems & Environment, 2019. 284 : p. 106594. Graham, K.K., et al., Pesticide risk to managed bees during blueberry pollination is primarily driven by off-farm exposures. Scientific Reports, 2022. 12 (1): p. 7189. Maus, C., A. Alix, and D. Schmehl, Assessing the impact of pesticides on pollinators . 2022. p. 183-224. Maienfisch, P., et al., Chemistry and biology of thiamethoxam: a second generation neonicotinoid. Pest Management Science, 2001. 57 (10): p. 906-913. Goulson, D., REVIEW: An overview of the environmental risks posed by neonicotinoid insecticides. Journal of Applied Ecology, 2013. 50 (4): p. 977-987. Wood, T.J. and D. Goulson, The environmental risks of neonicotinoid pesticides: a review of the evidence post 2013. Environmental science and pollution research international, 2017. 24 (21): p. 17285-17325. Matsuda, K., et al., Neonicotinoids: Insecticides Acting on Insect Nicotinic Acetylcholine Receptors. Trends in Pharmacological Sciences, 2001. 22 : p. 573-580. Iwasa, T., Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera. Crop Protection, 2004. 23 : p. 371-378. Tosi, S. and J.C. Nieh, A common neonicotinoid pesticide, thiamethoxam, alters honey bee activity, motor functions, and movement to light. Scientific Reports, 2017. 7 (1): p. 15132. Tavares, D.A., et al., Exposure of larvae to thiamethoxam affects the survival and physiology of the honey bee at post-embryonic stages. Environmental Pollution, 2017. 229 : p. 386-393. Qi, S., et al., Flumethrin at honey-relevant levels induces physiological stresses to honey bee larvae (Apis mellifera L.) in vitro. Ecotoxicology and environmental safety, 2019. 190 : p. 110101. Decio, P., et al., Thiamethoxam exposure deregulates short ORF gene expression in the honey bee and compromises immune response to bacteria. Scientific Reports, 2021. 11 (1): p. 1489. Everich, R., et al., Effects of Captan on Apis mellifera Brood Development Under Field Conditions in California Almond Orchards. Journal of economic entomology, 2009. 102 : p. 20-9. Heylen, K., et al., The effects of four crop protection products on the morphology and ultrastructure of the hypopharyngeal gland of the European honeybee, Apis mellifera. Apidologie, 2011. 42 : p. 103-116. Rondeau, S. and N.E. Raine, Fungicides and bees: a review of exposure and risk. Environment International, 2022. 165 : p. 107311. Atkins, E.L. and D. Kellum, Comparative Morphogenic and toxicity Studies on the Effect of Pesticides on Honeybee Brood. Journal of Apicultural Research, 1986. 25 (4): p. 242-255. USEPA, Captan Reregistration Eligibility Decision - EPA-738-F99-015 , P.a.T.S. Prevention, Editor. 1999. Iverson, A., et al., Synergistic effects of three sterol biosynthesis inhibiting fungicides on the toxicity of a pyrethroid and neonicotinoid insecticide to bumble bees. Apidologie, 2019. Johnson, R., Honey Bee Toxicology. Annual review of entomology, 2014. 60 . Wade, A., et al., Combined Toxicity of Insecticides and Fungicides Applied to California Almond Orchards to Honey Bee Larvae and Adults. Insects, 2019. 10 : p. 20. Zhu, W., et al., Four Common Pesticides, Their Mixtures and a Formulation Solvent in the Hive Environment Have High Oral Toxicity to Honey Bee Larvae. PloS one, 2014. 9 : p. e77547. Bushway, R.J. and A.R. Hanks, Pesticide inhibition of growth and macromolecular synthesis in the cellular slime mold Dictyostelium discoideum. Pesticide Biochemistry and Physiology, 1976. 6 (3): p. 254-260. Mussen, E., J. Lopez, and C. Peng, Effects of Selected Fungicides on Growth and Development of Larval Honey Bees, Apis mellifera L. (Hymenoptera: Apidae). Environmental Entomology, 2004. 33 : p. 1151-1154. Santos, D., L. Alberici, and K. Hartfelder, Mitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development . Vol. 73. 2016. Sepúlveda Friol, P., et al., Can the exposure of Apis mellifera (Hymenoptera, Apiadae) larvae to a field concentration of thiamethoxam affect newly emerged bees? Chemosphere, 2017. 185 . Tavares, D.A., et al., Exposure to thiamethoxam during the larval phase affects synapsin levels in the brain of the honey bee. Ecotoxicology and Environmental Safety, 2019. 169 : p. 523-528. Millar, N.S. and I. Denholm, Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invert Neurosci, 2007. 7 (1): p. 53-66. Di Prisco, G., et al., Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees. Proceedings of the National Academy of Sciences, 2013. 110 (46): p. 18466-18471. Coulon, M., et al., Influence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees. PLOS ONE, 2019. 14 : p. e0220703. Berenbaum, M.R. and R.M. Johnson, Xenobiotic detoxification pathways in honey bees. Current Opinion in Insect Science, 2015. 10 : p. 51-58. Schuhmann, A., et al., Interaction of Insecticides and Fungicides in Bees. Frontiers in Insect Science, 2022. 1 . Claudianos, C., et al., A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee. Insect Molecular Biology, 2006. 15 (5): p. 615-636. OECD, Guidance document No. 239: Honey Bee Larval Toxicity Test following Repeated Exposure . Vol. 34. 2016: ENV/JM/MONO. Schott, M., et al., Honeybee colonies compensate for pesticide-induced effects on royal jelly composition and brood survival with increased brood production. Scientific Reports, 2021. 11 (1): p. 62. Sandrock, C., et al., Impact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure. PLOS ONE, 2014. 9 (8): p. e103592. Tarpy, D.R., E. Talley, and B.N. Metz, Influence of brood pheromone on honey bee colony establishment and queen replacement. Journal of Apicultural Research, 2021: p. 1-9. Withrow, J.M., J.S. Pettis, and D.R. Tarpy, Effects of Temperature During Package Transportation on Queen Establishment and Survival in Honey Bees (Hymenoptera: Apidae). J Econ Entomol, 2019. Liao, L.-H., W.-Y. Wu, and M.R. Berenbaum, Behavioral responses of honey bees (Apis mellifera) to natural and synthetic xenobiotics in food. Scientific Reports, 2017. 7 (1): p. 15924. Lucchetti, M.A., et al., Nursing protects honeybee larvae from secondary metabolites of pollen. Proceedings of the Royal Society B: Biological Sciences, 2018. 285 (1875): p. 20172849. Ricke, D.F., C.-H. Lin, and R.M. Johnson, Pollen Treated with a Combination of Agrochemicals Commonly Applied During Almond Bloom Reduces the Emergence Rate and Longevity of Honey Bee (Hymenoptera: Apidae) Queens. Journal of Insect Science, 2021. 21 (6). Milone, J.P. and D.R. Tarpy, Effects of developmental exposure to pesticides in wax and pollen on honey bee (Apis mellifera) queen reproductive phenotypes. Scientific Reports, 2021. 11 (1): p. 1020. Dai, P., et al., Chronic toxicity of amitraz, coumaphos and fluvalinate to Apis mellifera L. larvae reared in vitro. Sci Rep, 2018. 8 (1): p. 5635. Wood, S., et al., Effects of chronic dietary thiamethoxam and prothioconazole exposure on Apis mellifera worker adults and brood . 2019. Schmehl, D., et al., Protocol for the in vitro rearing of honey bee (Apis mellifera L.) workers. Journal of Apicultural Research, 2016. Linksvayer, T., et al., Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism . Vol. 24. 2011. 1939-48. Hartfelder, K., et al., Chapter One - Old Threads Make New Tapestry—Rewiring of Signalling Pathways Underlies Caste Phenotypic Plasticity in the Honey Bee, Apis mellifera L , in Advances in Insect Physiology , A. Zayed and C.F. Kent, Editors. 2015, Academic Press. p. 1-36. OECD, Test No. 237: Honey Bee (Apis Mellifera) Larval Toxicity Test, Single Exposure . 2013. Michelette, E.R. and A.E.E. Soares, Characterization of preimaginal developmental stages in Africanized honey bee workers (Apis melliferaL.). Apidologie, 1993. 24 . Imdorf, A., et al., ÜBERPRÜFUNG DER SCHÄTZMETHODE ZUR ERMITTLUNG DER BRUTFLÄCHE UND DER ANZAHL ARBEITERINNEN IN FREIFLIEGENDEN BIENENVÖLKERN. Apidologie, 1987. 18 (2): p. 137-146. Bliss, C.I., The toxicity of poisons applied jointly. Annals of Applied Biology, 1939. 26 (3): p. 585-615. Abbott, W.S., A Method of Computing the Effectiveness of an Insecticide. Journal of Economic Entomology, 1925. 18 (2): p. 265-267. Sgolastra, F., et al., Synergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species. Pest Management Science, 2017. 73 (6): p. 1236-1243. R Core Team, R: A Language and Environment for Statistical Computing. 2018. Therneau, T.M.a.L., T, ‘survival’: Survival analysis. R package version 2.44-1.1. . 2019. Kassambara, A., M. Kosinski, and P. Biecek, survminer: Drawing Survival Curves using 'ggplot2'. R package version 0.4.6. 2019. Lenth, R., emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1.4.2. 2019. Andrade, B.B., lbreg: Log-Binomial Regression with Constrained Optimization. R package version 1.3. 2019. Magnusson, A., Skaug, H.J., Nielsen, A., Berg, C.W., Kristensen, K., Maechler. M., van Bentham, K., Bolker, B., and Brooks, M.E. , glmmTMB: Generalized linear mixed models using a template model builder. R package version 0.1 3. 2018. Heinze, G.a.P., M. , ‘logistf’: Firth's Bias-Reduced Logistic Regression. R package version 1.23. 2018. Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 May, 2024 Reviews received at journal 08 May, 2024 Reviewers agreed at journal 15 Apr, 2024 Reviews received at journal 29 Feb, 2024 Reviewers agreed at journal 22 Feb, 2024 Reviewers invited by journal 22 Feb, 2024 Editor assigned by journal 19 Feb, 2024 Editor invited by journal 19 Feb, 2024 Submission checks completed at journal 19 Feb, 2024 First submitted to journal 09 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3944102","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":273892320,"identity":"e155893d-b6fa-4a46-a8ee-35eeddc3fdaa","order_by":0,"name":"Daiana De Souza","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDCCA4wNUBbzASAhIUOKFrYEkBYeIrTAWTwGYJKgDr7jhxs/F9TUyRmcP/P51Y0aCx4G9sNHN+DTInkmsVl6xrHDxgYHzm6zzjkGdBhPWtoNfFoMDiQ2SPOwHUjccLB3m3EOG1CLBI8Zfi3nHzb/5vlXl7jhMM8z45x/xGi5kdgmzdvGnLjhGA/z49w2IrRI3njYZs3bd9hY8gybGXNunwQPGyG/8J1Pf3yb51udHN/5w48/5wAZ/OyHj+HVggzYJMAkscpBgPkDKapHwSgYBaNg5AAA6J9K6fEe20oAAAAASUVORK5CYII=","orcid":"","institution":"Cornell University","correspondingAuthor":true,"prefix":"","firstName":"Daiana","middleName":"","lastName":"De Souza","suffix":""},{"id":273892321,"identity":"b1631428-0218-45bc-b617-897671c299be","order_by":1,"name":"Christine M. Urbanowicz","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"M.","lastName":"Urbanowicz","suffix":""},{"id":273892322,"identity":"7a5539f7-d567-4f09-98bc-4020ca7d4e9b","order_by":2,"name":"Wee Hao Ng","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Wee","middleName":"Hao","lastName":"Ng","suffix":""},{"id":273892323,"identity":"ff2087cc-c480-4845-811d-dcb4d13a9f5a","order_by":3,"name":"Nicolas Baert","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Baert","suffix":""},{"id":273892324,"identity":"802878ca-dbce-480e-bed8-3cc8ba6d804e","order_by":4,"name":"Ashley A. Fersch","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Ashley","middleName":"A.","lastName":"Fersch","suffix":""},{"id":273892325,"identity":"285235db-ecb2-4702-8bd4-8b2416f9b93d","order_by":5,"name":"Michael L. Smith","email":"","orcid":"","institution":"Auburn University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"L.","lastName":"Smith","suffix":""},{"id":273892326,"identity":"7f934f57-0af6-44a8-a1a4-69e3b28f3316","order_by":6,"name":"Scott H. McArt","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Scott","middleName":"H.","lastName":"McArt","suffix":""}],"badges":[],"createdAt":"2024-02-09 20:30:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3944102/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3944102/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51419377,"identity":"960ffdb6-7cfe-4c0c-96cd-da2b30625f37","added_by":"auto","created_at":"2024-02-21 08:38:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":155773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival of honey bee larvae exposed to different doses of captan, thiamethoxam, or captan+thiamethoxam.\u003c/strong\u003e The exposure scenarios include: liquid diet with no additional chemicals (Control (-), \u003cem\u003en\u003c/em\u003e=235), exposure to the solvent acetone (Control (+), \u003cem\u003en\u003c/em\u003e=236), exposure to captan at concentrations of 0.10 ng/ul (Captan low, \u003cem\u003en\u003c/em\u003e=92), 0.50 ng/ul (Captan medium, \u003cem\u003en\u003c/em\u003e=96) and 2.0 ng/ul (Captan high, \u003cem\u003en\u003c/em\u003e=96); exposure to thiamethoxam at concentrations of 0.01 ng/ul \u0026nbsp;(Thiamethoxam low, \u003cem\u003en\u003c/em\u003e=94), 0.07 ng/ul (Thiamethoxam medium, \u003cem\u003en\u003c/em\u003e=95) and 1.44 ng/ul (Thiamethoxam high, \u003cem\u003en\u003c/em\u003e=94); and exposure to a combination of captan at a fixed concentration of 2.0 ng/ulblended with thiamethoxam at concentrations of 0.01 ng/ul (Capt.+Thiameth. low, \u003cem\u003en\u003c/em\u003e=109), 0.07 ng/ul (Capt.+Thiameth. medium, \u003cem\u003en\u003c/em\u003e=109) and 1.44 ng/ul(Capt.+Thiameth. high, \u003cem\u003en\u003c/em\u003e=103). Pesticide exposure started on Day 4 of larval development according to recommended protocols (Schmehl et al. 2016, OECD 2016). Significant pairwise differences in overall survival using Tukey’s post-hoc contrasts (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) are indicated using compact letter displays.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3944102/v1/72e58732ea4ad77c30636d2b.png"},{"id":51419376,"identity":"ef1b1ea8-2d75-431d-ad47-e47e78dfceeb","added_by":"auto","created_at":"2024-02-21 08:38:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMortality rates by developmental stage of honey bees exposed to different doses of captan, thiamethoxam, or captan+thiamethoxam.\u003c/strong\u003eDevelopmental stages: larva (until larvae finished eating the provided diet and assumed a vertical position, generally encompassing days 1-6), prepupa (larvae in pre-pupation stage, generally occurring during days 7-9) and pupa (the last ontogenetic stage of development, generally occurring between day 10-14). Significant differences in overall survival between treatment groups (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05) are indicated using compact letter displays.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3944102/v1/804fddfc17910ef9f71eb996.png"},{"id":51419383,"identity":"d3c3693d-d790-468f-b4f2-ed0148222c8d","added_by":"auto","created_at":"2024-02-21 08:38:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":134450,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of frames covered by adult bees (a), nectar cells (b), pollen cells (c), and worker brood cells (d) across four treatments. The proportion of each frame covered by adult bees, nectar cells, pollen cells and brood cells was visually estimated, and these proportions were summed across all frames in a hive. Different letters below month names indicate significant differences between months (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) according to post-hoc Tukey pairwise comparisons.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3944102/v1/93fd3cebc9f3702a6553755b.png"},{"id":51419384,"identity":"e9cfb5d7-cef6-4652-bdf4-573fc2faeef7","added_by":"auto","created_at":"2024-02-21 08:38:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148426,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curves for four pesticide treatments applied from June to October 2016. “+” indicates censored observations (i.e., percent of colonies surviving at 12 months, when experiment ended).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3944102/v1/0be582998c6863cd41b49904.png"},{"id":51419385,"identity":"6351cb2c-3792-4580-8409-ed2026c0ad1a","added_by":"auto","created_at":"2024-02-21 08:38:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":896027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944102/v1/79195137-e35f-4328-bcd2-2a34b35729a5.pdf"},{"id":51419382,"identity":"e4e31e50-15e6-4214-aeb9-aef2ae89c6b5","added_by":"auto","created_at":"2024-02-21 08:38:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":215137,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3944102/v1/1b539547e2d7ccc1f606021c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acute toxicity of the fungicide captan to honey bees and mixed evidence for synergism with the insecticide thiamethoxam","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe western honey bee, \u003cem\u003eApis mellifera\u003c/em\u003e, is the most important managed pollinator in the world, contributing approximately half of crop pollination services worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Unfortunately, in recent years high rates of honey bee colony losses have been observed [\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Lack of floral resources, exposure to pesticides, and pathogens/parasites are among the major stressors contributing to high colony loss rates and weakened pollinator populations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, pesticide residues in pollen and nectar collected by bees are often found at levels known to influence honey bee susceptibility to parasites and pathogens [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] foraging behaviors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and growth and survival of bees [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring crop pollination, honey bees are commonly co-exposed to multiple pesticides [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Fungicide residues are often the most abundant pesticides found in bee-collected pollen, bee bread, and other hive products [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This should not be surprising since fungicide applications are recommended during crop bloom, when honey bees and other pollinators are actively foraging at flowers. But honey bees are also exposed to insecticides and other pesticides during crop pollination. For example, honey bees conducting blueberry pollination in Michigan were simultaneously exposed to an average of 35 pesticides in bee-collected pollen [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] with the majority of risk coming from the organophosphate insecticide chlorpyrifos (max concentration in pollen\u0026thinsp;=\u0026thinsp;214 parts per billion; ppb), and the neonicotinoid insecticides clothianidin (max concentration\u0026thinsp;=\u0026thinsp;35 ppb), imidacloprid (max concentration\u0026thinsp;=\u0026thinsp;9 ppb), and thiamethoxam (max concentration\u0026thinsp;=\u0026thinsp;15 ppb; [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Honey bees conducting apple pollination in New York were also commonly exposed to insecticides; freshly collected beebread from hives at 24 of 30 orchards contained insecticides, with the majority of risk attributed to the oxadiazine insecticide indoxacarb (max concentration in pollen\u0026thinsp;=\u0026thinsp;918 ppb) and the neonicotinoid insecticide thiamethoxam (max concentration in pollen\u0026thinsp;=\u0026thinsp;48 ppb [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeonicotinoids contribute to honey bee risk during crop pollination, and a worldwide review of pesticide risk to bees found that the neonicotinoid thiamethoxam was one of the three highest-risk pesticides to honey bees [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thiamethoxam is a systemic and environmentally persistent insecticide, which increases the likelihood of exposure via pollen, nectar, plant guttation fluids, soils, and other environmental matrices for days, months, or even years [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thiamethoxam\u0026rsquo;s action as a neurotoxin can lead to paralysis and death of adult bees by binding to nicotinic acetylcholine receptors, and at sublethal exposure levels, can affect the ability of bees to perform important tasks inside and outside of the hive [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Exposure of larvae to thiamethoxam is also known to affect survival and physiology of the honey bee postembryonic stages [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough fungicide exposure is generally considered safe for bees, concern has recently been raised about the risk posed from some fungicides, including captan [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Captan is one of the most widely used fungicides in the United States and the most-sprayed fungicide during apple bloom in New York State [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Officially, captan is considered relatively non-toxic to adult honey bees [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, little is known regarding interactions between captan and any insecticides, including thiamethoxam. Such information is important since some fungicide-insecticide combinations lead to synergisms and some do not [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which can be useful in guiding recommendations regarding which fungicides are safest to apply during crop pollination.\u003c/p\u003e \u003cp\u003eHere, we investigate the individual and interactive effects of two commonly used pesticides, the neonicotinoid insecticide thiamethoxam and the fungicide captan, on honey bee larval development and full-colony growth and survival. In the lab, we exposed larvae to three field-realistic doses of each pesticide individually and in combination, monitoring survival and development. In the field, we exposed full colonies to each pesticide individually and in combination at the lowest dose used in the laboratory assays, monitoring colony performance and survival over a full calendar year. This study addresses three main questions: 1) What are the direct effects of captan and thiamethoxam on development and survival of individual larvae and full colonies? 2) Does captan-thiamethoxam co-exposure synergistically affect development and survival of individual larvae and full colonies? and 3) Do \u003cem\u003ein vitro\u003c/em\u003e assays with individual larvae scale up to predict colony-level outcomes?\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003elaboratory assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe assessed the impact of treatment on survival of immature bees in two ways: first using a Cox proportional hazards model for survivorship (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and second using a logistic model for the percentage mortality at the end of the trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Survival of immature bees in the positive and negative control groups (with and without acetone used as a solvent) were 74% and 69%, respectively, and were not significantly different in either model (Cox model \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003eTukey\u003c/em\u003e\u003c/sub\u003e= 0.95; logistic model \u003cem\u003ep\u003c/em\u003e\u003csub\u003eTukey\u003c/sub\u003e = 0.97). All pesticide treatments resulted in significantly greater mortality than the positive control (Tables S2 and S3). Treatment of larvae with the fungicide captan resulted in an 80\u0026ndash;90% increase in mortality compared to the positive controls, with no significant difference between dosages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Tables S2 and S3). Treatment of larvae with the insecticide thiamethoxam at the low, medium and high dosages increased the mortality rates by 90%, 120% and 150%, respectively, compared to the positive control, although again the differences between dosages were not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Tables S2 and S3). Finally, the combination of captan (highest dosage) with thiamethoxam at the low, medium and high dosages demonstrated 130%, 140% and 210% increase in mortality rates, respectively, compared to the positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Mortality from the combination with the high dose of thiamethoxam was significantly greater than mortality from the combination with the medium or low doses via the Cox model (Table S2), but these differences were not observed via the logistic model (Table S3).\u003c/p\u003e \u003cp\u003eWe developed an improved method to assess interactions between pesticides that identifies non-additive responses (synergism or antagonism) using a log-binomial generalized linear model (GLM) for survivorship. We did not detect any significant interactions on the overall survivorship (see Table S4). However, when survivorship within each developmental stage was analyzed separately, we detected statistically significant synergistic interactions (leading to higher-than-expected mortality) at the larval stage in the bioassay when captan was crossed with thiamethoxam at highest dose (LR\u0026thinsp;=\u0026thinsp;7.932, \u003cem\u003ep\u003c/em\u003e\u003csub\u003eHolm\u003c/sub\u003e = 0.044). For other developmental stages and chemical dosages, no significant interactions were observed (see Table S4).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eField trials with whole colonies\u003c/h2\u003e \u003cp\u003eWe assessed the impact of treatment using nine performance metrics in all colonies over a full year \u0026ndash; number of adult bees, nectar cells, pollen cells, worker brood cells, drone brood cells, varroa levels, chalkbrood presence, queen supersedure events, and colony survival. No significant difference was observed among treatment groups and there was no treatment \u0026times; month interaction for: number of adult bees, nectar cells, pollen cells, worker brood cells, or drone brood cells, but each of these performance metrics varied significantly among months (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplemental material Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLikelihood ratio test results for separate models to test the effect of pesticide treatment on five performance metrics - area of adult bees, nectar cells, pollen cells, worker brood cells, and drone brood cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDegrees of freedom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdult bees\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBee yard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment \u0026times; month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNectar cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea of adult bees\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBee yard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment \u0026times; month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePollen cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea of adult bees\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBee yard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment \u0026times; month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrood cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e159.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea of adult bees\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBee yard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment \u0026times; month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrone brood cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e62.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArea of adult bees\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBee yard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment \u0026times; month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConsumption of pollen patties containing the treatments (captan, thiamethoxam, captan\u0026thinsp;+\u0026thinsp;thiamethoxam, or control) did not vary by treatment, bee yard, or the treatment \u0026times; month interaction (Supplemental material Table S5), but consumption varied significantly among months and was positively correlated with the area of adult bees in a colony (β\u0026thinsp;=\u0026thinsp;4.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06; Table S5, Fig. S2).\u003c/p\u003e \u003cp\u003eQueen supersedure occurred in eleven of the 46 colonies, but supersedure was not related to treatment (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.43, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.093). Varroa levels varied significantly among months, with levels higher in September 2016 than in July 2016 or April 2017 (Supplemental material Table S6; Fig. S3), but did not vary by treatment. Chalkbrood was present in 25 of the 46 hives. The presence of chalkbrood at any point in the study was not related to treatment (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.54) or bee yard (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.26, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32). Finally, no significant difference in colony survival was observed (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e11\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.51, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur data indicate that exposure of honey bee larvae to field-realistic levels of the fungicide captan, neonicotinoid insecticide thiamethoxam, and the combination of captan\u0026thinsp;+\u0026thinsp;thiamethoxam significantly increased mortality. Perhaps most importantly, we observed that the 80\u0026ndash;90% increase in mortality of immature bees exposed to captan was dose-independent and similar in magnitude to the lowest dose of thiamethoxam. Furthermore, synergism between captan and thiamethoxam was observed to non-additively increase larval mortality at the highest dose of thiamethoxam, but there was no evidence of synergism or antagonism for other developmental stages or chemical dosages. At the full-colony level in the field, the effects of pesticide exposure at the lowest doses used in the laboratory assays did not translate to changes in colony performance as measured by bee population numbers, resistance to parasites/pathogens, or colony survivorship. Thus, we find that captan and thiamethoxam are each acutely toxic to immature honey bees, but whole colonies can potentially compensate for detrimental effects, at least at the low doses and duration used for our field trial.\u003c/p\u003e \u003cp\u003eFungicides are not intended to control insects, and their acute toxicity to honey bees is generally low[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. That said, a recent review shows that risk from some fungicide exposures can be high for bees [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Impacts of fungicides on honey bee brood have been observed [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], which is in agreement with our findings regarding captan\u0026rsquo;s acute toxicity to developing larvae/pupae. Captan inhibits respiratory pathways in fungal cells, but this mode of action may also be detrimental to animals such as honey bees since they use similar metabolic pathways [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The larval stages of honey bees may be particularly susceptible to captan due to the high rate of mitochondrial biogenesis and respiratory metabolism in response to the large ATP demand at this stage of development [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to the direct acute toxicity of captan by itself, we also addressed the potential interaction of this fungicide with the neonicotinoid insecticide thiamethoxam. The combination of captan with thiamethoxam at the highest concentration tested did synergistically increase mortality of larvae. However, synergisms at other concentrations and at other developmental stages were not observed. Thiamethoxam is an insecticide distinguished for its lethality to bees in both immature and adult stages [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Despite this neonicotinoid impacting neurologic [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and immune systems [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], the honey bee detoxification system does have some mechanisms to metabolize nicotine-like compounds [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Sterol biosynthesis-inhibiting (SBI) fungicides are known to interfere with these detoxification mechanisms [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], but captan does not have the same mode of action as SBI fungicides and therefore the mechanism underlying synergism is unclear. Due to their limited number of detoxification genes compared to other insects, honey bees may have difficulty processing multiple xenobiotics simultaneously [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the findings of the \u003cem\u003ein vitro\u003c/em\u003e assays, our field trial with full colonies did not observe impacts of pesticide exposure over one year at the low concentrations used in the study. The incongruity among lab and field toxicology studies of bees is important to consider. Three main factors that can lead to incongruence between laboratory-based and field-based toxicology studies: pesticide concentration, duration of exposure, and the capacity of foragers to choose between foraging sites in the field. Our choice of pesticide concentrations for the \u003cem\u003ein vitro\u003c/em\u003e lab assays (100, 500, and 2,000 ppb for captan, and 10, 70, and 1,440 ppb for thiamethoxam) were based on a careful search of pesticide residues found in field surveys of honey bee-collected pollen and beebread (see Tables S7 \u0026amp; S8) and the concentrations chosen provide a range of field-realistic exposure possibilities. In addition, in the \u003cem\u003ein vitro\u003c/em\u003e lab assay, larvae were exposed to pesticide treatments via diet from the fourth day of development until reaching adulthood. This protocol is what is advised by the Organization for Economic Cooperation and Development guidelines for toxicological studies (Test No. 239 (OECD, 2016)[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], indicating our laboratory bioassays were appropriate. Thus, our lab-based study was sound, and the field-based study shows that full colonies likely have mechanisms to compensate for increased larval mortality. For example, the queen may compensate for increased larval mortality by laying more eggs. Short-term colony resilience has been described among colonies exposed to sub-lethal dosages of neonicotinoids, whereby colonies increase brood initiation rate to compensate for increased brood mortality [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. While effective in the short term, this could lead to longer-term costs for the queen and an increased incidence of queen failure [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe degree to which nurse bees in honey bee colonies can detoxify xenobiotic substances in pollen before secreting jelly to the brood is poorly known, but the limited evidence to date suggests this can be part of an effective \u0026ldquo;social detoxification system\u0026rdquo; [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. For example, Ricke et al. (2021) found that less than 2% of pesticides in pollen fed to nurse bees made it into jelly excreted by the workers[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Moreover, royal jelly from colonies exposed to pesticide-contaminated pollen contained negligible residues, although queens reared with this jelly had reduced reproductive quality [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. At immature stages, exposure occurs transdermally and orally at the same time. This may be especially important given that early life stages of bees are generally more sensitive to contaminants relative to adult stages [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, our laboratory and field results show that field-realistic exposures to the fungicide captan greatly increase immature honey bee mortality, and synergism with the neonicotinoid thiamethoxam occurs, but synergism is dose-dependent and full colonies may be able to compensate for losses. Therefore, we suggest that further effort be placed in understanding how exposure to captan and co-exposure with other pesticides affects honey bee colony dynamics and mortality of solitary and sub-social wild bees, which lack the social detoxification capabilities of honey bees and can therefore be more susceptible to pesticides in the field. Notably, captan affected honey bee larval survivorship regardless of the concentration and at field-realistic levels. Most studies investigating captan describe effects after chronic exposure [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], while our study found an impact via a single exposure event. Since captan is actively applied to crops during bloom while honey bees and wild pollinators are carrying out critical pollination services, further investigations of this fungicide are warranted to ensure the sustainability of crop pollination and agricultural production.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eThe agrochemicals used for toxicity assays were the fungicide captan (CAS number 133-06-2, \u0026ge;\u0026thinsp;98% purity); and the insecticide thiamethoxam (CAS number 153719-23-4, \u0026ge;\u0026thinsp;98% purity), both from Sigma-Aldrich. The fungicide captan is one of the most-used fungicides in the United States and is the most-sprayed fungicide during apple bloom in New York [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thiamethoxam is a widely used neonicotinoid insecticide and has been identified as one of the highest-risk pesticides to honey bees worldwide [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].The agrochemicals used for toxicity assays were the fungicide captan (CAS number 133-06-2, \u0026ge;\u0026thinsp;98% purity); and the insecticide thiamethoxam (CAS number 153719-23-4, \u0026ge;\u0026thinsp;98% purity), both from Sigma-Aldrich. The fungicide captan is one of the most-used fungicides in the United States and is the most-sprayed fungicide during apple bloom in NY [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thiamethoxam is a widely used neonicotinoid insecticide [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and contributes to worldwide risk to honey bees [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003elaboratory assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe used an \u003cem\u003ein vitro\u003c/em\u003e rearing method to assess the impact of pesticide exposure on individual honey bees during development. Worker larvae were sampled from six honey bee colonies (unselected stock) located at the Dyce Lab for Honey Bee Studies at Cornell University in Ithaca, New York. Worker larvae at the first instar were grafted individually in commercial plastic queen cups, which were placed inside 96-well microcentrifuge tube racks containing equal numbers of wells filled with a saturated K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and using a feeding protocol previously described for honey bee worker rearing in an artificial environment [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] with slight modifications on the volume of liquid diet provided (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eLarval feeding regimen adopted to the\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003erearing system.\u003c/b\u003e The diet provided were A (44.25% of royal jelly, 5.3% of D-Glucose, 5.3% of D-Fructose, 0.9% of Yeast extract and 44.25 distilled water), B (42.94% of royal jelly, 6.4% of D-Glucose, 6.4% of D-Fructose, 1.3% of Yeast extract and 42.95 distilled water) or C (50% of royal jelly, 9% of D-Glucose, 9% of D-Fructose, 2% of Yeast extract and 30% distilled water).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarval progress\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmount of diet (\u0026micro;l)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 0 (at grafting)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en/a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGlucose (CAS Number: 50-99-7) and Fructose (CAS Number: 57-48-7) were obtained from Sigma-Aldrich (St. Louis, MO, USA), yeast extract (#288620) was obtained from Life Technologies Corp. (Sparks, MD, USA) and organic royal jelly was obtained from Glorybee (Eugene, OR, USA). Diets were prepared and provided in different amounts according to each larval stage. The larvae were kept in an incubator at 34.5 ˚C and 80% RH until the defecation stage, then at 70% RH during the pupal stage until emergence of the adults [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder normal development, worker larvae switch from an exclusive royal jelly diet to a mix of the same secretions with pollen and nectar between the fourth to the fifth larval instar [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Thus, we applied the pesticide treatments at this phase, following the OECD 239 study design [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Cautious attention was taken to select only larvae that had achieved the fourth larval instar [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], thus ensuring the next stages of developmental monitoring were accurate. Treatment solutions were mixed into the diet and supplied individually to larvae according to each treatment group. The concentrations used were based on sublethal concentrations previously found in the literature (see Tables S7 and S8). For the insecticide thiamethoxam, the concentrations were: low (10 ng/ml\u0026thinsp;=\u0026thinsp;10 parts per billion), medium (70 ng/ul) and high (1,440 ng/ml). For the fungicide captan, the concentrations were: low (100 ng/ml), medium (500 ng/ml) and high (2,000) ng/ml. We tested for potential interactions between these pesticides by using a blend of captan at high concentration (2,000 ng/ml) and thiamethoxam at low (10 ng/ml\u0026thinsp;=\u0026thinsp;10 parts per billion), medium (70 ng/ul) and high (1,440 ng/ml) concentrations. Both chemicals were solubilized in acetone before combining with the jelly diet. Positive (+\u0026thinsp;acetone) and negative (no acetone) control groups were tested to measure possible effects of the solvent. Three experimental trials were conducted at different times; see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the number of treated larvae in each treatment group and trial. The cups containing the diet with different treatments were randomly distributed in the racks to avoid site effects.\u003c/p\u003e \u003cp\u003eAll larvae were monitored twice a day and larvae that died (movement stopped, discoloring, drowned in the food) or if there was any evidence of contamination, were immediately recorded and removed. Survivorship and developmental advances were recorded during all ontogenic stages until each larva reached adulthood. Adults were considered viable if they successfully crossed the pupation stages and reached adulthood healthy, morphologically well-shaped, and actively walking around.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eField trials with whole colonies\u003c/h2\u003e \u003cp\u003eForty-eight nucleus honey bee colonies were acquired from a commercial beekeeper (Chuck Kutik, Norwich, NY). All nucleus colonies were transferred into a new 10-frame box with plastic foundation and allowed to draw comb for two weeks in a common location. Before starting the experiments, all colonies were checked for queen status, number of bees, and frame composition. Frames were redistributed among colonies such that all colonies had a similar composition of brood, bees, pollen and nectar immediately prior to enrollment in the field experiment. In addition, each colony was fitted with a pollen trap, placed at the entrance of the hive, to restrict pollen flow into the hive and therefore ensure consumption of the treated pollen patties [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwelve colonies were assigned to each of four treatments: control (no pesticides added), thiamethoxam (10 ppb), captan (1,000 ppb), and thiamethoxam\u0026thinsp;+\u0026thinsp;captan (10 ppb thiamethoxam\u0026thinsp;+\u0026thinsp;1,000 ppb captan). We used the lowest dose of thiamethoxam from our laboratory assays (10 ppb) and a medium/high dose of captan (1,000 ppb) because these contamination levels are commonly observed from our own studies during crop pollination [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]; and the broader literature (see Tables S7 and S8). Pollen patty treatments were prepared the day before they were fed to the bees using captan (CAS number 133-06-2, \u0026ge;\u0026thinsp;98% purity) and thiamethoxam (CAS number 153719-23-4, \u0026ge;\u0026thinsp;98% purity), both from Sigma-Aldrich (St. Louis, MO, USA), and Bee Pro from Mann Lake (Hackensack, MN, USA). The pollen patties were provided to each colony weekly from the first week of June to the first week in October 2016. Patties were kept on for 3\u0026ndash;7 days, with an average of 4 days, until they were almost entirely consumed. Two hives had patties left on for 14 days in one month. Because the colonies accumulated minimal honey during the summer, we supplementally fed each colony three liters of sugar syrup (30% by volume) from the third week of August until the first week first week of October. Sugar syrup was prepared weekly according to treatments: thiamethoxam (10 ppb), captan (1,000 ppb), and thiamethoxam\u0026thinsp;+\u0026thinsp;captan (10 ppb thiamethoxam\u0026thinsp;+\u0026thinsp;1,000 ppb captan). The colonies were equally distributed among three bee yards: Varna (42.463730, -76.440748), Turkey Hill (42.437293, -76.427063), and Sarkaria (42.443964, -76.452238); which are ~\u0026thinsp;2 km from each other. The colonies were not treated for varroa mites during the experiment, and overwintering preparation consisted of placing a moisture board and foam-insulated inner cover under the telescoping outer cover.\u003c/p\u003e \u003cp\u003ePerformance and survival of colonies were assessed via monthly inspections in June, July, August, and October 2016, and April 2017. During each visit, we estimated the number of adult bees, brood cells, nectar cells, and pollen cells of each surviving colony. We followed the Liebefelder method [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], visually estimating the proportion of each frame covered by adult bees or each type of cell and summing these proportions across all frames in a hive. Additionally, each colony had one drone comb frame, and the proportion of the frame covered by drone brood was recorded once per month in July, August, September, October 2016 and April 2017. The inspections also accounted for queen supersedure events, varroa levels (measured as mites per 300 bees using the sugar roll method) and chalkbrood presence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003elaboratory assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe assessed the impact of treatment on the survival of immature bees using a Cox proportional hazards model for the survivorship, as well as a logistic model for the percentage mortality at the end of the trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In each model, both the pesticide treatment and trial number were included as additive fixed effects, the latter to account for variation in natural mortality between trials. Post-hoc tests for pairwise differences between treatments, marginalized across trials, were performed for both models, adjusting for multiple comparisons using Tukey's HSD.\u003c/p\u003e \u003cp\u003eWe used Bliss' definition of independence to define synergism or antagonism between different pesticides [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Although Bliss independence is traditionally defined using mortality rates, it can also be defined using survival rates. Let \u003cem\u003es\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e be the control survival rate, \u003cem\u003es\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e the survival rate when exposed to pesticide A, and \u003cem\u003es\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e the survival rate when exposed to pesticide B; here \u003cem\u003es\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e and \u003cem\u003es\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e can be interpreted as Abbott-corrected survival rates [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. If pesticides A and B are Bliss-independent, then the survival rate when simultaneously exposed to both pesticides is given by \u003cem\u003es\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e; if the actual survival rate is lower, then the two pesticides interact synergistically, whereas if the reverse is true, then they interact antagonistically. Biologically, Bliss independence requires that the two pesticides have independent biological action, and also that individual susceptibilities to A and to B are uncorrelated. The second requirement is important but often overlooked: as an extreme example, if the susceptibilities were perfectly correlated, then all bees that survive pesticide A will also survive any additional exposure to pesticide B, so the survival rate remains at \u003cem\u003es\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e \u0026lt; \u003cem\u003es\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e\u0026middot;\u003cem\u003es\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e, leading to an apparent antagonism.\u003c/p\u003e \u003cp\u003eWe developed a new approach to assess Bliss independence between pesticides A and B, based on the well-established framework of the generalized linear model (GLM). The idea is as follows. We fitted \u003cem\u003esurvivorship\u003c/em\u003e using a binomial GLM with a \u003cem\u003elog\u003c/em\u003e link function (as opposed to typical binomial GLMs for \u003cem\u003emortality\u003c/em\u003e with \u003cem\u003elogit\u003c/em\u003e link functions), with binary (0/1) predictors \u003cem\u003ex\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e and \u003cem\u003ex\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e, representing exposure to pesticide A and to B, as well as the interaction \u003cem\u003ex\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e\u0026middot;\u003cem\u003ex\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e. The log link function means that log(p\u003csub\u003esurv\u003c/sub\u003e) = \u003cem\u003eβ\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e\u0026middot;\u003cem\u003ex\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e\u0026middot;\u003cem\u003ex\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eAB\u003c/sub\u003e\u0026middot;\u003cem\u003ex\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e\u0026middot;\u003cem\u003ex\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e, so the survival rates are exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e) for control, exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e) for pesticide A only, exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e) for pesticide B only, and exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eAB\u003c/sub\u003e) for simultaneous exposure. If pesticides A and B are Bliss-independent, then the survival rate for the latter should simply be exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e)\u0026middot;exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e), so we require that exp(\u003cem\u003eβ\u003c/em\u003e\u003csub\u003eAB\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1, or equivalently \u003cem\u003eβ\u003c/em\u003e\u003csub\u003eAB\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0. Therefore, Bliss independence is equivalent to the null hypothesis that the interaction \u003cem\u003ex\u003c/em\u003e\u003csub\u003eA\u003c/sub\u003e \u003cem\u003ex\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e has no effect, which can be evaluated using a likelihood ratio test. A significant negative \u003cem\u003eβ\u003c/em\u003e\u003csub\u003eAB\u003c/sub\u003e would imply synergism, and a significant positive \u003cem\u003eβ\u003c/em\u003e\u003csub\u003eAB\u003c/sub\u003e would imply antagonism. The use of a log-binomial GLM also allows us to include the trial number as an additional fixed effect to account for variations in natural mortality between trials. Hence our method improves upon the method in (Sgolastra et al., 2017) [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], not only by accounting for natural mortality, but also by allowing for variations between experimental replicates. We tested for interactions between the fungicide and the insecticide at three different chemical dosages on the overall survivorship, as well as the survivorship within each of three benchmarks of ontogenic development (5th larval instar, Pre-pupae and pupation). Holm correction was used to account for multiple testing.\u003c/p\u003e \u003cp\u003eAll analyses and graphical presentation were conducted in R version 3.5.1 [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], using the packages \"survival\" [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] and \u0026ldquo;survminer\u0026rdquo; [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] for the analysis of survival curves, \"emmeans\" [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e] for post-hoc pairwise tests between treatments, and \u0026ldquo;lbreg\u0026rdquo; [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e] for fitting log-binomial models in the Bliss independence analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eField trials with whole colonies\u003c/h2\u003e \u003cp\u003eFirst, we tested whether pollen patty consumption (grams consumed per day) varied across treatments. Treatment, month, the treatment \u0026times; month interaction, and bee yard were fixed effects, and colony identity was a random effect. We also included the estimated number of adult bees during performance assessments, as a covariate. Month was treated as a categorical predictor because the number of adult bees was not measured in September, we estimated this value by averaging the number of adult bees in October and August. Significance of model terms here and below was assessed with likelihood ratio tests. We used post-hoc Tukey pairwise comparisons to test for differences in pollen patty consumption between months.\u003c/p\u003e \u003cp\u003eRegarding the survival analysis, we performed a Kaplan-Meier survival curves for each treatment; \u003cem\u003esurv function\u003c/em\u003e, \u0026lsquo;survival\u0026rsquo; package [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. We assessed statistical differences in survival among treatments with a log-rank test, using treatment as the main effect and bee yard as a frailty term (\u003cem\u003esurvdiff function\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eTo examine the performance ratios, we used separate linear mixed models to test how treatment influenced the frame area of adult bees, nectar cells, pollen cells, and worker brood cells. Treatment, month, the treatment \u0026times; month interaction, and bee yard were fixed effects, and colony identity was a random effect. We also included the frame area of adult bees as a covariate when modeling the frame area of brood cells, nectar cells, and pollen cells. The proportion of the drone brood frame covered by drone brood was modeled with the same fixed and random effects using a generalized linear mixed model and a Tweedie error distribution; glmmTMB function, \u0026lsquo;TMB\u0026rsquo; package [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. When month was significant in any of the above performance metrics, we used post-hoc Tukey pairwise comparisons to test for differences between months.\u003c/p\u003e \u003cp\u003eThe possible event of supersedure of the original queen (yes/no) at any point in the study was modeled as a function of treatment and bee yard using penalized logistic regression due to small number of positive cases (logistf function, \u0026lsquo;logistf\u0026rsquo; package, [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. For parasites and pathogens, we assessed Varroa levels (mites per 300 bees, log(x\u0026thinsp;+\u0026thinsp;1) transformed) with a linear mixed model. Treatment, month, the treatment \u0026times; month interaction, and site (bee yards) were included as fixed effects, and colony identity was included as a random effect. We modeled the presence of chalkbrood (yes/no) at any point in the study as a function of treatment and bee yard using logistic regression.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Alina Xiao, Trebor Hall \u0026amp; David Lewis for help with the field experiment, and Connor Hinsley and Hailey Scofield for help with lab experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDDS, MLS, and SHM conceived and designed the experiments. DDS, MLS, AAF, and NB collected the data. DDS, CMU, WHN and SHM analyzed and/or interpreted the data. DDS and CMU drafted the manuscript. All authors revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by USDA-NIFA AFRI grant 2018-08603 and the New York State Environmental Protection Fund. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting Interests\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKleijn, D., et al., \u003cem\u003eDelivery of crop pollination services is an insufficient argument for wild pollinator conservation.\u003c/em\u003e Nature Communications, 2015. \u003cstrong\u003e6\u003c/strong\u003e(1): p. 7414.\u003c/li\u003e\n\u003cli\u003eVan der Zee, R., et al., \u003cem\u003eTitle: Managed honey bee colony losses in Canada, China, Europe, Israel and Turkey, for the winters of 2008-9 and 2009-10.\u003c/em\u003e Journal of Apicultural Research, 2012. \u003cstrong\u003e51\u003c/strong\u003e: p. 91-114.\u003c/li\u003e\n\u003cli\u003eTraynor, K., et al., \u003cem\u003eMultiyear survey targeting disease incidence in US honey bees.\u003c/em\u003e Apidologie, 2016. \u003cstrong\u003e47\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eTraynor, K.S., et al., \u003cem\u003eIn-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States.\u003c/em\u003e Scientific Reports, 2016. \u003cstrong\u003e6\u003c/strong\u003e: p. 33207.\u003c/li\u003e\n\u003cli\u003eSeitz, N., et al., \u003cem\u003eA national survey of managed honey bee 2014\u0026ndash;2015 annual colony losses in the USA.\u003c/em\u003e Journal of Apicultural Research, 2015. \u003cstrong\u003e54\u003c/strong\u003e(4): p. 292-304.\u003c/li\u003e\n\u003cli\u003eKulhanek, K., et al., \u003cem\u003eA national survey of managed honey bee 2015\u0026ndash;2016 annual colony losses in the USA.\u003c/em\u003e Journal of Apicultural Research, 2017. \u003cstrong\u003e56\u003c/strong\u003e(4): p. 328-340.\u003c/li\u003e\n\u003cli\u003eGoulson, D., et al., \u003cem\u003eBee declines driven by combined stress from parasites, pesticides, and lack of flowers.\u003c/em\u003e Science, 2015. \u003cstrong\u003e347\u003c/strong\u003e(6229): p. 1255957.\u003c/li\u003e\n\u003cli\u003eAlaux, C., et al., \u003cem\u003eInteractions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera).\u003c/em\u003e Environmental Microbiology, 2010. \u003cstrong\u003e12\u003c/strong\u003e(3): p. 774-782.\u003c/li\u003e\n\u003cli\u003ePettis, J.S., et al., \u003cem\u003ePesticide exposure in honey bees results in increased levels of the gut pathogen Nosema.\u003c/em\u003e Naturwissenschaften, 2012. \u003cstrong\u003e99\u003c/strong\u003e(2): p. 153-158.\u003c/li\u003e\n\u003cli\u003eWu, J.Y., et al., \u003cem\u003eHoney bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection.\u003c/em\u003e Journal of Invertebrate Pathology, 2012. \u003cstrong\u003e109\u003c/strong\u003e(3): p. 326-329.\u003c/li\u003e\n\u003cli\u003eHenry, M., et al., \u003cem\u003eA common pesticide decreases foraging success and survival in honey bees.\u003c/em\u003e Science, 2012. \u003cstrong\u003e336\u003c/strong\u003e(6079): p. 348-350.\u003c/li\u003e\n\u003cli\u003eStanley, D.A., et al., \u003cem\u003eNeonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees.\u003c/em\u003e Nature, 2015. \u003cstrong\u003e528\u003c/strong\u003e(7583): p. 548-550.\u003c/li\u003e\n\u003cli\u003eDesneux, N., A. Decourtye, and J.-M. Delpuech, \u003cem\u003eThe sublethal effects of pesticides on beneficial arthropods.\u003c/em\u003e Annual Review of Entomology, 2006. \u003cstrong\u003e52\u003c/strong\u003e(1): p. 81-106.\u003c/li\u003e\n\u003cli\u003eWhitehorn, P.R., et al., \u003cem\u003eNeonicotinoid pesticide reduces bumble bee colony growth and queen production.\u003c/em\u003e Science, 2012. \u003cstrong\u003e336\u003c/strong\u003e(6079): p. 351-352.\u003c/li\u003e\n\u003cli\u003eVidau, C., et al., \u003cem\u003eExposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae.\u003c/em\u003e PLoS ONE, 2011. \u003cstrong\u003e6\u003c/strong\u003e(6): p. e21550.\u003c/li\u003e\n\u003cli\u003eGraham, K.K., et al., \u003cem\u003eIdentities, concentrations, and sources of pesticide exposure in pollen collected by managed bees during blueberry pollination.\u003c/em\u003e Scientific Reports, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 16857.\u003c/li\u003e\n\u003cli\u003ePettis, J., et al., \u003cem\u003eCrop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae.\u003c/em\u003e PloS one, 2013. \u003cstrong\u003e8\u003c/strong\u003e: p. e70182.\u003c/li\u003e\n\u003cli\u003eFrazier, M., et al., \u003cem\u003eAssessing Honey Bee (Hymenoptera: Apidae) Foraging Populations and the Potential Impact of Pesticides on Eight U.S. Crops.\u003c/em\u003e Journal of Economic Entomology, 2015. \u003cstrong\u003e108\u003c/strong\u003e: p. tov195.\u003c/li\u003e\n\u003cli\u003eMcArt, S.H., et al., \u003cem\u003eHigh pesticide risk to honey bees despite low focal crop pollen collection during pollination of a mass blooming crop.\u003c/em\u003e Scientific Reports, 2017. \u003cstrong\u003e7\u003c/strong\u003e: p. 46554.\u003c/li\u003e\n\u003cli\u003eChauzat, M.-P., et al., \u003cem\u003eA Survey of Pesticide Residues in Pollen Loads Collected by Honey Bees in France.\u003c/em\u003e Journal of economic entomology, 2006. \u003cstrong\u003e99\u003c/strong\u003e: p. 253-62.\u003c/li\u003e\n\u003cli\u003eMullin, C.A., et al., \u003cem\u003eHigh Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health.\u003c/em\u003e PLOS ONE, 2010. \u003cstrong\u003e5\u003c/strong\u003e(3): p. e9754.\u003c/li\u003e\n\u003cli\u003eSanchez-Bayo, F. and K. Goka, \u003cem\u003ePesticide Residues and Bees \u0026ndash; A Risk Assessment.\u003c/em\u003e PLOS ONE, 2014. \u003cstrong\u003e9\u003c/strong\u003e(4): p. e94482.\u003c/li\u003e\n\u003cli\u003eMullin, C., et al., \u003cem\u003eThe formulation makes the honey bee poison.\u003c/em\u003e Pesticide Biochemistry and Physiology, 2015. \u003cstrong\u003e120\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eMilano, N., et al., \u003cem\u003eComparative survival and fitness of bumble bee colonies in natural, suburban, and agricultural landscapes.\u003c/em\u003e Agriculture, Ecosystems \u0026amp; Environment, 2019. \u003cstrong\u003e284\u003c/strong\u003e: p. 106594.\u003c/li\u003e\n\u003cli\u003eGraham, K.K., et al., \u003cem\u003ePesticide risk to managed bees during blueberry pollination is primarily driven by off-farm exposures.\u003c/em\u003e Scientific Reports, 2022. \u003cstrong\u003e12\u003c/strong\u003e(1): p. 7189.\u003c/li\u003e\n\u003cli\u003eMaus, C., A. Alix, and D. Schmehl, \u003cem\u003eAssessing the impact of pesticides on pollinators\u003c/em\u003e. 2022. p. 183-224.\u003c/li\u003e\n\u003cli\u003eMaienfisch, P., et al., \u003cem\u003eChemistry and biology of thiamethoxam: a second generation neonicotinoid.\u003c/em\u003e Pest Management Science, 2001. \u003cstrong\u003e57\u003c/strong\u003e(10): p. 906-913.\u003c/li\u003e\n\u003cli\u003eGoulson, D., \u003cem\u003eREVIEW: An overview of the environmental risks posed by neonicotinoid insecticides.\u003c/em\u003e Journal of Applied Ecology, 2013. \u003cstrong\u003e50\u003c/strong\u003e(4): p. 977-987.\u003c/li\u003e\n\u003cli\u003eWood, T.J. and D. Goulson, \u003cem\u003eThe environmental risks of neonicotinoid pesticides: a review of the evidence post 2013.\u003c/em\u003e Environmental science and pollution research international, 2017. \u003cstrong\u003e24\u003c/strong\u003e(21): p. 17285-17325.\u003c/li\u003e\n\u003cli\u003eMatsuda, K., et al., \u003cem\u003eNeonicotinoids: Insecticides Acting on Insect Nicotinic Acetylcholine Receptors.\u003c/em\u003e Trends in Pharmacological Sciences, 2001. \u003cstrong\u003e22\u003c/strong\u003e: p. 573-580.\u003c/li\u003e\n\u003cli\u003eIwasa, T., \u003cem\u003eMechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera.\u003c/em\u003e Crop Protection, 2004. \u003cstrong\u003e23\u003c/strong\u003e: p. 371-378.\u003c/li\u003e\n\u003cli\u003eTosi, S. and J.C. Nieh, \u003cem\u003eA common neonicotinoid pesticide, thiamethoxam, alters honey bee activity, motor functions, and movement to light.\u003c/em\u003e Scientific Reports, 2017. \u003cstrong\u003e7\u003c/strong\u003e(1): p. 15132.\u003c/li\u003e\n\u003cli\u003eTavares, D.A., et al., \u003cem\u003eExposure of larvae to thiamethoxam affects the survival and physiology of the honey bee at post-embryonic stages.\u003c/em\u003e Environmental Pollution, 2017. \u003cstrong\u003e229\u003c/strong\u003e: p. 386-393.\u003c/li\u003e\n\u003cli\u003eQi, S., et al., \u003cem\u003eFlumethrin at honey-relevant levels induces physiological stresses to honey bee larvae (Apis mellifera L.) in vitro.\u003c/em\u003e Ecotoxicology and environmental safety, 2019. \u003cstrong\u003e190\u003c/strong\u003e: p. 110101.\u003c/li\u003e\n\u003cli\u003eDecio, P., et al., \u003cem\u003eThiamethoxam exposure deregulates short ORF gene expression in the honey bee and compromises immune response to bacteria.\u003c/em\u003e Scientific Reports, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 1489.\u003c/li\u003e\n\u003cli\u003eEverich, R., et al., \u003cem\u003eEffects of Captan on Apis mellifera Brood Development Under Field Conditions in California Almond Orchards.\u003c/em\u003e Journal of economic entomology, 2009. \u003cstrong\u003e102\u003c/strong\u003e: p. 20-9.\u003c/li\u003e\n\u003cli\u003eHeylen, K., et al., \u003cem\u003eThe effects of four crop protection products on the morphology and ultrastructure of the hypopharyngeal gland of the European honeybee, Apis mellifera.\u003c/em\u003e Apidologie, 2011. \u003cstrong\u003e42\u003c/strong\u003e: p. 103-116.\u003c/li\u003e\n\u003cli\u003eRondeau, S. and N.E. Raine, \u003cem\u003eFungicides and bees: a review of exposure and risk.\u003c/em\u003e Environment International, 2022. \u003cstrong\u003e165\u003c/strong\u003e: p. 107311.\u003c/li\u003e\n\u003cli\u003eAtkins, E.L. and D. Kellum, \u003cem\u003eComparative Morphogenic and toxicity Studies on the Effect of Pesticides on Honeybee Brood.\u003c/em\u003e Journal of Apicultural Research, 1986. \u003cstrong\u003e25\u003c/strong\u003e(4): p. 242-255.\u003c/li\u003e\n\u003cli\u003eUSEPA, \u003cem\u003eCaptan Reregistration Eligibility Decision - EPA-738-F99-015\u003c/em\u003e, P.a.T.S. Prevention, Editor. 1999.\u003c/li\u003e\n\u003cli\u003eIverson, A., et al., \u003cem\u003eSynergistic effects of three sterol biosynthesis inhibiting fungicides on the toxicity of a pyrethroid and neonicotinoid insecticide to bumble bees.\u003c/em\u003e Apidologie, 2019.\u003c/li\u003e\n\u003cli\u003eJohnson, R., \u003cem\u003eHoney Bee Toxicology.\u003c/em\u003e Annual review of entomology, 2014. \u003cstrong\u003e60\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eWade, A., et al., \u003cem\u003eCombined Toxicity of Insecticides and Fungicides Applied to California Almond Orchards to Honey Bee Larvae and Adults.\u003c/em\u003e Insects, 2019. \u003cstrong\u003e10\u003c/strong\u003e: p. 20.\u003c/li\u003e\n\u003cli\u003eZhu, W., et al., \u003cem\u003eFour Common Pesticides, Their Mixtures and a Formulation Solvent in the Hive Environment Have High Oral Toxicity to Honey Bee Larvae.\u003c/em\u003e PloS one, 2014. \u003cstrong\u003e9\u003c/strong\u003e: p. e77547.\u003c/li\u003e\n\u003cli\u003eBushway, R.J. and A.R. Hanks, \u003cem\u003ePesticide inhibition of growth and macromolecular synthesis in the cellular slime mold Dictyostelium discoideum.\u003c/em\u003e Pesticide Biochemistry and Physiology, 1976. \u003cstrong\u003e6\u003c/strong\u003e(3): p. 254-260.\u003c/li\u003e\n\u003cli\u003eMussen, E., J. Lopez, and C. Peng, \u003cem\u003eEffects of Selected Fungicides on Growth and Development of Larval Honey Bees, Apis mellifera L. (Hymenoptera: Apidae).\u003c/em\u003e Environmental Entomology, 2004. \u003cstrong\u003e33\u003c/strong\u003e: p. 1151-1154.\u003c/li\u003e\n\u003cli\u003eSantos, D., L. Alberici, and K. Hartfelder, \u003cem\u003eMitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development\u003c/em\u003e. Vol. 73. 2016.\u003c/li\u003e\n\u003cli\u003eSep\u0026uacute;lveda Friol, P., et al., \u003cem\u003eCan the exposure of Apis mellifera (Hymenoptera, Apiadae) larvae to a field concentration of thiamethoxam affect newly emerged bees?\u003c/em\u003e Chemosphere, 2017. \u003cstrong\u003e185\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eTavares, D.A., et al., \u003cem\u003eExposure to thiamethoxam during the larval phase affects synapsin levels in the brain of the honey bee.\u003c/em\u003e Ecotoxicology and Environmental Safety, 2019. \u003cstrong\u003e169\u003c/strong\u003e: p. 523-528.\u003c/li\u003e\n\u003cli\u003eMillar, N.S. and I. Denholm, \u003cem\u003eNicotinic acetylcholine receptors: targets for commercially important insecticides.\u003c/em\u003e Invert Neurosci, 2007. \u003cstrong\u003e7\u003c/strong\u003e(1): p. 53-66.\u003c/li\u003e\n\u003cli\u003eDi Prisco, G., et al., \u003cem\u003eNeonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees.\u003c/em\u003e Proceedings of the National Academy of Sciences, 2013. \u003cstrong\u003e110\u003c/strong\u003e(46): p. 18466-18471.\u003c/li\u003e\n\u003cli\u003eCoulon, M., et al., \u003cem\u003eInfluence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees.\u003c/em\u003e PLOS ONE, 2019. \u003cstrong\u003e14\u003c/strong\u003e: p. e0220703.\u003c/li\u003e\n\u003cli\u003eBerenbaum, M.R. and R.M. Johnson, \u003cem\u003eXenobiotic detoxification pathways in honey bees.\u003c/em\u003e Current Opinion in Insect Science, 2015. \u003cstrong\u003e10\u003c/strong\u003e: p. 51-58.\u003c/li\u003e\n\u003cli\u003eSchuhmann, A., et al., \u003cem\u003eInteraction of Insecticides and Fungicides in Bees.\u003c/em\u003e Frontiers in Insect Science, 2022. \u003cstrong\u003e1\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eClaudianos, C., et al., \u003cem\u003eA deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee.\u003c/em\u003e Insect Molecular Biology, 2006. \u003cstrong\u003e15\u003c/strong\u003e(5): p. 615-636.\u003c/li\u003e\n\u003cli\u003eOECD, \u003cem\u003eGuidance document No. 239: Honey Bee Larval Toxicity Test following Repeated Exposure\u003c/em\u003e. Vol. 34. 2016: ENV/JM/MONO.\u003c/li\u003e\n\u003cli\u003eSchott, M., et al., \u003cem\u003eHoneybee colonies compensate for pesticide-induced effects on royal jelly composition and brood survival with increased brood production.\u003c/em\u003e Scientific Reports, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 62.\u003c/li\u003e\n\u003cli\u003eSandrock, C., et al., \u003cem\u003eImpact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure.\u003c/em\u003e PLOS ONE, 2014. \u003cstrong\u003e9\u003c/strong\u003e(8): p. e103592.\u003c/li\u003e\n\u003cli\u003eTarpy, D.R., E. Talley, and B.N. Metz, \u003cem\u003eInfluence of brood pheromone on honey bee colony establishment and queen replacement.\u003c/em\u003e Journal of Apicultural Research, 2021: p. 1-9.\u003c/li\u003e\n\u003cli\u003eWithrow, J.M., J.S. Pettis, and D.R. Tarpy, \u003cem\u003eEffects of Temperature During Package Transportation on Queen Establishment and Survival in Honey Bees (Hymenoptera: Apidae).\u003c/em\u003e J Econ Entomol, 2019.\u003c/li\u003e\n\u003cli\u003eLiao, L.-H., W.-Y. Wu, and M.R. Berenbaum, \u003cem\u003eBehavioral responses of honey bees (Apis mellifera) to natural and synthetic xenobiotics in food.\u003c/em\u003e Scientific Reports, 2017. \u003cstrong\u003e7\u003c/strong\u003e(1): p. 15924.\u003c/li\u003e\n\u003cli\u003eLucchetti, M.A., et al., \u003cem\u003eNursing protects honeybee larvae from secondary metabolites of pollen.\u003c/em\u003e Proceedings of the Royal Society B: Biological Sciences, 2018. \u003cstrong\u003e285\u003c/strong\u003e(1875): p. 20172849.\u003c/li\u003e\n\u003cli\u003eRicke, D.F., C.-H. Lin, and R.M. Johnson, \u003cem\u003ePollen Treated with a Combination of Agrochemicals Commonly Applied During Almond Bloom Reduces the Emergence Rate and Longevity of Honey Bee (Hymenoptera: Apidae) Queens.\u003c/em\u003e Journal of Insect Science, 2021. \u003cstrong\u003e21\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eMilone, J.P. and D.R. Tarpy, \u003cem\u003eEffects of developmental exposure to pesticides in wax and pollen on honey bee (Apis mellifera) queen reproductive phenotypes.\u003c/em\u003e Scientific Reports, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 1020.\u003c/li\u003e\n\u003cli\u003eDai, P., et al., \u003cem\u003eChronic toxicity of amitraz, coumaphos and fluvalinate to Apis mellifera L. larvae reared in vitro.\u003c/em\u003e Sci Rep, 2018. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 5635.\u003c/li\u003e\n\u003cli\u003eWood, S., et al., \u003cem\u003eEffects of chronic dietary thiamethoxam and prothioconazole exposure on Apis mellifera worker adults and brood\u003c/em\u003e. 2019.\u003c/li\u003e\n\u003cli\u003eSchmehl, D., et al., \u003cem\u003eProtocol for the in vitro rearing of honey bee (Apis mellifera L.) workers.\u003c/em\u003e Journal of Apicultural Research, 2016.\u003c/li\u003e\n\u003cli\u003eLinksvayer, T., et al., \u003cem\u003eLarval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism\u003c/em\u003e. Vol. 24. 2011. 1939-48.\u003c/li\u003e\n\u003cli\u003eHartfelder, K., et al., \u003cem\u003eChapter One - Old Threads Make New Tapestry\u0026mdash;Rewiring of Signalling Pathways Underlies Caste Phenotypic Plasticity in the Honey Bee, Apis mellifera L\u003c/em\u003e, in \u003cem\u003eAdvances in Insect Physiology\u003c/em\u003e, A. Zayed and C.F. Kent, Editors. 2015, Academic Press. p. 1-36.\u003c/li\u003e\n\u003cli\u003eOECD, \u003cem\u003eTest No. 237: Honey Bee (Apis Mellifera) Larval Toxicity Test, Single Exposure\u003c/em\u003e. 2013.\u003c/li\u003e\n\u003cli\u003eMichelette, E.R. and A.E.E. Soares, \u003cem\u003eCharacterization of preimaginal developmental stages in Africanized honey bee workers (Apis melliferaL.).\u003c/em\u003e Apidologie, 1993. \u003cstrong\u003e24\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eImdorf, A., et al., \u003cem\u003e\u0026Uuml;BERPR\u0026Uuml;FUNG DER SCH\u0026Auml;TZMETHODE ZUR ERMITTLUNG DER BRUTFL\u0026Auml;CHE UND DER ANZAHL ARBEITERINNEN IN FREIFLIEGENDEN BIENENV\u0026Ouml;LKERN.\u003c/em\u003e Apidologie, 1987. \u003cstrong\u003e18\u003c/strong\u003e(2): p. 137-146.\u003c/li\u003e\n\u003cli\u003eBliss, C.I., \u003cem\u003eThe toxicity of poisons applied jointly.\u003c/em\u003e Annals of Applied Biology, 1939. \u003cstrong\u003e26\u003c/strong\u003e(3): p. 585-615.\u003c/li\u003e\n\u003cli\u003eAbbott, W.S., \u003cem\u003eA Method of Computing the Effectiveness of an Insecticide.\u003c/em\u003e Journal of Economic Entomology, 1925. \u003cstrong\u003e18\u003c/strong\u003e(2): p. 265-267.\u003c/li\u003e\n\u003cli\u003eSgolastra, F., et al., \u003cem\u003eSynergistic mortality between a neonicotinoid insecticide and an ergosterol-biosynthesis-inhibiting fungicide in three bee species.\u003c/em\u003e Pest Management Science, 2017. \u003cstrong\u003e73\u003c/strong\u003e(6): p. 1236-1243.\u003c/li\u003e\n\u003cli\u003eR Core Team, \u003cem\u003eR: A Language and Environment for Statistical Computing.\u003c/em\u003e 2018.\u003c/li\u003e\n\u003cli\u003eTherneau, T.M.a.L., T, \u003cem\u003e\u0026lsquo;survival\u0026rsquo;: Survival analysis. R package version 2.44-1.1. .\u003c/em\u003e 2019.\u003c/li\u003e\n\u003cli\u003eKassambara, A., M. Kosinski, and P. Biecek, \u003cem\u003esurvminer: Drawing Survival Curves using \u0026apos;ggplot2\u0026apos;. R package version 0.4.6.\u003c/em\u003e 2019.\u003c/li\u003e\n\u003cli\u003eLenth, R., \u003cem\u003eemmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1.4.2.\u003c/em\u003e 2019.\u003c/li\u003e\n\u003cli\u003eAndrade, B.B., \u003cem\u003elbreg: Log-Binomial Regression with Constrained Optimization. R package version 1.3.\u003c/em\u003e 2019.\u003c/li\u003e\n\u003cli\u003eMagnusson, A., Skaug, H.J., Nielsen, A., Berg, C.W., Kristensen, K., Maechler. M., van Bentham, K., Bolker, B., and Brooks, M.E. , \u003cem\u003eglmmTMB: Generalized linear mixed models using a template model builder. R package version 0.1 3.\u003c/em\u003e 2018.\u003c/li\u003e\n\u003cli\u003eHeinze, G.a.P., M. ,\u003cem\u003e \u0026lsquo;logistf\u0026rsquo;: Firth\u0026apos;s Bias-Reduced Logistic Regression. R package version 1.23.\u003c/em\u003e 2018.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Apis mellifera, ecotoxicology, synergism, antagonism, in vitro rearing","lastPublishedDoi":"10.21203/rs.3.rs-3944102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3944102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHoney bees are commonly co-exposed to pesticides during crop pollination, including the fungicide captan and neonicotinoid insecticide thiamethoxam. We assessed the impact of exposure to these two pesticides individually and in combination, at a range of field-realistic doses. In laboratory assays, mortality of larvae/pupae treated with captan was 80\u0026ndash;90% greater than controls, dose-independent, and similar to mortality from the lowest dose of thiamethoxam. There was evidence of synergism (i.e., a non-additive response) from captan-thiamethoxam co-exposure at the highest dose of thiamethoxam, but not at lower doses. In the field, we exposed whole colonies to the lowest doses used in the laboratory. Exposure to captan and thiamethoxam individually and in combination resulted in minimal impacts on population growth or colony mortality, and there was no evidence of synergism or antagonism. These results suggest captan and thiamethoxam are each acutely toxic to immature honey bees, but whole colonies can potentially compensate for detrimental effects, at least at the low doses used in our field trial. Further work is needed to assess how compensation occurs, potentially via increased queen egg laying, and whether short-term compensation leads to long-term costs. Other crop pollinators that lack the social detoxification capabilities of honey bees may also be less resilient.\u003c/p\u003e","manuscriptTitle":"Acute toxicity of the fungicide captan to honey bees and mixed evidence for synergism with the insecticide thiamethoxam","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 08:38:18","doi":"10.21203/rs.3.rs-3944102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-09T03:28:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T10:15:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76df8e48-818e-4ef1-85d1-f2e4b5c91cda","date":"2024-04-15T04:38:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-29T16:20:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a09b8227-31ad-4c8f-ad44-069694deb9ca","date":"2024-02-22T16:18:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-22T16:12:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-20T00:22:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-19T18:00:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-19T17:41:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-09T20:29:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afe3814e-c669-4b7d-9262-f9769725898a","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-29T12:12:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-21 08:38:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3944102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3944102","identity":"rs-3944102","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}