DOI: 10.6086/d1k96q
title: Data from: Sugar content of diet does not buffer against chronic oral imidacloprid exposure in the alfalfa leafcutting bee, Megachile rotundata (Hymenoptera: Megachilidae)

description: Bees are economically critical pollinators, but are declining broadly due
 to several stressors, including non-target exposure to insecticides and
 deficiencies in nutrition. Understanding the simultaneous impact of
 stressors, particularly interactions between them, are critical to
 effectively conserving bees. While behavioral effects of pesticides like
 neonicotinoids have received some attention in solitary bees, our
 understanding of how they are modulated by diet quality is limited.
 Furthermore, scarce data exist on what concentrations of orally ingested
 neonicotinoids elicit mortality in solitary bees. In a controlled exposure
 laboratory experiment, we investigated how diet quality, as sugar
 concentration, and chronic oral exposure to imidacloprid impact adult
 alfalfa leafcutting bees, Megachile rotundata (Fabricius). We provided
 individuals ad libitum with either 20% or 50% (m/m) sucrose syrups
 containing either 0, 30, or 300 ppb imidacloprid (measuring 0, 27, and 209
 ppb via an ELISA assay). Over five weeks, we tracked behavior and
 survivorship of individuals. Imidacloprid decreased survivorship in a
 dose-dependent fashion, but sucrose content did not affect survivorship,
 even in bees not fed imidacloprid. In the high imidacloprid treatment, 45%
 of bees were observed in a motionless supine position while still alive,
 with this effect appearing to be buffered against by the higher sucrose
 diet. Our results suggest diets higher in sugar concentration may prevent
 an intermediate stage of poisoning, but does not ultimately extend
 longevity. In devising risk assessments for bees, it is important to
 consider that interactions between stressors may occur in the stages
 leading up to death even if survivorship is unaffected.
description: <strong>Experimental Design</strong>
 We acquired nest cells containing prepupae of alfalfa leafcutting
 bees, <em>M. rotundata</em>, originating in Canada (JWM
 Leafcutters, Inc., Nampa, ID) and stored them at 4°C until the experiment.
 We placed nest cells in an incubator at 30.3 ± 0.03°C and 57.2 ± 0.35%
 (mean ± SE) relative humidity to induce completion of development. Adult
 emergence began 21 d after the start of incubation and continued for
 several days. Adults were kept at the above temperature and humidity for
 the entire duration of the experiment. Upon emergence,
 each adult bee was isolated in its own container (Supp Fig. 1 [online
 only]) and randomly assigned to one of six treatments (Table 1). The day
 of emergence, assigned treatment, and sex were recorded upon isolation.
 Each container consisted of an inverted, clear 59-ml plastic soufflé cup
 with a 5.5-cm diameter circle of filter paper (Ahlstrom 610, 1.5-μm pore
 size, Thomas Scientific, Swedesboro, NJ) fitted inside as a substrate.
 Through a hole in the side of each cup, we inserted a 0.6-ml
 microcentrifuge tube with a 2-mm hole near the bottom. These tubes served
 as feeders through which we delivered each bee’s assigned diet. Each of
 the six treatments included 20 adult bees comprising both sexes (n = 120
 bees). About 29% of bees were female, which reflects sex ratios in managed
 populations of M. rotundata that are biased towards males (Pitts-Singer
 and James 2005). In a fully crossed factorial
 experiment, six diets were prepared (Table 1). Sugar solutions were
 prepared as either 20 or 50% sucrose (m/m) solutions. These concentrations
 are well within the range of total sugar concentrations naturally found in
 floral nectars and reflect the lower and higher sugar diets, respectively,
 used in a similar experiment on honey bees (Tosi et al. 2017). Premeasured
 quantities of Marathon 1% Granular (Olympic Horticultural Products,
 Mainland, PA) were added to achieve one of three concentrations of
 imidacloprid in solution: 0 (control), 30 (low), or 300 ppb (high). This
 formulation is 1% imidacloprid and 99% inert ingredients (up to 9% of
 which is crystalline silica) by mass. We chose to test the same
 insecticide concentrations as a previous study (Abbott et al. 2008),
 though these authors exposed M. rotundata larvae to imidacloprid via
 pollen. Although imidacloprid concentrations in floral resources can vary
 widely due to numerous factors (formulation, time since application,
 etc.), a concentration of 30 ppb (μg kg<sup>−1</sup> or ng
 g<sup>−1</sup>) is slightly above the ‘field-realistic’
 average for nectars of imidacloprid-treated plants (Bonmatin et al. 2015,
 Wood and Goulson 2017) but commensurate with levels that elicit sublethal
 effects in other bee species (Tan et al. 2014, Gregorc et al. 2018). The
 300 ppb treatment was intended as an upper bound for investigating rates
 of mortality and is closer to the maximum concentrations in nectar
 reported in reviews (Sánchez-Bayo and Goka 2014, Bonmatin et al.
 2015). Diet stock solutions were stored in the dark at
 5°C to minimize photodegradation of imidacloprid. We periodically removed
 100-μl aliquots of each diet stock solution to confirm imidacloprid
 concentrations using a commercial ELISA kit (QuantiPlate, catalog #EP-006,
 EnviroLogix, Portland, ME). ELISA results were quantified by measuring
 absorbance at 450 nm wavelength using a Multiskan GO 1.01.10 (Thermo
 Fisher Scientific, Waltham, MA) and SkanIt Software 4.1 for Microplate
 Readers RE v. 4.1.0.43. Absorbance readings for our control (no
 imidacloprid) diets indicated a lack of matrix effects in this ELISA
 (Byrne et al. 2005). For our low and high diets, respectively, the ELISA
 returned concentrations of 27 ± 5 and 209 ± 34 ppb (mean ± SE of multiple
 aliquots from the same stock solution), and these numbers did not decline
 significantly over the duration of the experiment. Mean ELISA readings for
 our high pesticide diet were 91 ppb lower on average than we had intended,
 perhaps due to interference from the inert materials in the insecticide
 formulation, but were still over seven times higher than the low pesticide
 treatment. We added a drop of yellow food coloring
 (glycerin, polysorbate 80, and turmeric) to 10-ml diet aliquots to
 facilitate visually tracking consumption. Bees were supplied 100-μl doses
 of their respective diets daily for the first week after emergence, and
 three times a week for the remainder of the experiment (ad libitum) as
 consumption tended to decrease over time. Until the last individual’s
 death, bee survivorship and any abnormal behaviors were documented.
 Feeders obstructed with precipitated solid sucrose were cleared with a
 clean needle when necessary. Upon each bee’s death, we recorded their date
 of death and measured the distance between tegulae (wing bases) as a proxy
 for body size (Cane 1987). <strong>Statistical
 Analyses</strong> All statistical analyses were
 performed in R version 3.3.3 (R Core Team 2019). We ran survival analyses
 with the package survival (Therneau 2015) using a Cox proportional hazards
 model (functions ‘Surv’ and ‘coxph’) to determine the effect of the fixed
 effects (sucrose and imidacloprid concentrations and the interaction
 between these two factors, bee sex, and bee body size) on bee
 survivorship, calculated as the number of days between an individual’s
 emergence and death. We checked for multicollinearity among fixed effects
 using the function ‘vif’ in the car package (Fox and Weisberg 2011) and
 ensured all variance inflation factors were &lt;2. We used the package
 survminer (Kassambara and Kosinski 2018) to generate a Forest plot
 (function ‘ggforest’) for this model. We examined residuals of this model
 using the function ‘cox.zph’ in survival and verified they met model
 assumptions (P &gt; 0.05). We created survival curve plots using the
 functions ‘survfit’ (survival) and ‘ggsurvplot’ (survminer).
 To determine whether a bee exhibited a behavior (dependent
 variable) that was related to the same fixed effects, we performed a
 logistic regression using the function ‘glm’ with a binomial distribution
 and logit link function in the package lme4 (Bates et al. 2014). We
 conducted posthoc pairwise Tukey tests via the functions ‘emmeans’ and
 ‘pairs’ in the package emmeans (Lenth 2019). To determine whether the
 amount of time that passed until this behavior was exhibited, or how long
 it took for bees to die after exhibiting this behavior, was related to our
 fixed effects, we constructed two separate linear models (function ‘lm’).
 Type III sums of squares and F- and P-values for our logistic regression
 and linear models were obtained using the function ‘Anova’ in the car
 package (Fox and Weisberg 2011). We created bar plots using the function
 ‘ggplot’ in the package ggplot2 (Wickham 2016).
description: The file includes a "metadata" tab describing all of
 the variables.