Wax syrup pots added.

<p><b>A,</b> Imidacloprid, Chi-square test = 10.23, DF = 4, p = 0.0368. <b>B,</b> Clothianidin, Chi-square test, F = 21.54, DF = 4, p<0.0002, Kruskal-Wallis, Wilcoxon Test.</p

Queen mortality at weeks 1–11.

<p><b>A,</b> Imidacloprid, Week 6: Chi-square test = 9.26, DF = 4, 235, p<0.055, week 11: Chi-square test = 75.49, DF = 4,435, p<0.001. <b>B,</b> Clothianidin, Week 6: Chi-square test = 22.87, DF = 4, 247, p<0.001, week 11: Chi-square test = 102.78, DF = 4, 457, p<0.001, Kruskal-Wallis, Wilcoxon Test.</p

Chronic Exposure of Imidacloprid and Clothianidin Reduce Queen Survival, Foraging, and Nectar Storing in Colonies of <i>Bombus impatiens</i>

<div><p>In an 11-week greenhouse study, caged queenright colonies of <i>Bombus impatiens</i> Cresson, were fed treatments of 0 (0 ppb actual residue I, imidacloprid; C, clothianidin), 10 (14 I, 9 C), 20 (16 I, 17C), 50 (71 I, 39 C) and 100 (127 I, 76 C) ppb imidacloprid or clothianidin in sugar syrup (50%). These treatments overlapped the residue levels found in pollen and nectar of many crops and landscape plants, which have higher residue levels than seed-treated crops (less than 10 ppb, corn, canola and sunflower). At 6 weeks, queen mortality was significantly higher in 50 ppb and 100 ppb and by 11 weeks in 20 ppb–100 ppb neonicotinyl-treated colonies. The largest impact for both neonicotinyls starting at 20 (16 I, 17 C) ppb was the statistically significant reduction in queen survival (37% I, 56% C) ppb, worker movement, colony consumption, and colony weight compared to 0 ppb treatments. Bees at feeders flew back to the nest box so it appears that only a few workers were collecting syrup in the flight box and returning the syrup to the nest. The majority of the workers sat immobilized for weeks on the floor of the flight box without moving to fed at sugar syrup feeders. Neonicotinyl residues were lower in wax pots in the nest than in the sugar syrup that was provided. At 10 (14) ppb I and 50 (39) ppb C, fewer males were produced by the workers, but queens continued to invest in queen production which was similar among treatments. Feeding on imidacloprid and clothianidin can cause changes in behavior (reduced worker movement, consumption, wax pot production, and nectar storage) that result in detrimental effects on colonies (queen survival and colony weight). Wild bumblebees depending on foraging workers can be negatively impacted by chronic neonicotinyl exposure at 20 ppb.</p></div

Colony consumption.

<p><b>A,</b> Imidacloprid, Week 2: F = 52.51, DF = 4, 16, p<0.001, Week 4: F = 27.40, DF = 4, 14, p<0.001, Week 6: F = 22.61, DF = 4, 12, p<0.001, Week 8: F = 7.67, DF = 3, 17, p = 0.002. <b>B,</b> Clothianidin, Week 2: F = 42.05, DF = 4, 17, p<0.001, Week 4: F = 91.96, DF = 4, 14, p<0.001, Week 6: F = 42.77, DF = 4, 28, p<0.001, Week 8: F = 48.52, DF = 4, 8, p<0.001, ANOVA, Tukey-Kramer MRT by treatment for each week are on the figures, ProcMixed showed a significant interaction for imidacloprid and clothianidin, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091573#pone.0091573.s002" target="_blank">Table S1</a>.</p

Total, dead, and alive brood.

<p><b>A,</b> Imidacloprid, Week 11: Total Brood: F = 2.99, DF = 4, 17, p = 0.049, Dead Brood: F = 1.67, DF = 4, 17, p = 0.205, Alive Brood: F = 5.74, DF = 4, 14, p = 0.006. <b>B,</b> Clothianidin, Week 11: Total Brood: F = 4.16, DF = 4,37, p = 0.007, Dead Brood: F = 1.83, DF = 4,37, p = 0.144, Alive Brood: F = 4.13, DF = 4,17, p = 0.016, ANOVA, Tukey-Kramer MRT.</p

Bee consumption.

<p><b>A,</b> Imidacloprid, Week 2: F = 30.97, DF = 4, 16, p<0.001, Week 4: F = 10.31, DF = 4, 33, p<0.001, Week 6: F = 0.89, DF = 4, 8, p = 0.513, Week 8: F = 2.51, DF = 3, 17, p = 0.093. <b>B,</b> Clothianidin, Week 2: F = 17.68, DF = 4, 17, p<0.001, Week 4: F = 32.73, DF = 4, 15, p<0.001, Week 6: F = 9.37, DF = 4, 28, p<0.001, Week 8: F = 4.32, DF = 4, 8, p = 0.035, ANOVA, Tukey-Kramer MRT by treatment for each week are on the figures to compare the 2 chemicals, but ProcMixed did not show a significant interaction for imidacloprid or clothianidin, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091573#pone.0091573.s002" target="_blank">Table S1</a>.</p

Colony weight and syrup weight in wax pots.

<p><b>A,</b> Imidacloprid, colony weight, Week 0: F = 1.84, DF = 4, 16, p = 0.170, Week 11: F = 16.20, DF = 4, 35, p<0.001; syrup weight, Week 11: F = 4.83, DF = 4, 15, p = 0.011. <b>B,</b> Clothianidin, colony weight, Week 0: F = 0.87, DF = 4, 37, p = 0.492, Week 11: F = 16.10, DF = 4, 37, p<0.001; syrup weight Week 11: F = 6.83, DF = 4, 16, p = 0.002, ANOVA, Tukey-Kramer MRT.</p

Imidacloprid and clothianidin residue (ppb) in sugar syrup stock solutions (50%) from one sample in each replicate experiment and from stored syrup in wax pots (3 colonies mixed) from replicate 1(1 sample) and replicate 2 (2 samples) experiment, residue was determined by the standard USDA method, USDA, AMS, Gastonia, NC.

<p>Imidacloprid and clothianidin residue (ppb) in sugar syrup stock solutions (50%) from one sample in each replicate experiment and from stored syrup in wax pots (3 colonies mixed) from replicate 1(1 sample) and replicate 2 (2 samples) experiment, residue was determined by the standard USDA method, USDA, AMS, Gastonia, NC.</p

Worker, male, and queen production.

<p><b>A,</b> Imidacloprid, Week 11: All Castes: F = 4.62, DF = 4, 35, p = 0.004, Workers: F = 1.92, DF = 4, 35, p = 0.129, Males: F = 4.59, DF = 4, 14, p = 0.014, Queens: F = 0.19, DF = 4, 35, p = 0.945. <b>B,</b> Clothianidin, Week 11: All Castes: F = 5.12, DF = 4, 37, p = 0.002, Workers: F = 2.15, DF = 4, 37, p = 0.094, Males: F = 7.44, DF = 4, 16, p = 0.002, Queens: F = 2.23, DF = 4, 37, p = 0.085, ANOVA, Tukey-Kramer MRT.</p

Survival of four species of adult lady beetles that were fed Mexican milkweed, <i>Asclepias curassavica</i>, flowers that that were untreated (C), treated with label rate (1X), or twice label rate (2X) of soil-applied imidacloprid (Marathon 1%G).

<p>Means in the same column followed by different letters are significantly different, Tukey-Kramer MRT,α = 0.05.</p><p>Survival of four species of adult lady beetles that were fed Mexican milkweed, <i>Asclepias curassavica</i>, flowers that that were untreated (C), treated with label rate (1X), or twice label rate (2X) of soil-applied imidacloprid (Marathon 1%G).</p