156 Naturwissenschaften (2012) 99:153–158 Table 1 Levels of imidacloprid in bee bread (stored pollen/protein patties) and random-aged worker bees from experimental colonies and average weights of newly emerged bees from the three colony treatments for trials 1 and 2 in July and August 2008, respectively (n020 bees/hive) Imidacloprid levelsa (mean ±SEM, ppb) Treatment colonies Control Low 5 ppb High 20 ppb Bee bread from coloniesb 0.20±0.22 1.62±0.68 3.49±1.55 Random-aged bees from coloniesb 0.6±0.31 1.58±0.68 3.67±1.48 Imid. level emerged beesc Not detected Not detected Not detected Emerged bee weight (mean±SEM, n020 beesd) Emerged bee weight (g) July trial 1 Emerged bee weight (g) Aug. Trial 2 0.115±0.0014 0.112±0.0016 *0.106±0.0017 0.116±0.0018 0.116±0.0017 0.116±0.0020 *P<0.05 (indicate significant differences in emerged bee weights when compared to control hives in the same trial) a Limit of detection is 0.1 ppb b Sample size n010 patties sampled c Newly emerged bees from both trials were tested for imidacloprid d One control cage, July trial, had only 11 bees available to weigh, all others n020 bees control, 5 and 20 ppb colonies, respectively, and was not significantly different among treatments (ANOVA, F 0 0.83, d.f. 0 2, 7, P 0 0.39). Residues of imidacloprid were found in bee bread and bees from exposed colonies and increased in direct and expected proportion to the concentrations in the treated protein patties (Table 1). We also found traces of imidacloprid in bees and bee bread collected from control colonies. This imidacloprid exposure in our controls may have been the result of bee drift between experimental colo- nies and/or from exposure resulting from foraging on treated agricultural crops in the vicinity. Newly emerged bees tested negative for imidacloprid and bees from the higher 20-ppb pesticide exposure colonies were significantly lighter in weight in the July trial (Table 1). Similar effects of reduced weight were documented in queen bees exposed to another pesticide, coumaphos (Pettis et al. 2004). Bees reared in colonies exposed to imidacloprid and fed sucrose solutions dosed with Nosema spores had significantly higher levels of Nosema infection as compared to bees originating from con- trol colonies (Figs. 1 and 3). The concentration of imidaclo- prid in protein patties fed to colonies did not have an effect on final Nosema spore counts in 12-day-old bees. Similarly, the dose of Nosema spores provided to emerged bees (0.1 or 1 million spores per milliliter of sucrose water) did not affect total spore count in 12-day-old bees (Fig. 2). The group of 30 colonies that provided experimental bees for cage experiments were in good health as indicated by low mite and disease levels. Only three of these colonies tested positive for Nosema at the beginning of the trial and those three colonies were excluded from use. At the end of 10 weeks, eight of 30 tested positive for Nosema but there was surprisingly no relationship between Nosema infection and imidacloprid treatment which would have been pre- dicted by the lab study. Three control, three 5 ppb, and two 20 ppb colonies tested Nosema positive, with average spore counts of 4.3, 2.9, and 0.5 million spores per bee, respectively. Varroa mite counts were low with an average mite count of 0.36 mites/100 bees, well below damaging levels which range from five to ten mites per 100 bees, with 27 of 30 colonies testing positive using 300 adult bees (Lee et al. 2010). No colonies tested were found to have tracheal mites. Discussion We clearly demonstrate an interaction between sub-lethal exposure to imidacloprid at the colony level and the spore production in individual bees of honey bee gut parasite Nosema. Two similar studies have just been published that directly treated individual bees with imidacloprid, fipronil, or thiacloprid and then challenged them with Nosema with similar synergistic interaction between Nosema and pesti- cide exposure (Alaux et al. 2010; Vidau et al. 2011). Our study differs in several significant ways; (1) our study employed sub-lethal colony-level pesticide chronic expo- sure instead of laboratory direct exposure to experimental bees, (2) no imidacloprid residues could be found in the newly emerged worker bees challenged in our study (Table 1), (3) our test bees could only have received pesticide exposure during larval development, thus (4) pesticide exposure to test bees could only have been indirectly from brood food from nurse bees (Winston 1987) that were exposed as they fed on imidacloprid-spiked protein. We can only speculate as to why sub-lethal exposure in brood food to larva resulted in in- creased spore production in adult bees. Since Nosema initiates infection in the mid-gut then perhaps in the larval stages the mid-gut ways altered or weakened in some manner that resulted in increased Nosema infection in adult bees. Vidau et al. (2011) failed to demonstrate a change in the pesticide detoxification system in adult bees yet still demonstrated an increase in mortality in adult bees when pesticides and