154 2009a). A myriad of abiotic and biotic factors can adversely affect the ability of honey bees to fight infection, including interactions between disease agents, mite parasitism, poor nutrition, and sub-lethal exposure to pesticides (Johnson et al. 2009; vanEngelsdorp et al. 2009a). Systemic pesticides such as imidacloprid pose a new route of exposure in pollen and nectar and have been demonstrated to have negative effects on learning in adult bees (Decourtye et al. 2004). Here we test the hypothesis that bees exposed to sub-lethal levels of pesticide are more susceptible to disease. We ex- posed colonies of honey bees to the insecticide imidacloprid at sub-lethal levels and then challenged newly emerged workers from those colonies with the gut parasite Nosema spp., two species (Nosema apis and Nosema ceranae) of which are known to adversely affect colony health (Higes et al. 2008; Kauko et al. 2003). The pesticide dosages used were below levels demonstrated to cause effects on longevity or foraging in adult honey bees (Maus et al. 2003; Bonmatin et al. 2007; Deseneux et al. 2007). Materials and methods For 10 weeks, we continually exposed full sized colonies of bees (30–40,000 adults) to 5 and 20 ppb imidacloprid by provisioning colonies with protein supplement patties spiked with the pesticide. After 5 and 8 weeks of exposure (ca. 1.5 and 2.5 generations of exposure), wax combs with emerging brood were taken into the laboratory and groups of newly emerged adult bees from selected colonies were removed and either used to determine fresh weight or caged and fed a suspension containing spores of the known bee pathogens N. apis and N. ceranae over the first 2 days of adult life. Ten days later, bees were sacrificed and the development of Nosema infection in individual bees determined. Chronic imidacloprid exposure to colonies Thirty honey bee colonies were used and divided into three treatment groups of ten colonies each. Colonies were estab- lished in April 2008 in five apiaries, approximately 0.5 km apart containing two colonies from each treatment group (total colonies per apiary 0 6). Packages of bees (1.8 kg) were installed in new hive equipment including frames with wax- coated plastic foundation. All queens established in study colonies came from the same genetic source. All colonies were managed to limit the levels of other pests and pathogens. All colonies were fed equal amounts of sucrose syrup until natural forage was abundant in May. Treatments consisted of untreated Megabee® protein patties (100 g each) or patties containing 5 and 20 ppb of imidacloprid made by mixing neat material with the sucrose solution used to make the protein patties. Samples were taken of fresh treated protein patties and Naturwissenschaften (2012) 99:153–158 analyzed for imidacloprid content to insure proper delivery of the target dose. Beginning in May 2008, each colony received four 80-g patties per week for 10 weeks. Unconsumed patties were removed after 7 days, weighed to measure consumption, and replaced with new treatment protein patties. Adult bees and stored pollen “bee bread” were collected 1 week after the 10-week feeding period and analyzed for imidacloprid levels. Samples (>5 g) of newly emerged bees and random-aged bees from each experimental hive and protein patties from each treatment group were all analyzed for imidacloprid levels at the USDA-AMS laboratory in Gastonia, NC, using GC/MS with a limit of detection of 0.1 ppb. Experimental bees and pathogen challenge Five weeks into treatment in July 2008, a single brood frame with emerging worker bees were removed from each of three to four colonies treatment and held in an incubator at 34°C overnight. Newly emerged bees were collected the next morn- ing and placed in groups of 30 bees per cage for Nosema challenge. An additional 20 newly emerged bees per colony were weighed to determine average emerged bee weight. A Nosema spore suspension was made by taking the mid-guts of ten workers from a single Nosema-infected colony and mac- erating the guts in 10 ml of water and determining the total spore count using the methods of Cantwell (1970) and a hemocytometer slide. The spore suspension was then centri- fuged for 10 min at 3,000 rpm, the supernatant discarded, and the spore pellet resuspended using a 50:50 volume to volume sucrose solution to reach a final spore concentration of one million spores per milliliter of solution. Ten milliliters of this solution was fed to each cage of 30 bees over the first 2 days of adult life and after complete consumption of the 10 ml each bee received an approximate dose of 333,333 spores over 2 days. Treated bees were allowed to age in cages for 12 days, sacrificed, and spore levels determined for each bee using standard light microscopy and a hemocytometer after the methods of Cantwell (1970). Nosema spore data was log transformed prior to analysis to meet statistical assumptions. Mortality was monitored daily and no cage exceeded 20% over 12 days. At day 12, ten randomly selected live bees were used for spore determination on an individual bee basis. A second trial was conducted in August, 8 weeks into treatment and this time however emerged bees were feed 10 ml of sugar solution containing either 0 (control), 0.1, or 1 million spores per milliliter to explore potential effects of inoculum dose of pathogen growth. The Nosema spore suspension was made using bees from the same source colony as before. Mortality in the cages averaged less than 20% after 12 days. Samples of the Nosema spore suspensions from both dates were analyzed for species composition using established molecular methods (Chen et al. 2008) and found to be predominantly N. ceranae but a weak band corresponding to N. apis was also detected