Naturwissenschaften (2012) 99:153–158 Nosema were combined. Alaux et al. (2010) showed that co- exposure to imidacloprid and Nosema weakened bees but they also showed a trend toward a slight decrease in spore produc- tion with pesticide exposure. Individual bees in our study showed a marked increase in Nosema spore production in the laboratory but the parent colonies failed to show increased Nosema levels over time. Our understanding of N. ceranae at the colony level is still very limited as colony-level spore counts can be highly variable. Perhaps spore production is not the proper way to measure N. ceranae infection as has always been the case with N. apis? Taken together these three studies clearly demonstrate synergism between pesticides and Nosema. The current study, with the more robust chronic sub- lethal pesticide exposure at the colony level, clearly demon- strates that such interactions are possible in the real world, not just in a laboratory setting. Additional research is needed to understand the underlying mechanisms of pesticide pathogen interactions. This is especially true in our study in trying to determine how the pesticide moved in the colony, affected nurse bees, and what level of exposure that larvae in these colonies actually received. The pesticide was consumed equally among the two dosages and house bees contained the pesticide but the pharmacokinetics within the colony is yet to be determined. Past studies have found that chronic, sub-lethal exposure to pesticides can have an adverse effect on colonies (Bendahou et al. 1999) and has been associated with overt disease out- breaks in honey bees (Morse et al. 1965). Conversely, further infection with the chronic bee paralysis virus can affect honey bee tolerance to agricultural pesticides (Bendahou et al. 1997). Recent increases in colony losses in the USA and Europe have drawn particular attention to one relatively new class of sys- temic pesticides, the neonicotinoids, of which imidacloprid is a member. Beekeeper conviction that imidacloprid is respon- sible for colony losses in France has resulted in the withdrawal of its registration (under the trade name Gaucho, Bayer, Leverkusen, Germany) as a seed treatment for sunflowers and corn Ministere de l’Agriculture et de la Peche (1999 and 2004). Similar pressure in California has resulted in a reeval- uation of four neonicotinoids, including imidacloprid, because of elevated levels of residues in leaves and blossoms of ornamental plants after imidacloprid applications. The published levels of imidacloprid expressing acute and chronic toxicity on bees are variable and conflicting (Nguyen et al. 2009). Most studies suggest that imidacloprid can cause disorientation and associative learning problems in honey bees at exposure levels above 20 ppb (Decourtye et al. 2004; Deseneux et al. 2007). However, crop residue studies have detected imidacloprid at levels of 2–5 ppb in pollen and >1.5 ppb in nectar of seed-treated corn, sun- flowers, and rape (Kauko et al. 2003) well below the 20 ppb level documented to cause acute and chronic toxicity effects. To our knowledge no studies have examined chronic 157 effects of dietary exposure to imidacloprid in functional colonies over multiple brood cycles and potential synergistic effects of pesticide and disease interactions. Our results suggest that the current methods used to evaluate the poten- tial negative effect of pesticides are inadequate. This is not the first study to note a complex and unexpected interaction between low pesticide exposure and pathogen loads. Trem- atodes levels in amphibian populations are driven by atra- zine in the aquatic environment (Rohr et al. 2008). Elevated levels of the fungicide chlorothalonil in honey bees have been associated with “entombed pollen” which is linked with increased risk of colony mortality (vanEngelsdorp et al. 2009b). The call for a reevaluation of pesticide test protocols required for the registration of products is not new (Colin et al. 2004; Halm et al. 2006). These proposed new standards utilize the Predicated No Effect Concentration which is determined using chronic and acute toxicity data and not potentially indirect effects of pesticide exposure, such as increased susceptibility to pathogens. With the wide variety of pesticides that have been documented in failing beehives (Mullen et al. 2010), it is imperative that we understand both the synergistic effects these compound may have and the interactions with other variables, like pathogens, involved in bee health. We suggest new pesticide testing standards be devised that incorporate increased pathogen susceptibility into the test protocols. Lastly, we believe that subtle interactions between pesticides and pathogens, such as demonstrated here, could be a major contributor to increased mortality of honey bee colonies worldwide. Acknowledgments These studies were made possible with the help of Nathan Rice, Vic Levi, Mike Embry, Terry Patton, and Amy Miller. Financial support was provided to JSP from the USDA-ARS Areawide Project to improve bee health and to DVE from the Pennsylvania Department of Agriculture, and GD from the North American Pollinator Protection Campaign and Foundation from the Preservation of Honey Bees. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 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