Secondary biomarkers of insecticide-induced stress of honey bee colonies and their relevance for overwintering strength Jakob Wegener a,n , Haike Ruhnke a , Kathrin Milchreit a,b , Katharina Kleebaum b , Monique Franke b , Sebastian Mispagel c , Gabriela Bischoff d,1 , Günter Kamp c , Kaspar Bienefeld a a Bee Research Institute Hohen Neuendorf, F.-Engels-Straße 32, 16540 Hohen Neuendorf, Germany b BioChem Agrar GmbH, Kupferstraße 6, 04827 Machern, Germany c AMP-Lab GmbH, Becherweg 9-11, 55128 Mainz, Germany d Julius-Kühn-Institute, Institute for Bee Protection, Königin-Luise-Straße 19,14195 Berlin, Germany article info Article history: Received 29 February 2016 Received in revised form 21 June 2016 Accepted 21 June 2016 Keywords: Apis mellifera Fenoxycarb Imidacloprid Overwintering strength Field test Sublethal effects abstract The evaluation of pesticide side-effects on honeybees is hampered by a lack of colony-level bioassays that not only are sensitive to physiological changes, but also allow predictions about the consequences of exposure for longer-term colony productivity and survival. Here we measured 28 biometrical, bio- chemical and behavioural indicators in a field study with 63 colonies and 3 apiaries. Colonies were stressed in early summer by feeding them for five days with either the carbamate growth regulator fenoxycarb or the neurotoxic neonicotinoid imidacloprid, or left untreated. Candidate stress indicators were measured 8–64 days later. We determined which of the indicators were influenced by the treat- ments, and which could be used as predictors in regression analyses of overwintering strength. Among the indicators influenced by fenoxycarb were the amount of brood in colonies as well as the learning performance and 24 h-memory of bees, and the concentration of the brood food component 10HDA in head extracts. Imidacloprid significantly affected honey production, total number of bees and activity of the immune-related enzyme phenoloxidase in forager bee extracts. Indicators predictive of over- wintering strength but unrelated to insecticide feeding included vitellogenin titer and glucose oxidase- activity in haemolymph/whole body-extracts of hive bees. Apart from variables that were themselves components of colony strength (numbers of bees/brood cells), the only indicator that was both influ- enced by an insecticide and predictive of overwintering strength was the concentration of 10HDA in worker bee heads. Our results show that physiological and biochemical bioassays can be used to study effects of insecticides at the colony level and assess the vitality of bee colonies. At the same time, most bioassays evaluated here appear of limited use for predicting pesticide effects on colony overwintering strength, because those that were sensitive to the insecticides were not identical with those that were predictive of colony overwintering. Our study therefore illustrates the difficulties involved in evaluating the economic/ecological significance of pesticide-induced stress in honey bee field studies. & 2016 Elsevier Inc. All rights reserved. 1. Introduction In most countries, pesticides whose intended use implies a possible exposure of honeybees (Apis mellifera) have to be tested for negative effects on this economically and ecologically im- portant species in order to achieve homologation (see for example European Union regulation 1107/2009; US-OCSPP-guidelines 850- 3020 to 850-340). In recent years, the adequacy of existing testing schemes for plant protection products on honeybees has been questioned, both because of risen public awareness and of new scientific results highlighting the extent and importance of sub- lethal and/or delayed effects (Abramson et al., 2004; Decourtye et al., 2004; Di Prisco et al., 2013; Dively et al., 2015; Rondeau et al., 2014). Efforts are therefore being made to improve them (EPA, 2012; EFSA, 2013 (revised 2014)). Studies on entire honey bee colonies are of special importance for pesticide testing. They reflect the most realistic scenario of exposure of the different life stages, and integrate the social stress buffering mechanisms of the species. They therefore are the ulti- mate way to judge whether effects observed in individual larvae or adults are economically and ecologically relevant (reviewed for the Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ecoenv Ecotoxicology and Environmental Safety http://dx.doi.org/10.1016/j.ecoenv.2016.06.038 0147-6513/& 2016 Elsevier Inc. All rights reserved. n Corresponding author. E-mail address: wegenerj@hu-berlin.de (J. Wegener). 1 Past address: Julius-Kühn Institute, Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Germany. Ecotoxicology and Environmental Safety 132 (2016) 379–389