1. Introduction

Honey bees are important pollinators and are responsible for much of the world’s agricultural production and the conservation of biodiversity (Klein et al., 2007; Gallai et al., 2009). In many regions of the world, the number of honey bee colonies is declining, thus possibly endangering pollination (Aizen et al., 2009; VanEngelsdorp and Meixner, 2010; van der Zee et al., 2012). Parasites, pathogens and pesticides are three of the major threats to honey bees and are believed to be partially responsible for the abovementioned declines (Neumann and Carreck, 2010). Therefore, the effects of these handicaps and the combination of two or more sublethal effects are extensively investigated (Mullin et al., 2010; Moritz et al., 2010; Genersch et al., 2010). An important tool for this research is the rearing of honey bee larvae in vitro (i.e. in the laboratory and in the absence of nurse bees) because it allows more controllable conditions compared to in vivo (i.e. in the hive by nurse bees). However, artificial larval rearing can also be regarded as an in vivo method conducted in an in vitro system. In particular, the testing of the toxicity of plant protection products on brood can be conducted in a reproducible and standardized way only in the laboratory, because a defined uptake of food containing the testing compound is not feasible using in-hive methods (Wittmann and Engels, 1981; Oomen et al., 1992; Schuur et al., 2003; Becker et al., 2009; Aupinel et al., 2007a).

The first attempt at mass application and standardization of in vitro rearing of honey bee larvae and testing plant protection products was a ring test, in which seven different laboratories assessed the LD50 for dimethoate 48 hours after acute larval exposure (Aupinel et al., 2009). This experiment underlined the variability in results and the importance of further investigation of factors such as colony origin of larvae, effect of season and larval heterogeneity at grafting.

In this paper, we give an overview of existing in vitro protocols, present the crucial points of rearing honey bee larvae in the laboratory, and discuss where further research or standardization of methods might be useful or necessary. The aim of this paper is not to present a rigorous recipe for rearing larvae in vitro (as these are mostly well documented in individual papers), but rather to discuss advantages and disadvantages of different protocols and give recommendations where necessary and meaningful.