1. Introduction

Since the description of Nosema apis in the early part of the last century (Zander, 1909), nosema disease, or nosemosis of honey bees has been regarded as a serious obstacle for profitable beekeeping in temperate climates (Fries, 1993). With the detection of the new parasite Nosema ceranae, originally described from the Asian honey bee Apis cerana (Fries et al., 1996), in European honey bees (Higes et al., 2006) the need for research in this field has become urgent. In particular, since early reports on the effects of this new parasite suggested a more severe impact on colony health compared to infections by N. apis  (Higes et al., 2008a). Following the COLOSS workshop “Nosema disease: lack of knowledge and work standardization” in Guadalajara (19-22 October, 2009) data were collected on the heterogeneity of methods used in Nosema research in different laboratories in Europe and in the USA. This survey showed the widely heterogeneous experimental conditions applied in the nine different participating laboratories. Even if common sense implies that some conditions should be applied in all cases, their costs and the availability of these analyses have to be taken into account. For example, one could be tempted to assert that virus presence has to be checked in colonies providing honey bees for Nosema experiments, but some laboratories are not equipped with virus diagnostics, and this could restrict these teams from performing experiments on Nosema without controlling for cofounding viral infections. The level and extent of diagnosis should also be specified: which viruses should be studied, is viral detection sufficient, or should virus quantification be included? 

Here we attempt to standardize study of the microsporidians Nosema apis and Nosema ceranaeNosema apis, the historical microsporidian parasite of European honey bees, can decrease worker longevity and cause considerable winter colony losses (Fries, 1993), whilst N. ceranae, probably introduced into European honey bees from its Asian congener (Apis cerana) within the last few decades (Higes et al., 2006; Martín-Hernández et al., 2007; Klee et al., 2007; Paxton et al., 2007; Chen et al., 2008; Williams et al., 2008; Invernizzi et al., 2009; Currie et al., 2010; Botías et al., 2011), is associated with colony depopulation and collapse in warmer areas of Europe (Higes et al., 2008a), but not in northern parts of Europe (Gisder et al., 2010a), in North America (Guzman-Nova, 2010; Williams et al., 2010) or in South America (Invernizzi et al., 2009). Yet because detection of N. ceranae in European honey bees coincided with recent large-scale honey bee colony losses throughout the world, data on the pathology and management of this parasite are of significant interest. 

When working in the field with full-sized colonies, several considerations need to be made, such as where to sample, how often to sample and also the size of samples. We find, for example, that sample sizes are often too small to satisfy a statistically reasonable level of diagnostic precision (Fries et al., 1984). Please refer to the section on statistics in the BEEBOOK paper on miscellaneous methods (Human et al., 2013) to determine sample size.

Similarly, laboratory tests using bees in cages are often employed to investigate Nosema intra-host development (e.g. Higes et al., 2007; 2010; Martín-Hernández et al., 2009; Forsgren and Fries, 2010), effects of parasitism on host mortality (e.g. Paxton et al., 2007), immunity (e.g. Antúnez et al., 2009), and physiology (e.g. Dussaubat et al., 2010; Mayack and Naug, 2009; Martín-Hernández et al., 2011), as well as for testing the efficacy of potential control treatments (e.g. Maistrello et al., 2008; Higes et al., 2011). When designing cage experiments, researchers typically must control for a number of variables, ranging from selection of study subjects (e.g. parasite and host strains) to experimental environment (e.g. growth chamber conditions, food quality and quantity).  Although decisions typically do not jeopardize the scientific rigor of a study, they may profoundly affect results, and may make comparisons with similar, independent studies difficult.  An important consideration is that most current data on Nosema were collected from experiments with N. apis. The same research is now needed for N. ceranae in order to assess the similarities and differences between the two species.

Here we discuss some important factors that researchers must consider when studying the Nosema-honey bee system using field as well as laboratory cage experiments.  This will allow researchers to make informed choices when developing experimental protocols and will increase confidence when comparing results among studies.