2.1.4. Storing dead adults

When post-mortem examinations, or necropsies, are to be performed for a particular study it is imperative that honey bees to be examined are maintained under appropriate conditions to ensure degradation does not occur. Ideally, samples should be placed under optimal preservation conditions as soon as possible after death if analyses or examination does not occur immediately. Storage conditions, as well as the materials to be preserved, will largely depend upon the question being asked.

Generally, freezing is the best and most commonly used strategy for maintaining well preserved samples; however, when this is not available certain chemical stabilisers (e.g. RNALater® (Qiagen, Hilden, Germany), and TN, Kiev and TRIS-NaCL buffers) may provide alternative options, at least in the short term (Table 2). Careful attention must be paid during examination of easily degradable material, such as DNA and in particular RNA because of its single stranded architecture and because of endogenous RNases that occur ubiquitous in organisms and the environment (Chen et al., 2007; Winnebeck et al., 2010; Dainat et al., 2011). Additionally, pheromone, pesticide residue, and whole tissue examination also require appropriate preservation (Table 2).

Ideally, samples should be preserved at -80°C; however, freezing at -20°C or less should be sufficient for relatively short-term storage. More in depth discussions on sample preservation can be found in respective papers of the BEEBOOK, such as de Miranda et al. (2013) for viruses, Fries et al., (2013) for nosema, and Medrzycki et al. (2013) for toxicology.

Table 2. Examples of methods used to store honey bees and selected bee products depending on purpose of the study.

Method of storage

Storage description

Body part stored and purpose

Reference

Cold

-20 °C

Adult worker ventriculi for Nosema qPCR quantification

Forsgren and Fries (2010)

 

-20 °C

Whole adult workers for Nosema species identification

Williams et al. (2008; 2011)

 

-20 °C

Adult workers, honey & beeswax for gas chromatography (GC)-tandem mass spectrometry (MS/MS) & liquid chromatography (LC-MS/MS) chemical residue analyses

Nguyen et al. (2009)

 

-20 °C

Beebread, brood, adult workers for LC/MS-MS and GC/MS pesticides residue analyses

Mullin et al. (2010)

 

-80 °C

Mature queen spermathecal fluid protein profiling using gel electrophoresis

Baer et al. (2009)

 

-80 °C

Mature queen ovaries & eviscerated abdomens (cuticle with attached fat bodies) for quantitative real-time PCR of Vitellogenin gene expression

Kocher et al. (2008)

 

-80 °C

Adult drone antennae for microarray and qPCR sex pheromone gene expression quantification

Wanner et al. (2007)

 

-80 °C

Whole adult workers for quantitative real-time PCR of immune gene expression

Antúnez et al. (2009)

 

-80 °C

Extracted RNA from adult workers, eggs, queen faeces & queen tissues for RT-PCR analyses of viruses

Chen et al. (2006)

 

-80 °C

Brood comb (beeswax, beebread and brood) and adult workers for LC/MS-MS and GC/MS pesticides residue analyses

Mullin et al. (2010)

Cold & chemical

-20 °C & Kiev buffer

Queen spermathecae for sperm counting

Kocher et al. (2008)

 

-80 °C & RNALater®

Worker honey bee RT-PCR virus analyses

Williams et al. (2009)