4.8. Incubation conditions
- Clean environment is needed (à Section 5.3. Sterile environment)
- Prepare saturated solutions of K2SO4 and NaCl, place in open dishes in desiccator
- Incubate larvae at 34.5°C and 95 % RH for the first 6 days
- On day 7 change
humidity to 80 % RH
Honey bee larvae may be kept in hermetic plexiglass desiccators or other air-tight plastic containers (to facilitate humidity control, e.g. Tupperware) placed inside incubators. Incubators alone are also adequate when the necessary humidity can be maintained in the chamber. It is important to maintain the desired rearing temperature of 34.5°C with maximum precision (± 0.5°C), as suboptimal larval temperature affects adult bee longevity and adult bee resistance to dimethoate and induces malformed wings (Medrzycki et al., 2010). Furthermore, pathogens may increase or lower in pathogenicity according to temperature (Vojvodic et al., 2011a). The deviation of incubation temperature must be kept as low as possible and temperature should be verified with data loggers to help explain any possible problems with mortality. Beside temperature, honey bee larvae also require constant and high humidity (Human et al., 2006). Most researchers propose a humidity of 95-96 % RH during the first 6 days followed by a reduction to 70-80 % RH, which proved to be appropriate (Rembold and Lackner, 1981; Vandenberg and Shimanuki, 1987; Peng et al., 1992; Aupinel et al., 2005). The humidity adjustment is accomplished by first placing a dish with a saturated solution of K2SO4 (to achieve 95% RH) and later a saturated solution of NaCl (to achieve 80% RH) on the bottom of the desiccator. More on preparation of these solutions can be found in section 6.3 “Relative Humidity” of the BEEBOOK paper on maintaining adult bees in the laboratory (Williams et al., 2013). Humidity should also be measured with data loggers regularly to verify accuracy. If glycerol soaked dental rolls are used (see 3. Sterile environment) they have to be removed at day 7 (Aupinel et al., 2005). Alternatively, good humidity results have been obtained with a much easier approach by just filling up to one fourth of the wells of the plate with water, closing the plate with the accompanying lid, and placing the plates in bacterial incubators, i.e. incubators without CO2 and humidity adjustment (Genersch et al., 2005; 2006).
We recommend protecting larvae and reducing the handling of their rearing dishes to reduce mortality. The exposure of larvae to temperature and humidity conditions other than those in the incubator increase bacterial or fungal infections and mortality. Larvae should be weighed only if necessary for experiments. Transferring to new dishes or carefully cleaning larvae at the beginning of or after defecation (Smith, 1959; Shuel and Dixon, 1986, Genersch et al., 2005; 2006) is not performed in many methods, although it is reported to increase successful pupation and reduce pupal mortality (Vandenberg and Shimanuki, 1987).