3.4.2. Feeding site
Varroa mothers pierce the cuticle of honey bee larvae and pupae on which they (Fig. 7a) and their offspring feed (Fig. 7b). In late pupae, the wound can be seen under the binocular thanks to the scarring process of the cuticle (Fig. 7c). It can also be located by observation of the feeding mites, events that are relatively rare and need an artificial in vitro system to be observed (see section 3.2.2. ‘Breeding mites in the laboratory’). In most cases, no wound can be seen on larvae or pupae and a staining method is necessary to find it (Fig. 7d). The ability to locate the wound might be necessary for behavioural studies of feeding behaviour or reproduction or for secondary disease transmission studies (Kanbar and Engels, 2003). By extension, this method can also be used for all cases in which a perforation of the cuticle of immature honey bees has to be made visible (e.g. injection of pathogens or hormones, Kanbar and Engels, 2005).
Kanbar and Engels (2004) designed a vital staining method that allows the visualisation of feeding sites. They used Trypan blue, a dye that enters damaged cells (Roche, 1999), i.e. cells around the hole pierced by mother mites in the late 5th instar larvae, prepupae or pupae cuticle. Feeding sites could thus be stained durably on live individuals and observed over time (Herrmann et al., 2005). Staining can be detected until the stage when the cuticle darkens to the point of hiding the dyed blue cells. At this point the dyeing is not any longer necessary.
1. Sample larva, pre-pupae or pupae to be stained from varroa infested cells (see section 3.1. ‘Collecting mites’).
2. Immerse them for 30 min in a volume of Ringer-based staining medium sufficient to cover the major part of the body surface.
The larvae and pupae survive this treatment (Kanbar and Engels, 2003).
Ringer solution: see Table 1 of the BEEBOOK paper on cell cultures (Genersch et al., 2013) for a recipe.
Vital staining medium: 100 ml Ringer solution 0.01 g Trypan blue adjusted to pH 6.8 with KOH (0.1M).
3. Rinse in pure Ringer solution for 3 min.
Fig. 7. a. Adult varroa mite sucking haemolymph of a pupa at the feeding site. b. varroa nymph sucking haemolymph of a pupa as the feeding site. c. feeding site with melanisation (arrow). It is visible without staining. Such instances are more frequent in older pupae. d. a feeding site (arrow) on a white pupa after staining with Trypan blue. Photos: Swiss Bee Research Institute.