Visualisation by vital staining with trypan blue of wounds punctured by Varroa destructor mites in pupae of the honey bee (Apis mellifera)

Abstract

-After invading a honey bee brood cell shortly before capping, female Varroa destructor mites puncture the integument of the host bee in order to suck haemolymph. The perforations used as feeding sites are difficult to detect. We developed a method of vital staining with trypan blue to visualise the wounds. The dye is taken up by damaged epidermal cells in the margin of repeatedly used punctures. This new coloration method allows localisation of wounds in prepupal and especially in all pupal stages of workers and of drones, but the staining failed in early postfeeding 5th instar larvae. The low-cost method is easy to use. Because secondary infestations by viral and bacterial pathogens are assumed to enter the bees through these wounds, the trypan blue method may be useful in studies on parasite-host relations, especially concerning vector functions. Since pupae survive the vital staining without apparent harm, they can be used subsequently for other investigations.Apis mellifera / Varroa destructor / pupal infestation / integumental puncture / vital staining / trypan blue

Survival of honey bee (Apis mellifera) pupae after trypan blue staining of wounds caused by Varroa destructor mites or artificial perforation

“…The pupae were perforated at sites also punctured by female mites, the thorax and in particular the 2nd abdominal sternite (Kanbar and Engels, 2004b). Vital staining with 0.01% trypan blue solution in insect ringer (5.0 g NaCl, 0.42 g KCl, 0.25 g CaCl 2 in 1 L of distilled water adjusted to pH 6.8 with 0.1 molar KOH) was performed for 30 min at 35°C, followed by rinsing the pupae in ringer (modified after Kanbar and Engels, 2004a). Parasitised pupae with Varroa-made perforations as well as uninfested and non-punctured pupae also were stained.…”

“…The increase in mortality of about 5% or less was observed in specimens with any type of perforation and also in the controls without integumental lesions. At least the application of trypan blue in 0.01% solution according to the vital staining protocol (Kanbar and Engels, 2004a) apparently has no severe toxic effect (Falbe and Regitz, 1995). As shown by our SEM micrographs, the stained wounds scarred normally.…”

“…which the wounds can be visualised (Kanbar and Engels, 2004a). In subsequent observations of the infested pupae, any influence of the vital staining on the vitality of the pupa has to be considered.…”

Comportamiento del ácaro <i>Varroa destructor</i> en celdas, con cría de obrera, de abejas africanizadas <i>(Apis mellifera)</i>

“…Poco se ha reportado sobre la localización y el número de heridas causadas por varroa al alimentarse. Estas lesiones son difíciles de detectar a simple vista (Donzé & Guerin 1997), por lo cual, se emplea la tinción con azul de tripano, para determinar con certeza el punto de alimentación en la pupa (Kanbar & Engels 2004a). …”

“…Este sitio se usa, repetidamente, por el ácaro madre para alimentarse y mantenerlo activo; así, ser usado por su progenie. Diferentes investigaciones señalan los segmentos abdominales de la pupa como los lugares en los cuales el ácaro establece su sitio de alimentación, debido a que son las áreas menos queratinizadas (Kanbar & Engels 2004a).…”