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.