The damage to western honey bee, Apis mellifera, colonies caused by the originally Asian ectoparasitic mite Varroa destructor is mainly a consequence of the infestation of host bee pupae. In the capped brood cell, female mites puncture the host's integument at preferred sites in order to suck haemolymph. Due to repeated feeding by the mother mite and her progeny, these perforations are kept open until shortly before the imaginal moult of the bee. Thereafter scarring takes place, thus preventing microbial infection after the adult bee has emerged from the protected environment of the sealed brood cell. However, colonies of various bacteria were found in the open wounds of about 15-30% of all inspected host pupae with an abundance depended on the level of host brood cell infestation by the mite. The small punctures of the pupal integument are difficult to detect but, by vital staining with trypan blue, the wounds can be visualised. The ultrastructure of the pupal wounds, the bacterial colonies and the scarring process are documented by a series of scanning electron micrographs.
Abstract. The wounds inflicted on pupae in capped brood cells of the honey bee, Apis mellifera, infested with a single female of the ectoparasitic mite, Varroa destructor, were investigated after visualisation by vital staining with trypan blue. On average the mites made two integumental perforations for feeding on prepupae and one on pupae. Most of the punctures were on particular ventral sites on the abdomen. Possible reasons for this pronounced preference and the evolutionary aspects of this highly specialised parasite-host relationship are discussed.
-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
-The recently described technique of vital staining with trypan blue to visualise pupal wounds of honey bees, originating from punctures made by Varroa destructor mites, was applied to artificial perforations performed with a fine needle. The stained pupae were subsequently reared in vitro until eclosion of the adult bees. Their mortality was recorded daily. The survival of the treated pupae was only moderately affected by the staining procedure. No obvious toxic effects caused by the compound trypan blue were observed. The process of wound healing was normal. Pupae with previously trypan blue stained integumental wounds can therefore be used for long lasting experiments. This is of particular interest for future studies on the assumed vector function of Varroa mites in the transfer of bacterial and viral pathogens in honey bee diseases.Apis mellifera / Varroa destructor / pupal wounds / vital staining / trypan blue / survival
From wounds of honey bee pupae, caused by the mite Varroa destructor, coccoid bacteria were isolated and identified as Melissococcus pluton. The bacterial isolate was grown anaerobically in sorbitol medium to produce a toxic compound that was purified on XAD columns, gelfiltration and preparative HPLC. The toxic agent was identified by GC-MS and FTICR-MS as tyramine. The toxicity of the isolated tyramine was tested by a novel mobility test using the protozoon Stylonychia lemnae. A concentration of 0.2 mg/ml led to immediate inhibition of mobility. In addition the toxicity was studied on honey bee larvae by feeding tyramine/water mixtures added to the larval jelly. The lethal dosis of tyramine on 4-5 days old bee larvae was determined as 0.3 mg/larvae when added as a volume of 20 microl to the larval food in brood cells. Several other biogenic amines, such as phenylethylamine, histamine, spermine, cadaverine, putrescine and trimethylamine, were tested as their hydrochloric salts for comparison and were found to be inhibitory in the Stylonychia mobility test at similar concentrations. A quantitative hemolysis test with human red blood cells revealed that tyramine and histamine showed the highest membranolytic activity, followed by the phenylethylamine, trimethylamine and spermine, while the linear diamines, cadaverine and putrescine, showed a significantly lower hemolysis when calculated on a molar amine basis. The results indicate that tyramine which is a characteristic amine produced by M. pluton in culture, is the causative agent of the observed toxic symptoms in bee larvae. Thus this disease, known as European foulbrood, is possibly an infection transmitted by the Varroa destructor mite.
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