SummaryDiet in the winter has a vital effect on the survival and condition of a honey bee (Apis mellifera) colony in the spring. The effect of supplementation of the diet with vitamin C (ascorbic acid) on the total antioxidant status (TAS), glutathione content, and activity of 4 antioxidative enzymes: superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and glutathione transferase (GST) of honey bee brood developing in the spring was studied. Twelve stages, from newly hatched larvae to emerging adult worker bees were studied, allowing changes in the antioxidant profile during brood development to be determined for the first time. It was shown that bees are more exposed to oxidative stress after emergence. In workers emerging in colonies after supplementation with vitamin C, higher contents of protein and glutathione, and higher activities of peroxidase, catalase, and glutathione transferase were observed. Vitamin C did not alter brood weight increase, and the level of protein in emerged workers was higher than in the control group. The mean of bee losses over winter were about 33% lower in colonies receiving vitamin C.Suplementación de la dieta de la abeja de la miel con vitamina C: El efecto sobre el sistema antioxidante de la cría de Apis mellifera carnica en diferentes etapas Resumen La dieta durante el invierno tiene un efecto importante en la supervivencia y el estado de las colmenas de la abeja de la miel (Apis mellifera) durante la primavera. Se ha estudiado el efecto de la suplementación de la dieta con vitamina C (ácido ascórbico) sobre el estado antioxidante total (TAS), el contenido de glutatión y la actividad de cuatro enzimas antioxidantes: superóxido dismutasa (SOD), peroxidasa (POX), catalasa (CAT), y glutatión transferasa (GST) en la cría de abejas en desarrollo durante la primavera. Se han estudiado doce etapas, desde larvas recién nacidas hasta la emersión de las abejas obreras adultas, lo que permite determinar por primera vez los cambios en el perfil antioxidante durante el desarrollo de la cría. Se ha demostrado que las abejas están más expuestas a estrés oxidativo después de la emergencia. Los mayores contenidos de proteína y glutatión, y las mayores actividades de peroxidasa, catalasa y glutatión transferasa se observaron en las obreras que emergen en las colmenas tras la suplementación con vitamina C. La vitamina C no alteró el aumento del peso de la cría, y el nivel de proteína en las obreras emergentes fue mayor que en el grupo control. La media de las pérdidas de abejas durante el invierno fue un 33% menor en las colmenas que recibieron la vitamina C.

This paper describes a new technique for noninvasive diagnostic analysis of metabolic regulation and the microcirculation. Flow Mediated Skin Fluorescence (FMSF) is based on monitoring the intensity of NADH fluorescence emitted from skin tissue on the forearm. The principles of the technique are discussed, in particular, concerning experimental procedures and the definition and interpretation of the measured parameters. The unique features and potential avenues for development of the FMSF technique are also outlined.

A b s t r a c t The colony collapse disorder is a growing problem world-wide. For this reason, we were prompted to search for natural and harmless agents that could improve the living conditions of honey bees. This group of agents includes exogenous antioxidants, such as ascorbic acid, which boost natural immunity. We analysed the effect of vitamin C supplementation on carbohydrate metabolism in the developing honey bee worker brood. The total carbohydrate content and the concentrations of glycogen, trehalose, maltotriose, fructose, and glucose were estimated. The correlations between sugar content and the activity of the main hydrolases of carbohydrate metabolism -α-amylase, glucoamylase, trehalase, maltase, and sucrase -were determined. The addition of vitamin C to the diet of wintering bees did not impair their sugar metabolism. Vitamin C supplements exerted a positive effect by signifi cantly increasing glycogen and trehalose concentrations in the initial phase of development and in newly emerged workers. Vitamin C did not induce signifi cant changes in the developmental profi le of carbohydrate degrading enzymes, except for the earliest stage of larval development when enzyme activity levels were below those noted in the control group.

We studied a total of eight developmental stages of capped brood and newly emerged workers of Apis mellifera carnica colonies naturally parasitized with Varroa destructor. During winter and early spring four colonies were fed syrup containing 1.8 mg vitamin C kg(-1) (ascorbic acid group; group AA) while four colonies were fed syrup without the vitamin C (control group C). Selected elements of the antioxidative system were analysed including total antioxidant status (TAS), glutathione content and antioxidative enzyme activities (superoxide dismutase, catalase, peroxidase and glutathione S-transferase). Body weight, protein content and indices of infestation were also determined. The prevalence (8.11%) and intensity (1·15 parasite per bee) of the infestation were lower in group AA compared with group C (11.3% and 1.21, respectively). Changes in the indicators of antioxidative stress were evidence for the strengthening of the antioxidative system in the brood by administration of vitamin C. In freshly emerged worker bees of group AA, despite the infestation, protein content, TAS, and the activity of all antioxidative enzymes had significantly higher values in relation to group C.

The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.

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Varroa destructor is an ectoparasite that causes serious damage to the population of the honeybee. Increasing resistance of the parasite to acaricides is related, among others, to metabolic adaptations of its esterases to facilitate decomposition of the chemicals used. Esterases are a large heterogeneous group of enzymes that metabolize a number of endogenous and exogenous substrates with ester binding. The aim of the present study was to determine the activity of esterases in the body extracts (BE) and excretion/secretion products (E/SP) of the mite. The enzymes contained in the E/SP should originate mainly from the salivary glands and the alimentary system and they may play a particularly important role in the first line of defence of the mite against acaricides. Activity of cholinesterases (ChEs) [acetylcholinesterase (AChE) and butyrylcholinesterase], carboxylesterases (CEs) and phosphatases [alkaline phosphatase (AP) and acid phosphatase (AcP)] was investigated. The activity of all the enzymes except AChE was higher in the E/SP than in the BE. ChEs from the BE and from the E/SP reacted differently on eserine, a ChE inhibitor. Eserine inhibited both enzymes from the BE, increased decomposition of acetylcholine, but did not influence hydrolysis of butyrylcholine by the E/SP. Activity of the CEs from the BE in relation to the esters of carboxylic acids can be presented in the following series: C10 > C12 > C14 > C8 > C2 > C4 = C16, while activity of the CEs from the E/SP was: C4 > C8 > C2 > C14 > C10 > C12 > C16. The inhibitor of CEs, triphenyl phosphate, reduced the activity of esterases C2–C8 and C14–C16; however, it acted in the opposite way to CEs C10 and C12. The activity of both phosphatases was higher in the E/SP than in the BE (AcP about twofold and AP about 2.6-fold); the activities of AP and AcP in the same material were similar. Given the role of esterases in resistance to pesticides, further studies are necessary to obtain complete biochemical characteristics of the enzymes currently present in V. destructor.