Krystyna Żółtowska

Preliminary evidence associating oxidative stress in honey bee drone brood with Varroa destructor

SUMMARY Significantly high activity of three antioxidant enzymes isolated from tissues of Varroa destructor-infested drone prepupae suggest that oxidative stress may be one of the pathogenic pathways of varroosis. The average activity of superoxide dismutase (SOD), glutathione peroxidase (GPX) and plasma protein ceruloplasmin (CP) in infested prepupae were approximately 2–4 times (i.e. significantly) higher than levels in mite-free prepupae. All prepupae were taken from a naturally-infested colony where 47% of prepupae were infested with from 1 to 3 mother varroa mites.

Supplementation of the honey bee diet with vitamin C: The effect on the antioxidative system of Apis mellifera carnica brood at different stages

Summary Diet 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.

Purification and characterization of acid trehalase from muscle of Ascaris suum (Nematoda)

Acid trehalase (EC 3.2.1.28) was isolated from muscle of Ascaris suum by fractionating with ammonium sulfate, acetone and column chromatography on DEAE-cellulose and phenyl sepharose CL-4B. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 76 kDa and of 38 kDa on SDS-PAGE. The enzyme has the optimum pH 4.9, pI 4.3, Km of 6.6 mM and Vmax=34.5 nM min(-1) x mg(-1). Besides trehalose, it hydrolyses sucrose, isomaltose and maltose and, to a lesser degree melezitose, and it does not act on cellobiose and lactose. Acid trehalase was activated by MgCl2, KNO3, NaCl, CaCl2, CH2ICOOH and p-chloromercuribenzoate and inhibited by EDTA, ZnSO4 and FeCl3.

The activity of carbohydrate-degrading enzymes in the development of brood and newly emerged workers and drones of the Carniolan honeybee, Apis mellifera carnica.

Abstract The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes &agr;-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides &agr;-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods.

Variations in antioxidant defense during the development of the solitary bee Osmia bicornis

An efficient antioxidant system is of particular importance for insects whose high metabolic rates promote increased formation of reactive oxygen species (ROS). The amount of ROS can be additionally increased by environmental factors. This study investigates the ability of red mason bees (Osmia bicornis L.) to inactivate free radicals during insect development. Both male and female bees were studied, from the larval to the active imago stage. The activity of four antioxidant enzymes, superoxide dismutase, catalase, peroxidase and glutathione S-transferase, was measured; and glutathione content and total antioxidant status were determined. The highest values of the examined parameters were found in feeding stages-in larvae and in active imagines of both genders. Significant differences between genders were noted mainly in catalase activity, which was lower in overwintering imagines and active females than in males. Most differences were observed between females and males after emergence.

Proteolytic activity in the extracts and in the excretory/secretory products from Varroa destructor parasitic mite of honeybee

Proteolytic activity of the extracts and excretory–secretory (E/S) products from parasitic mite Varroa destructor of honeybees was determined and partially characterized. Proteolytic activity of mite extract was the highest at pH 3.5; the second smaller peak was at pH 5.0. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 23 protein fractions (4.3–210 kDa) in mite extract and 15 protein fractions in E/S products were revealed. Three of them (50, 52 and 54 kDa) in mite extract and two (50 and 54 kDa) in E/S products were active towards albumin and gelatin. Peptidases from E/S products had higher activity (per 1 mg protein) than enzymes from whole mite extract. They more efficiently hydrolysed albumin and haemoglobin than gelatin and casein. Peptidases from both materials were sensitive to aspartyl and cysteine proteinase inhibitors (peptidase inhibitors, or PIs). Pepstatin A (aspartyl PI) acted the strongest and diminished the activity at pH 3.5 by 76.5% and by 86.8% at pH 5, l-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (broad cysteine PI) by 20.1 and 33.7%, respectively. Proteolysis was slightly reduced only at pH 5 by ethylene diamine tetraacetic acid (metalloproteinase inhibitor) and serine peptidases, Kunitz and soybean trypsin inhibitors. These results suggested that the enzymes of V. destructor belong mainly to aspartyl and cysteine peptidases.

The mutual influence of proteins from Varroa destructor extracts and from honeybee haemolymph on their proteolytic activity--in vitro study.

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.

The antioxidant system in diapausing and active red mason bee Osmia bicornis

The red mason bee Osmia bicornis L. (Hymenoptera: Megachilidae) belongs to a group of insects that undergo an obligatory diapause in the imago stage. Red mason bees (O. bicornis) consume oxygen during overwintering diapause, and reactive oxygen species can be produced despite substantial inhibition of metabolism. These are first studies to investigate the antioxidant system in diapausing red mason bees and to compare the antioxidant systems of overwintering and active imagines. The present study analyzes total antioxidant status, glutathione and ascorbic acid levels, and the activity of superoxide dismutase, catalase and peroxidase in overwintering (October to March) and active (April) female and male O. bicornis. Diapause phases (prediapause, diapause and post‐diapause) cannot be distinguished based on the parameters of the antioxidant system of the bees. During overwintering, a significant decrease is noted only in ascorbic acid content. The remaining antioxidants remain fairly stable, which indicates the absence of oxidative stress in diapausing specimens. The analyzed parameters distinguish diapausing bees from active insects. Excluding total antioxidant status, the evaluated parameters are significantly higher in active individuals than in overwintering specimens. Sex‐related differences are found only for catalase, the level of which is consistently higher in males than in females.

The body composition and enzymes of carbohydrate metabolism of Varroa destructor

Abstract The aim of the present research was to determine the basic composition of the organic compounds present in extracts from Varroa destructor, a parasitic mite of the honeybee. The total protein content was 9.16 ± 0.82 mg/100 mg of body weight, lipid content was 9.81 ± 1.99 mg/100 mg, and carbohydrate content was 26.67 ± 4.52 mg/100 mg. The triacylglycerol content was 2.40 ± 0.86 mg/100 mg and the cholesterol content was 0.14 ± 0.02 mg/100 mg. Thin layer chromatography indicated that phospholipids comprised the major part of the lipid component; cephalins (78%), cerebrosides (16%), and lecithins (6%) were identified in the phospholipid pool. Glucose (23.6 ± 4.52 mg/100 mg) was the main carbohydrate, followed by glycogen (5.43 ± 1.23 mg/100 mg) and trehalose (0.35 ± 0.07 mg/100 mg). Enzyme-linked immunosorbent assays detected two major glycogen metabolism enzymes, glycogen phosphorylase and glycogen synthase. Among the enzymes metabolising disaccharides, maltase (24.7 ± 2.38 μmol/mg protein) and trehalase (14.81 ± 5.21 μmol/mg protein) presented the highest activity. Saccharose and lactose were hydrolysed to a minor extent. These are the first measurements of the basic composition of the mite body. Although these data are not exhaustive, they may serve as the basis for further research on the metabolism of V. destructor, particularly concerning lipid metabolism.

Fatty acid composition of the parasitic mite Varroa destructor and its host the worker prepupae of Apis mellifera

The present study analyzes the fatty acid (FA) profile of lipids isolated from Varroa destructor Anderson & Trueman, a parasitic mite of the honey bee (Apis mellifera L.), uninfected and infected worker prepupae of the Carnolian subspecies Apis mellifera carnica Pollmann, and bee bread fed to the worker brood. Significant differences are observed in the FA profiles of lipids isolated from parasites, hosts and bee bread. Parasitism by V. destructor (henceforth, varroosis) induces visible changes in the lipid profile of worker prepupae. In infected prepupae, the percentage of total saturated FAs is lower and the percentage of unsaturated FAs is higher than in uninfected insects. These differences result from significant changes in the percentages of FAs that are most abundant in the evaluated groups (i.e. C16:0, C18:1 9c, C18:2n‐6 and C18:3n‐3 FAs). In mites and in uninfected and infected prepupae, the predominant FAs are oleic acid (41.07 ± 2.26%, 42.79 ± 1.21% and 45 ± 0.20%, respectively) and palmitic acid (22.62 ± 0.87%, 39.48 ± 0.43% and 36.84 ± 0.22%, respectively). Highly significant differences in FA composition are noted between bee bread and worker brood. The results suggest specific mechanisms of FA uptake, accumulation and metabolism in the food chain of this parasitic association, beginning from the food processed by nurse bees for larval feeding, through host organisms (worker brood) to V. destructor mites.