Małgorzata Dmitryjuk

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.

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 In Vitro Effect of Ivermectin on the Activity of Trehalose Synthesis Pathway Enzymes and Their mRNA Expression in the Muscle of Adult Female Ascaris suum (Nematoda)

The in vitro effect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, and tpp genes) in the muscle of adult female Ascaris suum was investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression of tps1 and tps2, and unchanged tpp expression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression of tps2 gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case of tpp gene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway.

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.

Effects of Varroa destructor on sugar levels and their respective carbohydrate hydrolase activities in honey bee drone prepupae

Summary Varroa mite feeding caused a significant decrease in glycogen and trehalose levels during the prepupal stage of drone honey bees (Apis mellifera carnica). At the same time, the two groups were not statistically different with regards to free glucose concentration. Concentrations of glycogen, trehalose and total sugars in varroa-infested drone prepupae were (32.4 mg/g, 15.4 mg/g, and 66.8 mg/g), respectively, while levels in uninfested prepupae were (49.3 mg/g, 21.3 mg/g and 114.5 mg/g, respectively). The activities of glucoamylase (0.84 u/mg) and trehalase (1.37 u/mg) in varroa-infested prepupae were also significantly higher in the infested prepupae than in non-infested controls (0.54 u/mg and 0.78 u/mg, respectively). Interestingly, the activity of invertase was significantly lower in infested prepupae.

Expression of Genes Encoding the Enzymes for Glycogen and Trehalose Metabolism in L3 and L4 Larvae of Anisakis simplex

Trehalose and glycogen metabolism plays an important role in supporting life processes in many nematodes, including Anisakis simplex. Nematodes, cosmopolitan helminths parasitizing sea mammals and humans, cause a disease known as anisakiasis. The aim of this study was to investigate the expression of genes encoding the enzymes involved in the metabolism of trehalose and glycogen—trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), glycogen synthase (GS), and glycogen phosphorylase (GP)—in stage L3 and stage L4 larvae of A. simplex. The expression of mRNA all four genes, tps, tpp, gs, and gp, was examined by real-time polymerase chain reaction. The A. simplex ribosomal gene (18S) was used as a reference gene. Enzymatic activity was determined. The expression of trehalose enzyme genes was higher in L3 than in L4 larvae, but an inverse relationship was noted for the expression of gs and gp genes.

The gene expression and the activity of enzyme synthesis of trehalose during development of Ascaris suum (Nematoda) eggs

Abstract The activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) and their mRNA expression was determined in Ascaris suum eggs developing from a zygote to invasive larvae in an in vitro culture. The stages of nematode embryonic development, i.e. cleavage, gastrulation and formation of larvae, were always accompanied by an increase in TPS activity. An increase in TPP activity, the second enzyme of the analysed pathway, followed in subsequent developmental stages. Key changes in trehalose metabolism were observed in the tadpole stage. The development of moving larvae was accompanied by the highest mRNA expression and activity level of TPP, which converts metabolic intermediate T6P to trehalose. The trehalose synthesis enzymes activity was positively correlated with their mRNA expression. For three genes: tps1, tps2 (TPS coding) and tpp (TPP coding) there was very strong and statistically significant correlation with the activity of a specific enzyme. The Pearson’s coefficient was r = 0.903714 (p < 0.001) for tps1 gene, r = 0.803012 ( < 0.01) for tps2 and r = 0.856244 (p < 0.01) for tpp over development time.