Honey Bee Enzyme Research Articles

Unexpected Value of Honey Color for Prediction of a Non-Enzymatic H2O2 Production and Honey Antibacterial Activity: A Perspective.

Hydrogen peroxide is the principal antibacterial compound of honey and its concentration determines honey bacteriostatic (MIC) and bactericidal (MBC) potencies. Levels of H2O2 produced are highly relevant to honey therapeutic potential, but they vary extensively among honey with reasons not immediately apparent. According to a traditional view, H2O2 is produced as a by-product of glucose oxidation by the honey bee enzyme, glucose oxidase; however, significant levels of H2O2 could be produced in a non-enzymatic way via polyphenol autooxidation. The aim of this study was to evaluate the potential for such an alternative pathway by re-examining evidence from many experimental and correlative studies in order to identify factors and compounds required for pro-oxidant activity. Unexpectedly, the color intensity was found to be the main indicator separating honey varieties based on the quantitative differences in the polyphenolic content, antioxidant activity and the content of transition metals, Fe, Cu and Mn, the main factors required for pro-oxidant effects. The color-impeding polyphenolics and their oxidation products (semiquinones and quinones) further contributed to color development through multiple chemical conjugations with proteins, phenolic oxidative polymerization, chelation or the reduction of metal ions. Moreover, quinones, as an intrinsic part of polyphenol redox activity, play an active role in the formation of higher-order structures, melanoidins and colloids in honey. The latter structures are also known to chelate metal ions, potentially contributing to H2O2 production. Thus, the color intensity appears as a major parameter that integrates polyphenol-dependent pro-oxidant reactions resulting in H2O2 generation.

Activating transcription factor 2 (AccATF2) regulates tolerance to oxidative stress in Apis cerana cerana

Activating transcription factor 2 (ATF2), a basic leucine zipper (bZIP) transcription factor, plays a crucial role in immune and DNA damage response in mammals. However, the function of ATF2 in insects remains unknown. Here, we isolated the ATF2 gene from Apis cerana cerana (AccATF2) and found that AccATF2 was a main regulator of the honeybee response to oxidative stress. Our results showed that AccATF2 was highly expressed in the head, thorax and integument. AccATF2 was expressed throughout the development period of honeybees, and the highest AccATF2 transcript level was noted in brown-eyed pupae, indicating its indispensable roles in honeybee survival. Antioxidant function analysis showed that AccATF2 expression was markedly induced in response to oxidative stress caused by various environmental stresses. AccATF2 overexpression substantially enhanced the tolerance to oxidative stress of Escherichia coli cells compared with control cells. AccATF2 knockdown significantly increased the production of malondialdehyde (MDA), the transcription of antioxidant genes and the activity of antioxidant enzymes in honeybees, suggesting that AccATF2 knockdown resulted in oxidative damage to honeybees. Moreover, AccATF2 knockdown decreased honeybee resistance to oxidative stress caused by high temperature. Overall, AccATF2 plays an important role in maintaining redox homeostasis and protecting honeybees from oxidative stress caused by various environmental stimuli. Our discoveries add to a growing understanding of how honeybees cope with various adverse environmental conditions to ensure their survival.

Infectivity of Nosema ceranae isolates from different hosts and immune response in honey bees Apis mellifera and Apis cerana

The microsporidium, Nosema ceranae, was first discovered in Apis cerana. Currently, N. ceranae is a highly prevalent parasite in A. mellifera colonies worldwide. The pathology of N. ceranae and how bees defend themselves from this pathogen have been reported. Here, we investigated the infectivity of two N. ceranae isolates from different honey bee species, A. mellifera and A. cerana. Our results showed that the N. ceranae isolate from A. mellifera had a significantly higher infectivity than another isolate from A. cerana in all experimental cages of A. mellifera and A. cerana. Moreover, when comparing the infectivity of each N. ceranae isolate between the original and new hosts, it seems that no significant difference was obtained. The immunity-related enzymes in honey bees following N. ceranae inoculation were also analyzed at day six after spore feeding. Phenol oxidase (PO) transcript levels were up-regulated in A. cerana when inoculated by both N. ceranae isolates, but were not significantly different. However, the transcript level for PO in one experimental cage of A. mellifera was significantly down-regulated when inoculated with N. ceranae. Significant glucose dehydrogenase suppression occurred in only one cage of each honey bee species.

Ecologically appropriate xenobiotics induce cytochrome P450s in Apis mellifera.

BackgroundHoney bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored.Methodology/Principal FindingsWe used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey.Conclusions/SignificanceThese results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey.

Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA

Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 °C, respectively. Activity was fully stable for 30 min at 50 °C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 μM) of Ca 2+, Mg 2+, and Mn 2+ had a stimulatory effect on the activity. Single Michaelis–Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for l-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.

Energy metabolism during insect flight: biochemical design and physiological performance.

Flying insects achieve the highest known mass-specific rates of O(2) consumption in the animal kingdom. Because the flight muscles account for >90% of the organismal O(2) uptake, accurate estimates of metabolic flux rates (J) in the muscles can be made. In steady state, these are equal to the net forward flux rates (v) at individual steps and can be compared with flux capacities (V(max)) measured in vitro. In flying honeybees, hexokinase and phosphofructokinase, both nonequilibrium reactions in glycolysis, operate at large fractions of their maximum capacities (i.e., they operate at high v/V(max)). Phosphoglucoisomerase is a reversible reaction that operates near equilibrium. Despite V(max) values more than 20-fold greater than the net forward flux rates during flight, a close match is found between the V(max) required in vivo (estimated using the Haldane relationship) to maintain near equilibrium and this net forward flux rate and the V(max) measured in vitro under simulated physiological conditions. Rates of organismal O(2) consumption and difference spectroscopy were used to estimate electron transfer rates per molecule of respiratory chain enzyme during flight. These are much higher than those estimated in mammalian muscles. Current evidence indicates that metabolic enzymes in honeybees do not display higher catalytic efficiencies than the homologous enzymes in mammals, and the high electron transfer rates do not appear to be the result of higher enzyme densities per unit cristae surface area. A number of possible mechanistic explanations for the higher rates of electron transfer are proposed.

Oxygen Consumption During Hovering is Associated with Genetic Variation of Enzymes in Honey-Bees

Despite a general paucity of genetic variation, colonies of honey-bees, Apis mellifera L., usually contain genetic variation for the soluble form of the enzyme malate dehydrogenase (s-MDH). To determine whether this genetic variation influenced the physiology of honey-bees, we examined the maximal metabolic rate (V02max) during hovering flight of the common s-MDH genotypes. Among workers, heterozygotes and slow homozygotes had similar, low V02maxs and the fast homozygote had the highest. In drones, the pattern was reversed: the V02max of the slow haplotype exceeded that of the fast haplotype. In both males and females, the variances of V02max were heteroscedastic among s-MDH genotypes and the genotype with the highest V02max also had the highest variance. The data suggest that there may be kinetic differences among s-MDH allozymes. Drones had significantly higher thoracic temperatures than workers. The reversal of favoured genotypes in workers and drones may be related to the difference in thoracic temperature between

Studies on the inhibition of honey bee cholinesterase by carbamates

The kinetic constants for the inhibition (I50, Ka, K2c, -Ki, K3, and Ka' values) of honey bee cholinesterase by furadan and its two analogues were determined and evaluated. Furadan proved to be a potent inhibitor of honey bee enzyme. All the three carbamates showed a high affinity for honey-bee ChE. The affinity constants ranged from 0.22 to 2 .6 8 x 1 0 -- M. There was little variation among the carbamylation constants, indicating a minor role for the carbamylation step in the inhibition process. The significant variation in the bimolecular reaction constants of the three carbamates could be attributed to the variations in the affinity constants rather than to the carbamylation constants. In most cases, the average apparent affinity constants were comparable with the Ka values and increased progressively with the concentration of inhibitor.

Changes in sulfhydryl groups of honeybee glyceraldehyde phosphate dehydrogenase associated with generation of the intermediate plateau in its saturation kinetics.

Honeybee and rabbit muscle GPDH were studied to obtain information at the chemical level regarding anomolous saturation kinetics of the honeybee enzyme. Results demonstrate that the enzyme's sulfhydryl groups are implicated in the process. Measured by DTNB titration, native honeybee GPDH has one less active SH than the native rabbit muscle enzyme and displays changes in overall sulfhydryl reactivity after preincubation with G-3-P or G-3-P plus NAD+. The total DTNB reactive sulfhydryls of rabbit muscle GPDH are not changed by preincubation with NAD+ or G-3-P; honeybee GPDH, under certain conductions of preincubation with these ligands, shows a decrease of two total DTNB reactive SH groups. This difference has been confirmed by an independent experiment in which the two enzymes were carboxymethylated with C-14 bromoacetic acid.

Immunochemical studies with glycerol 3-phosphate dehydrogenase in bees and wasps

Rabbit antibodies were prepared against glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) purified from the flight muscle of honeybees, one bumblebee species, and yellow jackets. Crossreactivity tests by means of quantitative microcomplement fixation were conducted with extracts from several hymenopteran species. The degree of cross-reaction with thoracic extracts was, in general, compatible with the accepted phylogenetic relationships of the species tested. The one exception was the lower-than-expected reaction of the honeybee enzyme with anti-bumblebee glycerol 3-phosphate dehydrogenase. The cross-reactivity of the two types of isozymes of bumblebee glycerol 3-phosphate dehydrogenase was measured. The alarmuscular variants, which occur mainly in flight muscle, reacted much more strongly with anti-honeybee or anti-bumblebee sera than did the omniregional variants.

Glutamic acid decarboxylase and the GABA shunt in the supraoesophageal ganglion of the honey-bee, Apis mellifera

The presence of l-glutamic acid decarboxylase in homogenates of the honey-bee brain has been confirmed. The enzyme is most active between pH 6·0 to 9·0. At pH 6·8 K m and V max were 0·06 M and 91 μmoles/hr per g tissue respectively. The honey-bee enzyme is inhibited by high concentrations of GABA and some evidence for substrate inhibition by l-glutamic acid was found. No absolute requirement for the cofactor, pyridoxal-5-phosphate, could be detected. Inhibition by hydroxylamine and p-hydroxymercuribenzoate were competitive and non-competitive respectively. The major portion of the enzyme was bound to cellular particulate fractions sedimenting in a wide range of centrifugal forces. Results of sucrose gradient centrifugations indicated that the enzyme was not bound to subcellular particles of ‘synaptosome’ size. Enzymes capable of metabolizing GABA to succinic acid were also found in honey-bee brain homogenates. The results of this study are discussed in relation to the rôle of l-glutamic acid and GABA as possible neurotransmitter agents.

The molecular weights of glycerol-3-P dehydrogenases from chicken, rabbit, and honey bee

The molecular weights of liver and muscle type glycerol-3-P dehydrogenase isozymes (EC 1.1.1.8) from chicken have been reexamined and found to be about 68,000. This figure is sustained in equilibrium ultracentrifugation, gel filtration, and amino acid-composition studies. Direct comparison by gel filtration of solutions containing a mixture of chicken muscle and honey bee glycerol-3-P dehydrogenases indicate that the honey bee enzyme is heavier and has a molecular weight near 76,000. A similar comparison of rabbit muscle and honey bee glycerol-3-P dehydrogenases also indicated that the insect enzyme was heavier and that the glycerol-3-P dehydrogenases of chicken and rabbit have similar molecular weights. Previous estimates of the molecular weights of crystalline glycerol-3-P dehydrogenases from chicken muscle, rabbit muscle, and honey bee thorax do not agree with present observations.

Enzyme activity in cryobiological systems: III. Low-temperature properties of honeybee glyceraldehydephosphate dehydrogenase

Certain properties of honeybee and rabbit muscle GPDHs have been examined in vitro. While incubation of rabbit muscle GPDH at 0 °C in the presence of 4 m m ATP results in its dissociation, the enzyme from honeybees retains its native tetrameric structure. Furthermore all adenine nucleotides tested caused some inactivation of rabbit muscle GPDH but had either only a slight effect or none at all on the honeybee enzyme. However, both enzymes are competitively inhibited by ATP, ADP, and AMP and 3′,5′-AMP. In the absence of ammonium sulfate and ATP, honeybee GPDH was partially inactivated at 0 °C while the rabbit muscle enzyme was not affected. Inactivation of honeybee GPDH could be presented by low concentrations of ammonium sulfate. The honeybee enzyme retains essentially full activity over the pH range 6.5–8.0 when incubated with 10 m m ATP at 0 °C, while the rabbit muscle enzyme has been shown to be completely inactivated over this pH range in the presence of 2 m m ATP at 0 °C. On the basis of studies with adenine nucleotides it is concluded that insect GPDHs probably have evolved a capability for catalytic function at low temperature while retaining the ability to have this function controlled by these compounds.

Glycerol 3-Phosphate Dehydrogenases in Social Bees

Electrophoretic analysis of extracts of 12 bumblebee species revealed complex patterns of extramitochondrial diphosphopyridine nucleotide-dependent glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) variants. Most Bombus species show one pattern, while Bombus nevadensis has a second and Bombus occidentalis a third pattern. All three Psithyrus (i.e. parasitic bumblebee) species tested have a fourth pattern. All patterns share one feature, i.e. there is both a slow and fast migrating set of two to four bands each. The slow set, as a whole, stains more intensely than the fast set in flight muscle extracts, whereas the opposite is true for extracts of heads and abdomens. The slow sets have been termed alar-muscular, the fast sets omniregional. The major alar-muscular isozyme has been crystallized from thoraces of B. nevadensis. The omniregional isozymes of most, but not all, bumblebees utilize triphosphopyridine nucleotide, whereas none of the alarmuscular variants utilize this nucleotide. Electrophoretic experiments indicate that most, if not all, the variants differ in primary structure. In contrast to bumblebees, honeybees have only one form of glycerol 3-phosphate dehydrogenase. Rabbit antibodies directed against glycerol 3-phosphate dehydrogenase of honeybees and of B. nevadensis were prepared. Each of these antisera precipitated the honeybee enzyme and all of the bumblebee isozymes. Immunological experiments using the micro-complement fixation technique with anti-B. nevadensis glycerol 3-phosphate dehydrogenase reveal that thoracic extracts of all other bumblebees tested cross-reacted very strongly, with Bombus bifarius and B. occidentalis extracts showing slightly less reactivity than that of the other Bombus and Psithyrus species. Honeybee extracts cross-reacted much more weakly. Micro-complement fixation studies with anti-honeybee glycerol 3-phosphate dehydrogenase suggest that extracts of different bumblebee species are immunochemically indistinguishable from one another.

The sedimentation properties of cholinesterase from a mayfly ( Hexagenia bilineata (Say); Ephemeroptera) and the honey bee ( Apis mellifera L.)

1. 1. Solubilized mayfly and honey bee cholinesterase have the same sedimentation coefficient (7·3 S) at pH. 8·0. 2. 2. At pH 6·8 the mayfly enzyme sediments in 7·3 S, 10·2 S, and larger forms; the honey bee enzyme is polydispene at pH. 6·8. 3. 3. The mayfly ChE shifts from heavier forms to 7·3 S following dialysis, dilution, or an increase in pH from 6·8 to 8·0.

Glyceraldehydephosphate dehydrogenase: Crystallization from honeybees; Quantitative immunochemical and electrophoretic comparisons of the enzyme in other insects

Glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) was crystallized from honeybee ( Apis mellifera) thoraces. The yellow colour of the crystals is due to diphosphopyridine nucleotide bound to the enzyme. The sedimentation coefficient of the honeybee enzyme is the same as that reported for the enzyme from other species. A small amount of the dehydrogenase was also crystallized from bumblebee ( Bombus nevadensis) thoraces. Rabbit antibodies directed against the pure honeybee enzyme were prepared, and the cross-reaction with extracts of other insects quantitatively measured with the micro-complement fixation technique. The order of decreasing cross-reaction is: honeybee, leafcutting bee, bumblebee (three species)—lobster—mining bee, flesh fly. The order: honeybee, leafcutting bee, bumblebee, was also observed in two-dimensional immunodiffusion and in a precipitin test. The relative cross-reaction of this enzyme, thus, varies considerably from the classical taxonomy of the species tested. The anomalous immunochemical properties of the glyceraldehyde-P dehydrogenases may be due to the relatively conservative changes in primary structure of this protein during evolution. Electrophoretic patterns of glyceraldehyde-P dehydrogenase activity reveal one band in extracts of honeybees and bumblebees, three bands in extracts of leafcutting bees and flesh flies, and up to five bands in extracts of mining bees.