Bee Enzymes Research Articles

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.

The effects of short- and long-term diet supplementation with Iranian propolis on the growth and immunity in rainbow trout (Oncorhynchus mykiss)

Summary Propolis, a resinous substance collected by Apis mellifera bees from various plant sources is transformed in the presence of bee enzymes. Short- and long-term effects of diet supplementation with ethanol extract of Iranian propolis (EEIP) was investigated on growth and immunity in juvenile rainbow trout. The fish (mean body weight 30 ± 3.2 g) were fed a commercial diet supplemented with 0 (control), 1, 2 and 5 g/kg EEIP for 96 h and 0, 0.5, 1 and 2 g/kg EEIP for a 45-days. Rainbow trout growth performance significantly (P<0.05) increased by the dietary supplementation of Iranian propolis. Our results showed that significant increase in serum lysozyme activity, complement activity and total immunoglobulin were seen in all treatment groups during short- and longterm feeding trial when compared to the control group. On the basis of our findings, propolis improved rainbow trout growth performance and some immune parameters.

Portuguese propolis disturbs glycolytic metabolism of human colorectal cancer in vitro

BackgroundPropolis is a resin collected by bees from plant buds and exudates, which is further processed through the activity of bee enzymes. Propolis has been shown to possess many biological and pharmacological properties, such as antimicrobial, antioxidant, immunostimulant and antitumor activities. Due to this bioactivity profile, this resin can become an alternative, economic and safe source of natural bioactive compounds.Antitumor action has been reported in vitro and in vivo for propolis extracts or its isolated compounds; however, Portuguese propolis has been little explored. The aim of this work was to evaluate the in vitro antitumor activity of Portuguese propolis on the human colon carcinoma cell line HCT-15, assessing the effect of different fractions (hexane, chloroform and ethanol residual) of a propolis ethanol extract on cell viability, proliferation, metabolism and death.MethodsPropolis from Angra do Heroísmo (Azores) was extracted with ethanol and sequentially fractionated in solvents with increasing polarity, n-hexane and chloroform. To assess cell viability, cell proliferation and cell death, Sulforhodamine B, BrDU incorporation assay and Anexin V/Propidium iodide were used, respectively. Glycolytic metabolism was estimated using specific kits.ResultsAll propolis samples exhibited a cytotoxic effect against tumor cells, in a dose- and time-dependent way. Chloroform fraction, the most enriched in phenolic compounds, appears to be the most active, both in terms of inhibition of viability and cell death. Data also show that this cytotoxicity involves disturbance in tumor cell glycolytic metabolism, seen by a decrease in glucose consumption and lactate production.ConclusionOur results show that Portuguese propolis from Angra do Heroísmo (Azores) can be a potential therapeutic agent against human colorectal cancer.

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.

Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

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.

Exploring the antibacterial properties of honey and its potential

Exploring the antibacterial properties of honey and its potential

Sequence analysis and phylogenetic relationship of genes encoding heterodimeric phospholipases A2 from the venom of the scorpion Anuroctonus phaiodactylus

Some scorpion venom contain heterodimeric phospholipases A2. They were shown to be toxic to insects and to cause edema and/or hemolysis of mammalian erythrocytes. This manuscript describes the results of cDNA cloning of five different heterodimeric phospholipases from the venomous glands of the Mexican scorpion Anuroctonus phaiodactylus. The amino acid sequence deduced from the heterodimeric phospholipases open reading frames corresponds in each case to a different isoform. The nucleotide sequences corresponding to two of these genes were also obtained by directly sequencing genomic DNA. The cDNA isoforms show high similarity with the heterodimeric phospholipase Phaiodactylipin purified from the same scorpion. However, similar phospholipases were also found in scorpions from other species and the sequences available were used to construct a phylogenetic tree. In order to understand better the gene structure and phylogeny of these enzymes we analyzed their sequences and compared them with secretory phospholipases of other sources from groups I, II and III. The genomic DNA sequence of a similar phospholipase from bee venomous glands was also cloned. The information available on a Drosophila phospholipase was included in this analysis. The phospholipases of groups I and II contain a conserved exon–intron structure (four or five exons of the mature segment of the enzyme are separated by three or four introns). Also, the gene structure of the phospholipases from A. phaiodactylus and that of the bee venom, belonging to group III phospholipases, are interrupted by three introns. The mature peptide of the bee enzyme is a single polypeptide chain, coded by four exons, whereas those from the scorpion studied here although having four exons, showed the presence of two different polypeptides in its native state. The mature protein is processed after synthesis, producing the heterodimeric structure: a long and a short-peptide chain, linked by a disulfide bridge. The small subunit is the one coded by the fourth exon. The human phospholipase A2 and that of Drosophila, also classified into the group III phospholipases, have a considerably different exon–intron organization.

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.

Floral nectar phenolics as biochemical markers for the botanical origin of heather honey

In order to find out biochemical markers for the botanical origin of heather (Erica) honey, the phenolic metabolites present in heather floral nectar, collected from the honey-stomach of bees gathering nectar from these flowers, were analysed. The flavonoid fraction of nectar contained four main flavonoids. Two of them were quercetin and kaempferol 3-rhamnosides, and the other two were tentatively identified as myricetin 3′-methyl ether and isorhamnetin 3-rhamnosides. Since the natural glycosides are hydrolysed by bee enzymes to render the corresponding aglycones, which are the metabolites detected in honey, acid hydrolysis of the nectar glycosides was achieved. The aglycones quercetin, myricetin 3′-methyl ether, kaempferol and isorhamnetin were identified, as well as the gallic acid derivative ellagic acid. The analysis of Portuguese heather honey samples showed that ellagic acid was present in all the samples in significant amounts ranging between 100 μg and 600 μg per 100 g honey. The other nectar-derived flavonoids were also present, although some of them in very variable amounts. Ellagic acid and myricetin 3′-methyl ether, which have not been detected in any of the monofloral honey samples investigated so far, with the only exception being a French honey sample of the botanically relatedCalluna (Ericaceae) which also contained ellagic acid, seem to be the most useful potential markers for the floral origin of heather honey. However, more detailed and extensive investigations are needed to prove the utility of these markers.

The influence of colony population and brood rearing intensity on the activity of detoxifying enzymes in worker honey bees

Abstract. Cohorts of worker honey bees from a single parental hive, cross‐fostered into colonies which differed in population and other colony parameters, were assessed for activities of enzymes involved in metabolic detoxication. Activities of glutathione S‐transferase and mixed‐function oxidase enzymes were negatively correlated with foster colony population, but positively correlated with the ratio of larvae (brood)/adult workers. Worker bees which had begun foraging had enzyme activity levels higher than any found in bees which were still performing in‐hive duties. Elevated levels of detoxifying enzymes in colonies with low populations and high ratios of larvae/adults may be a protective mechanism to prevent poisoning of larvae by toxins brought to the colony by foragers.

Hesperetin: A marker of the floral origin of citrus honey

AbstractSeventeen flavonoid aglycones were identified in various experimental and commercial citrus honey samples by HPLC analysis. The flavanone hesperetin was detected in all samples. This flavanone was not detected in any of the honey samples, from diverse floral origin (including rosemary, lavender, sunflower, almond, sweet chestnut, white clover, Erisarum, Robinia, Rhododendron, Tilia, Prosopis, Eucalyptus and Calluna honeys) previously investigated. The analysis of the flavonoids present in orange nectar revealed that the flavanone hesperidin (hesperetin‐7‐rutinoside) was the major flavonoid detected and, therefore, this should be the main source of the hesperetin found in citrus honey. Hesperetin should be produced by hydrolysis of hesperidin by the bee enzymes present in honey. These results suggest that hesperetin could be used as a marker for the botanical origin of citrus honey.

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.

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.

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.

Insect extramitochondrial glycerophosphate dehydrogenase I. Crystallization and physical properties of the enzyme from honeybee (Apis mellifera) thoraces

Extramitochondrial glycerophosphate dehydrogenase (L-glycerol-3-phosphate: DPN+ oxidoreductase, EC 1.1.1.8) was purified and crystallized from honeybee (Apis mellifera) thoraces. The isolation procedure is quite simple, since it consists merely of a series of (NH4)2SO4 precipitations. The crystalline enzyme is homogeneous according to the following criteria: recrystallization to constant specific activity, electrophoresis on cellulose acetate strips, absence of 3 other glycolytic enzyme activities, chromatography on Sephadex G-200, and immunodiffusion on cellulose acetate strips. Sedimentation velocity studies indicate that the native protein readily associates at moderate protein concentrations. The minimum molecular weight as determined by Sephadex G-200 chromatography is around 67 000. Absorbance and fluorescence spectra show that neither DPNH nor adenosine diphosphate ribose is bound to the enzyme. The bee enzyme differs from the crystalline rabbit-muscle enzyme in electrophoretic, ultracentrifugal, and immunological properties.