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PubMed1998

Arginine kinase is highly expressed in the compound eye of the honey bee, Apis mellifera.

We have cloned and sequenced a 1.68-kb cDNA encoding arginine kinase in the honey bee, Apis mellifera. The predicted protein shows a high level of identity to known arginine kinases in invertebrates and to other proteins belonging to the conserved family of ATP: guanidino phospho-transferases. The pattern of expression of arginine kinase has been investigated for the first time in various tissues including the brain, antennae and compound eye. Our results show that three isoforms of arginine kinase, transcribed from a single gene, are expressed in a characteristic pattern in major tissues of the honey bee. Arginine kinase mRNA is relatively abundant in the central nervous system and in the antennae. However, the highest level of expression, that is at least two to three times higher than in the brain, is found in the compound eye of the bee. By contrast, the levels of mRNAs encoding another metabolically important enzyme, alpha-glycerolphosphate dehydrogenase (alpha-GPDH), are low in the eye. These findings suggest that arginine kinase is an important component of the energy releasing mechanism in the visual system that has high and fluctuating energy demands. Furthermore, our results support the role of phosphagen kinases in energy transport in polarised cells and are consistent with the role of arginine kinase as an energy shuttle that delivers ATP generated by mitochondria to high energy-requiring processes, such as massive membrane turnover and pigment regeneration in the retina.

Amino Acid Sequence↗

PubMed2000

In vitro biotransformation of the new antipsychotic agent, RWJ-46344 in rat hepatic S9 fraction: API-MS/MS/MS identification of metabolites.

The in vitro biotransformation of the antipsychotic agent, RWJ-46344 was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-46344 (approximately 37% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS/MS data. The proposed metabolic pathways for RWJ-46344 are proposed, and the six metabolic pathways are 1, O-dealkylation; 2, piperidinyl oxidation; 3, N-debenzylation; 4, phenyl hydroxylation; 5, dehydration; and 6, reduction. Pathways 1 to 3 formed O-desisopropyl RWJ-46344 (M3, approximately 13% of the sample) and its hydroxy-metabolite (M5, approximately 8%), hydroxypiperidinyl RWJ-46344 (M1, approximately 5%) and a phenylpiperidinyl metabolite (M8, approximately 24%) as major and moderate metabolites. Eight minor metabolites (each < 2%) were formed via a combination of six steps. RWJ-46344 is metabolized substantially by this rat hepatic system.

Animals↗

PubMed2002

The in vitro metabolism of thalicarpine, an aporphine-benzyltetrahydroisoquinoline alkaloid, in the rat. API-MS/MS identification of thalicarpine and metabolites.

The in vitro metabolism of an antitumor, hypotensive, and antimicrobial aporphine-benzyltetrahydroisoquinoline alkaloid, thalicarpine was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged thalicarpine (46% of the sample) plus eight metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS/MS data. The proposed metabolic pathways for thalicarpine are proposed, and the three metabolic pathways are: (1) N-demethylation; (2) aporphine ring oxidation; and (3) benzylic oxidation/reduction. Pathway 1 formed N-desmethyl thalicarpine (M1, 6%). Pathway 2 produced three minor keto/hydroxy metabolites (M2-M4, each 2-7%). Pathway 3 formed a major (M6, 28%) and three minor (M5, M7 and M8, each 2-3%) benzylic-cleavage metabolites. Thalicarpine is substantially metabolized by this rat hepatic system.

Animals↗

PubMed2003

In vitro metabolism of the new anxiolytic agent, RWJ-52763 in human hepatic S9 fraction-API-MS/MS identification of metabolites.

The in vitro metabolism of the anxiolytic agent, RWJ-52763 was studied after incubation with human hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-52763 (64% of the sample) plus six metabolites (M1-M6) were profiled, quantified, and tentatively identified on the basis of API-MS/MS data. The metabolic pathways for RWJ-52763 are proposed, and the two metabolic pathways are: (1) N/O-dealkylation, and (2) phenylhydroxylation. Pathway 1 formed a major N-dealkylated metabolite, N-desethoxy-RWJ-52763 (M1, 22% of the sample) and 2 minor N/O-dealkylated metabolites, O-desmethyl-RWJ-52763 (M2; 2%) and N,N-didesethoxymethyl-RWJ-52763 (M3; 3%). Pathway 2 produced two hydroxyphenyl metabolites, hydroxydifluorophenyl-RWJ-52763 (M4; 4%) and hydroxyphenyl-pyrido-RWJ-52763 (M5; 3%) in small amounts, and in conjunction with step 1 formed a minor N-desethoxymethyl-M4 (M6; 1%). RWJ-52763 is substantially metabolized by this human hepatic S9.

Anti-Anxiety Agents↗

PubMed1993

Antimicrobial susceptibility testing of Actinomyces pyogenes: comparison of disk diffusion test and Api ATB Strep system with the agar dilution method.

The use of the disk diffusion and the Api ATB Strep system or a related technique for a few antibiotics was compared with the agar dilution method for antimicrobial susceptibility testing of 103 clinical isolates of Actinomyces pyogenes. There was complete agreement between disk diffusion and MIC in 98.8%, with minor errors, in 0.7% and with very large errors, in 0.5% of cases. The common criteria for the interpretation of zones could be kept for penicillin G, amoxicillin, methicillin, cephalothin, erythromycin, lincomycin, pristinamycin, vancomycin, rifampin, streptomycin, kanamycin, gentamicin and chloramphenicol. The proposed breakpoints for a tetracycline 30 IU disk were > or = 22 mm for susceptibility and < 22 mm for resistance. There was complete agreement between the ATB strip system and MIC in 98.3% of cases, with minor errors in 0.9% and with very large errors, in 0.8% of cases. All the dissimilarities were due to streptomycin and tetracycline and results could not be reported for these 2 antibiotics when using this system. Wells with kanamycin and gentamicin at low concentrations as well as chloramphenicol should be included by the manufacturer.

Actinomyces↗

PubMed1993

Antibiogram typing of methicillin-resistant Staphylococcus aureus: a comparison with phage typing, biotyping and API Staph.

68 strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MRSA) have been characterized by four different methods. First, by their production of lecithinase, lipase, pigment and sheep haemolysin. Second, by API Staph code. Third, by their sensitivity to 9 antibiotics. Fourth, by phage typing using the International Set and Supplementary phages. The third method was the most discriminatory. The combination of the first three techniques provides a highly effective, cheap and simple system to type MRSA. 80 separate MRSA strains from 26 countries were found to belong to a wide variety of phage types. Most were of group III. The most commonly found types were 85 (6 strains), 84 (4 strains) and 47 (3 strains).

Bacterial Typing Techniques↗

PubMed2002

Utilization of waste materials for outdoor wintering of Apis mellifera L. colonies.

The studies were conducted for two years during 1996-1997 and 1997-1998 to evaluate the impact of different waste materials wheat bhusa (chopped wheat straw), paddy straw, sawdust and thermocole sheet (a type of polystyrene board) as inner packing, and in combination with polythene sheets as outer packing, on the brood rearing activity of honeybee (Apis mellifera L.) colonies. The increase in brood area showed that thermocole alone as well as in combination with polythene sheets was superior to all other treatments; followed by paddy straw, wheat bhusa and saw dust, respectively. Considering the economic feasibility and easy availability, the paddy straw packing is recommended for outdoor wintering of honeybee colonies.

Animals↗

PubMedDate not supplied

Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L. ): developmental changes in sensitivity to JH precursor and allatotropin.

Juvenile hormone (JH) is considered the prime endogenous signal for the induction of queen development in honey bees (Apis mellifera L.). At the beginning of the last (5th) larval stadium, worker corpora allata synthesize less JH than queen corpora allata as a consequence of a limited production of JH precursors and a caste- and stage-specific block of the terminal step in JH biosynthesis. As previously shown, the Manduca sexta allatotropin stimulates JH biosynthesis in honey bee corpora allata in a dose-dependent and reversible manner, but can not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for worker early 5th instars. In experiments with M. sexta allatotropin and with the JH precursor farnesoic acid, we found characteristic stage-specific differences in their effects on JH biosynthesis. From the end of the spinning stage on, corpora allata could be stimulated by farnesoic acid to a much higher extent than in earlier developmental stages, suggesting a sudden increase in epoxidase activity. Manduca sexta allatotropin, however, stimulated corpora allata activity until the end of the spinning stage, at which time the corpora allata become suddenly insensitive. These data suggest that in worker larvae, important changes in the regulation of the terminal enzymatic steps in JH biosynthesis occur at the transition from the spinning stage to the prepupal stage. However, the analysis of in vitro activities of the involved enzymes, O-methyltransferase and methyl farnesoate epoxidase, remained inconclusive.

Animals↗

PubMed2001

Variations in chemical mimicry by the ectoparasitic mite Varroa jacobsoni according to the developmental stage of the host honey-bee Apis mellifera.

The ectoparasitic mite Varroa jacobsoni poses a major threat to the survival of European honey-bee populations. Development of effective control methods is therefore much needed. Study of interspecific chemical communication between the parasite and host is a particularly promising avenue of research. Previous study has shown that the cuticular hydrocarbons of the parasite mite Varroa jacobsoni are qualitatively identical to those of its honey-bee host Apis mellifera (Nation J.L., Sanford M.T., Milne K., 1992. Cuticular hydrocarbons from Varroa jacobsoni. Experimental and Applied Acarology 16, 331-344). The purpose of the present study was to compare the cuticular hydrocarbon patterns of the two species at different stages of bee development. Cuticular components were identified by gas chromatography/mass spectrometry. The proportion of each component was calculated at three stages of bee development (larvae, pupa, emerging bee). The degree of chemical mimicry between the parasite and host was evaluated by multivariate analyses using the resulting proportions for each category of individuals. There were four main findings. The first was that the proportions of some components are different at the larval, pupal and imago stage of bee development. Second, Varroa profiles vary depending on the developmental stage of the host. Third, the cuticular profile of adult mites is more similar to that of the stage of the host than that of later and/or earlier stages except for parasites collected from emerging adult bees. Fourth, the degree of mimicry by Varroa is greater during larval and pupal stages than during the emerging adult bee stages. The role of chemical mimicry - although it is not perfect - in enabling parasites to infest bee colonies by the parasite is discussed.

Animals↗

PubMed2002

Purification and characterization of beta-glucosidase from honey bees (Apis mellifera).

beta-glucosidase has been purified from the ventriculus and honey sac of Apis mellifera using a combination of anion- and cation-exchange, hydroxyapatite and gel-permeation chromatography. In addition, beta-glucosidase from the hypopharyngeal glands has been partially purified using anion-exchange and gel-permeation chromatography. The purified beta-glucosidase gave a positive result by glycoprotein staining. This beta-glucosidase consists of only one subunit and has M(r) of 72 kDa as determined by SDS-PAGE. IEF-PAGE showed several bands with pIs ranging from 4.5 to 4.8. These multiform proteins have been proposed as having different degrees of glycosylation. The pH optimum of the purified beta-glucosidase from the ventriculus and honey sac are 5.0. These enzymes were stable at temperatures up to 50 degrees C and have a relatively wide pH stability range of 4.0 to 9.0. MALDI-TOF-MS peptide mass maps of purified beta-glucosidase from the ventriculus, honey sac and hypopharyngeal glands showed six matching masses. These results indicate that the beta-glucosidase isolated from the hypopharyngeal glands, honey sac and ventriculus is the same. It is proposed that beta-glucosidase is produced in the hypopharyngeal glands, secreted into the mouth during feeding and then passes to the honey sac. From the honey sac, this enzyme is transferred into honeycomb cells and the ventriculus.

Animals↗

PubMed2003

The vitellogenin of the honey bee, Apis mellifera: structural analysis of the cDNA and expression studies.

The cDNA of Apis mellifera vitellogenin was cloned and sequenced. It is 5440 bp long and contains an ORF of 1770 amino acids (including a putative signal peptide of 16 residues). The deduced amino acid sequence shows significant similarity with other hymenopteran vitellogenins (58% with Pimpla nipponica and 54% with Athalia rosae). The alignment with 19 insect vitellogenins shows a high number of conserved motifs; for example, close to the C-terminus there is a GL/ICG motif followed by nine cysteines, as occurs in all hymenopteran species, and, as in other insect vitellogenins, a DGXR motif is located 18 residues upstream the GL/ICG motif. Phylogenetic analysis of vitellogenin sequences available in insects gave a tree that is congruent with the currently accepted insect phylogenetic schemes. Using two fragments of the vitellogenin cDNA as probes, we analyzed by Northern blot the sex- and caste-specific patterns of vitellogenin expression in pupae and adults of A. mellifera. In queens, vitellogenin mRNA was first detected in mid-late pupal stage, whereas in workers it was first detected in late pupal stage. Vitellogenin mRNA was also observed in drones, although it was first detected not in pupae but in freshly molted adults.

Amino Acid Sequence↗

PubMedDate not supplied

Identification and developmental profiles of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera.

Four distinct hexamerin subunits (referred to as "hexamerins" in the following text) have been identified in the developing honeybee, Apis mellifera, by N-terminal protein sequencing. Hexamerins are abundant in the hemolymph of late larval and early pupal stages, and gradually decline during metamorphosis and adult development. Three hexamerins in the 70 kDa range have been found (Hex70a, Hex70b, Hex70c). In worker and drone, Hex70a is the only hexamerin present in large amount in later adult stages. Hex70b and c exhibit a similar developmental profile, disappearing in the drone just before adult emergence, and in the worker just after. Hex70b or Hex70c are still detectable in the adult queen. Hex80/110 likely exist in at least 3 different subunits, 1 of 110 kDa, and 2 of around 80 kDa, which all share a common N-terminus. They disappear during metamorphosis earlier than Hex70b and c. All these hexamerins have been found also in the antenna, suggesting their utilization in building up of antennal cuticle structures.

Amino Acid Sequence↗

PubMed1999

Molecular characterization of MRJP3, highly polymorphic protein of honeybee (Apis mellifera) royal jelly.

Major proteins of honey bee (Apis mellifera) royal jelly are members of the MRJP protein family. One MRJP protein termed MRJP3 exhibits a size polymorphism as detected by SDS-PAGE. In this report we show that polymorphism of the MRJP3 protein is a consequence of the polymorphism of a region with a variable number of tandem repeats (VNTR) located at the C-terminal part of the MRJP3 coding region. We present the characterization of five polymorphic alleles of MRJP3 by DNA sequencing. By PCR analyses, at least 10 alleles of distinct sizes were found in randomly sampled bees. Studies with nurse bees from a single honeybee colony revealed both Mendelian inheritance and very high variability of the MRJP3 genomic locus. The high variability and simple detection of the MRJP3 polymorphism may be useful for genotyping of individuals in studies of the honeybee.

Alleles↗

PubMed2002

Ferritin in iron containing granules from the fat body of the honeybees Apis mellifera and Scaptotrigona postica.

It is already known that the behaviour of the honeybee Apis mellifera is influenced by the Earth's magnetic field. Recently it has been proposed that iron-rich granules found inside the fat body cells of this honeybee had small magnetite crystals that were responsible for this behaviour. In the present work, we studied the iron containing granules from queens of two species of honeybees (A. mellifera and Scaptotrigona postica) by electron microscopy methods in order to clarify this point. The granules were found inside rough endoplasmic reticulum cisternae. Energy dispersive X-ray analysis of granules from A. mellifera showed the presence of iron, phosphorus and calcium. The same analysis performed on the granules of S. postica also indicated the presence of these elements along with the additional element magnesium. The granules of A. mellifera were composed of apoferritin-like particles in the periphery while in the core, clusters of organised particles resembling holoferritin were seen. The larger and more mineralised granules of S. postica presented structures resembling ferritin cores in the periphery, and smaller electron dense particles inside the bulk. Electron spectroscopic images of the granules from A. mellifera showed that iron, oxygen and phosphorus were co-localised in the ferritin-like deposits. These results indicate that the iron-rich granules of these honeybees are formed by accumulation of ferritin and its degraded forms together with elements present inside the rough endoplasmic reticulum, such as phosphorus, calcium and magnesium. It is suggested that the high level of phosphate in the milieu would prevent the crystallisation of iron oxides in these structures, making very unlikely their participation in magnetoreception mechanisms. They are most probably involved in iron homeostasis.

Animals↗

PubMed2001

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.

Abdomen↗

PubMed2003

Triazines facilitate neurotransmitter release of synaptic terminals located in hearts of frog (Rana ridibunda) and honeybee (Apis mellifera) and in the ventral nerve cord of a beetle (Tenebrio molitor).

Three triazine herbicides, atrazine, simazine and metribuzine, and some of their major metabolites (cyanuric acid and 6-azauracil) were investigated for their action on synaptic terminals using three different isolated tissue preparations from the atria of the frog, Rana ridibunda, the heart of the honeybee, Apis mellifera macedonica, and the ventral nerve cord of the beetle, Tenebrio molitor. The results indicate that triazines facilitate the release of neurotransmitters from nerve terminals, as already reported for the mammalian central nervous system. The no observed effect concentration, the maximum concentration of the herbicide diluted in the saline that has no effect on the physiological properties of the isolated tissue, was estimated for each individual preparation. According to their relative potency, the three triazines tested can be ranked as follows: atrazine (cyanuric acid), simazine>metribuzine (6-azauracil). The action of these compounds on the cholinergic (amphibians, insects), adrenergic (amphibian) and octopaminergic (insects) synaptic terminals is discussed.

Animals↗

PubMed2001

Modifications of the cuticular hydrocarbon profile of Apis mellifera worker bees in the presence of the ectoparasitic mite Varroa jacobsoni in brood cells.

Varroa jacobsoni is an ectoparasite of Apis mellifera which invades brood cells, on 8-day-old larvae several hours before cell capping. Reproduction of the parasite takes place in the capped brood cells during the nymphose of the bee. Cuticular hydrocarbons of unparasitized bees and of bees parasitized by Varroa jacobsoni were extracted and analysed by gas chromatography (GC) coupled with mass spectrometry (GC-MS). Three developmental stages of worker honey bees were studied: larvae, pupae and emergent adults. The comparison between unparasitized and parasitized hosts was performed with Principal Components Analysis coupled with a multivariate variance analysis. The cuticular hydrocarbon profiles of honey bees were qualitatively similar, for the 3 developmental stages and regardless of the presence of Varroa in the cells. Nevertheless, comparison of the relative proportions of hydrocarbons showed that the cuticular profiles of pupae and emergent adults parasitized by 1 mite and of larvae parasitized by 2 mites were significantly different from the corresponding unparasitized individuals. Such modifications could be regarded (i) as a cause of the multi-infestation in larvae during invasion of brood and (ii) as a consequence of stress and/or removal of proteins contained in the haemolymph of the host during its development.

Animals↗

PubMed2006

Effects of parasitization by Varroa destructor on survivorship and physiological traits of Apis mellifera in correlation with viral incidence and microbial challenge.

Varroa mites (Varroa destructor) are serious ectoparasites of honey bees (Apis mellifera). This research addresses the impact of varroa mites on survivorship, viral incidence, and physiological traits of newly-emerged worker bees. RT-PCR confirmed our previous finding that varroa parasitization was linked to high levels of deformed wing virus (DWV). In non-treatment bees, varroa parasitization combined with increased viral levels altered survivorship curves from long-survival to shorter-survival types. After challenge with live Escherichia coli, the survivorship of mite-parasitized bees was significantly lower than mite-free bees. Deformed-wing, mite-parasitized bees died on average within 1 day, even without E. coli challenge. This was correlated with the absence of an important enzyme activity in insect immunity, phenol oxidase, lacking even in those bees challenged with immuno-elicitors. The lack of inducible phenol oxidase activity indicated that the bee immune system is not fully competent upon adult emergence. Varroa parasitism also significantly reduced body weight of the parasitized bees, but body weight was not significantly correlated with the survivorship of mite-parasitized bees. Our research indicates that the combination of mite parasitization, the interaction of DWV and microbes, and a developmental immune incompetency attribute to decreased worker survivorship and have a negative impact on colony fitness.

Animals↗