Determination of Pseudomonas aeruginosa susceptibility to aminoglycosides using API and Autobac semi-automated systems.
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Aminoglycosides↗
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Detection of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci with the semi-automated API (ATB-plus) system.
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Methicillin↗
Analysis of cocaine, benzoylecgonine, ecogonine methyl ester, and ecgonine by high-pressure liquid chromatography-API mass spectrometry and application to a short-term degradation study of cocaine in plasma.
A method for the determination of cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and ecgonine (ECG) in plasma by liquid chromatography-mass spectrometry (LC-MS-MS) was developed. The analytes were isolated from human plasma by subsequent solid-phase extraction and were separated on a Zorbax Eclipse XDB-C8 narrow-bore column using an ammonium acetate buffer/acetonitrile/methanol gradient. A Turbolonspray source was used for ionization. The analytes were characterized by their particular molecular ion and several fragments. Multiple reaction monitoring (MRM) was used for isolation and quantitation. The assay was rapid, highly sensitive, and reliable. The method was applied to monitor the in vitro degradation of cocaine in plasma. Fresh unpreserved and preserved (0.25% KF) plasma samples were spiked with 1,000 ng cocaine/mL. Aliquots of both series were stored at 4 degrees C and 20 degrees C and were analyzed at selected storage times of up to 15 days. In all samples, degradation of cocaine that was dependent on storage time and temperature and on sample preservation could be observed. The formation of BE did not occur to a significant extent (< 12%, referred to the initial concentration of COC), and its concentration was slightly higher in preserved compared with unpreserved plasma at both storage temperatures chosen. EME was formed in considerably higher amounts compared to BE. As already observed for COC, its formation and degradation were dependent on storage time, temperature, and preservation. EME is suggested to be the major source of ECG, which was detectable in all samples after 1-2 days of storage. Although the degradation of COC was shown to be highly dynamic in nature, the sum of all hydrolysis products of COC accounted for the initial COC concentration at any particular time of storage. Therefore, production of hitherto unknown degradation products of COC seems unlikely. Moreover, the common transformation product of BE and EME appeared to be stable, and ECG is suggested as a promising postcollection artifact.
Artifacts↗
Genetic dissection of honeybee (Apis mellifera L.) foraging behavior.
We demonstrate the effects of a new quantitative trait locus (QTL), designated pln3, that was mapped in a backcross population derived from strains of bees selected for the amount of pollen they store in combs. We independently confirmed pln3 by demonstrating its effects on individual foraging behavior, as we did previously for QTLs pln1 and pln2 (Hunt et al. 1995). QTL pln2 is very robust in its effects on foraging behavior. In this study, pln2 was again shown to affect individual foraging behavior of workers derived from a hybrid backcross of the selected strains. In addition, pln2 was shown to affect the amount of pollen stored in combs of colonies derived from a wide cross of European and Africanized honeybees. This is noteworthy because it demonstrates that we can map QTLs for behavior in interstrain crosses derived from selective breeding and study their effects in unselected, natural populations. The results we present also demonstrate the repeatability of finding QTLs with measurable effects, even after outcrossing selected strains, suggesting that there is a relatively small subset of QTLs with major effects segregating in the population from which we selected our founding breeding populations. The different QTLs, pln1, pln2, and pln3, appear to have different effects, revealing the complex genetic architecture of honeybee foraging behavior.
Animals↗
Transcriptional profiling reveals multifunctional roles for transferrin in the honeybee, Apis mellifera.
Transferrins belong to a family of iron-binding proteins that have been implicated in innate immunity and in vitellogenesis in insects. Here we have sequenced and characterized a full-length cDNA encoding a putative iron-binding transferrin (AmTRF) in the honeybee. AmTRF shows high level of sequence identity with transferrins in both vertebrates and insects (26-46%) suggesting that the primary function of the predicted 712 amino acid protein is binding and transporting of iron. AmTRF is expressed ubiquitously, but particularly high levels of its mRNA are found in the central brain and in the compound eye. Using northern blotting and a microarray based approach we have examined the levels of AmTRF mRNA by expression profiling under a wide range of conditions including developmental stages, septic injury and juvenile hormone treatment. Increased expression of AmTRF is seen during early pupal stages, in the brain of mature foragers and in the abdomen of virgin queens, whereas treatment with juvenile hormone leads to a decrease of AmTRF levels in the abdomen. We show that a transcriptional response of transferrin to septic injury with E. coli is relatively moderate as compared to a dramatic up-regulation of an antibacterial polypeptide, Hymenoptaecin, under similar conditions. We conclude that major fluctuations of AmTRF mRNA in time and space are consistent with context-dependent functional significance and suggest broader multifunctional roles for transferrin in insects.
Amino Acid Sequence↗
Effects of carbon dioxide narcosis on ovary activation and gene expression in worker honeybees, Apis mellifera.
In an effort to uncover genes associated with ovary activation in honey bee workers, the extent to which eight candidate genes co-varied in their expression with experimentally-induced changes in worker reproductive state was examined. Groups of caged, queenless workers narcotized with CO(2) on consecutive days early in adult life showed a significantly lower level of ovary activation than did groups of untreated workers. This same experimental treatment, by contrast, is known to accelerate ovary activation and induce egg laying in virgin honey bee queens--an observation that suggests that CO(2) narcosis has contrasting effects in queen versus worker ovary activation. Experimentally-induced changes to worker reproductive state were associated with changes in gene expression. Vitellogenin, an egg yolk precursor, and transferrin, an iron transporter, were two transcripts found to be significantly down-regulated as a function of the ovary-inhibiting treatment. CO(2) narcosis did not effect the expression of six other genes selected as putative markers for processes that may underlie ovary activation. The show that the expression of vitellogenin and transferrin is correlated with ovary activation in workers, and may therefore be part of the gene network involved in the regulatory control of functional sterility in honeybees.
Animals↗
Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role.
The roles of adult worker honeybees change with age; young workers nurse brood by secreting bee milk (royal jelly), and older workers forage for nectar and pollen and process nectar into honey. The electrophoretic profile of worker hypopharyngeal-gland proteins changes with age and/or role. Immunoblotting analysis using affinity-purified antibodies against three major proteins (50, 56, and 64 kDa) of the nurse-bee gland showed that they are synthesized selectively and secreted as bee-milk proteins. Immunofluorescence study showed that the proteins condense in the duct after secretion from acini. However, a major 70-kDa protein synthesized specifically in the forager-bee hypopharyngeal gland was identified as an alpha-glucosidase. Therefore, the hypopharyngeal gland seems to have two distinct states differentiated by synthesizing of different major proteins depending on the age-dependent role change.
Aging↗
Laranja--an additional eye color gene in the snow series of Apis mellifera L.
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Alleles↗
Size homoplasy and mutational processes of interrupted microsatellites in two bee species, Apis mellifera and Bombus terrestris (Apidae).
Similar microsatellite electromorphs (PCR products of the same size) can arise from independent mutational events. Such alleles are not identical by descent. This phenomenon, termed size homoplasy, was studied by sequencing electromorphs of two microsatellite loci in which the stretch of basic repeats is interrupted by different short (1-2 bp) DNA motifs. The number and position of these interruptions were established for electromorphs from closely and distantly related populations of honeybees and bumblebees. No sequence difference was found when electromorphs came from the same subspecies or from closely related subspecies, suggesting that they were probably identical by descent. In contrast, sequence differences were often detected in distantly related subspecies, showing that size homoplasy frequently occurs at this level of population differentiation. Size homoplasy is increased by limits to free length variation of alleles, a phenomenon that seems to act on interrupted microsatellites when comparing distantly related taxa, that is, honeybee subspecies from different evolutionary lineages. Electromorph sequences suggest that, within the scope of these limits, large mutation events have occurred frequently at both interrupted loci studied. In good agreement with the molecular data, computations based on the observed heterozygosity and number of electromorphs and simulation studies showed that neither locus fits the one-step stepwise mutant model (SMM). We speculate that interrupted microsatellites in general could be characterized by a higher variance in repeat number and consequently a lower homoplasy rate than pure ones. Hence, interrupted microsatellites should be most appropriate for investigating population differentiation and evolutionary relationship between relatively distant populations.
Alleles↗
The effects of age on olfactory learning and memory in the honey bee Apis mellifera.
THE proboscis extension reflex (PER) has been used extensively in studies of bee learning. This paper reports the effect of age of bees on conditioning of the response. An initial experiment investigated acquisition and retention of PER conditioning in bees emerged from a sealed brood in a laboratory incubator. Reliable acquisition and retention of the learning was not achieved until bees were 6 days of age. With comparable learning and memory to mature forager bees only at day 10. In a second study, bees from the same colonies were reared in a full hive colony, and a much slower development of this behaviour was seen. The results suggest that the behavioural role of the bee in the hive colony is as important as brain maturation for olfactory learning. This was explored further by producing precocious learning abilities in young (3-day-old) bees by preventing their normal nurse bee activities. The results are discussed in terms of the anatomical maturation of the bee nervous system and behavioral demands on the olfactory system throughout adult development.
Aging↗
Honeybee workers (Apis mellifera capensis) compete for producing queen-like pheromone signals.
Physical fights are the usual means of establishing dominance hierarchies in animal societies. This form of dominance behaviour is most strongly expressed in honeybee queens who engage in fights to the death to establish themselves in the colony. Workers can also compete for reproductive dominance resulting in the establishment of stable hierarchies. They do not engage each other physically, but use pheromones that mimic those produced by queens. The dynamics of pheromone production in paired workers suggests that they engage in a pheromonal contest. Because queen pheromones suppress ovary activation, the contest results in the sterility of the loser.
Animals↗
A possible pitfall in the identification of Pasteurella spp. with the API system.
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Culture Media↗
Misidentification of Brucella melitensis as Ochrobactrum anthropi by API 20NE.
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Bacteremia↗
RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor).
Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.
Abdomen↗
Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera).
The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of two large protein families, G protein-coupled olfactory receptors (ORs) and odorant binding proteins (OBPs). In olfactory sensilla, OBPs deliver hydrophobic airborne molecules to ORs, but their expression in nonolfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. Here we used bioinformatic and experimental approaches to characterize the OBP-like gene family in a highly social insect, the Western honey bee. Comparison with other insects shows that the honey bee has the smallest set of these genes, consisting of only 21 OBPs. This number stands in stark contrast to the more than 70 OBPs in Anopheles gambiae and 51 in Drosophila melanogaster. In the honey bee as in the two dipterans, these genes are organized in clusters. We show that the evolution of their structure involved frequent intron losses. We describe a monophyletic subfamily of OBPs where the diversification of some amino acids appears to have been accelerated by positive selection. Expression profiling under a wide range of conditions shows that in the honey bee only nine OBPs are antenna-specific. The remaining genes are expressed either ubiquitously or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs are not restricted to olfaction and are likely to be involved in broader physiological functions.
Amino Acid Sequence↗
Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock.
The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.
Amino Acid Motifs↗
The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape.
We examined the possible role of hybridization in the invasion process of the African honeybee by testing two hypotheses regarding fluctuating asymmetry (FA), a measure of developmental stability, in wing characteristics: (1) FA should be higher in hybrid versus parental genotypes of African and European races; (2) FA should be lower in African bees compared to hybrid and European workers. Parental and reciprocal hybrid worker genotypes were cross fostered in common-hive rearing environments. We did not find greater FA for wing size and shape in the hybrids compared to both parental types. However, we did find significantly lower FA of shape in the African workers compared to the European and hybrid workers, suggesting that European bees and their hybrids may have compromised fitness relative to African bees. We also found that the two hybrid genotypes significantly differed in overall wing size and shape. If these differences affect wing aerodynamics, then the paternity of hybrids may influence worker performance and could potentially contribute to the loss of European matrilines. Hybridization had few consistent effects on directional asymmetry for wing size and shape. Genotypic factors played a far greater role in determining the effect of hybridization on wing morphology than did differences in rearing environment. Thus, African bees may have lower FA for wing shape (and by inference greater developmental stability) relative to European and hybrid workers, which may contribute to the ability of African bees to displace European honeybee races in invaded regions.
Africa↗
Expression of insulin pathway genes during the period of caste determination in the honey bee, Apis mellifera.
Female honeybees have two castes, queens and workers. Developmental fate is determined by larval diet. Coding sequences made available through the Honey Bee Genome Sequencing Consortium allow for a pathway-based approach to understanding caste determination. We examined the expression of several genes of the insulin signalling pathway, which is central to regulation of growth based on nutrition. We found one insulin-like peptide expressed at very high levels in queen but not worker larvae. Also, the gene for an insulin receptor was expressed at higher levels in queen larvae during the 2nd larval instar. These results demonstrate that the insulin pathway is a compelling candidate for pursing the relationship between diet and downstream signals involved in caste determination and differentiation.
Animals↗