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Whole-genome scan in thelytokous-laying workers of the Cape honeybee (Apis mellifera capensis): central fusion, reduced recombination rates and centromere mapping using half-tetrad analysis.

While workers of almost all subspecies of honeybee are able to lay only haploid male eggs, Apis mellifera capensis workers are able to produce diploid female eggs by thelytokous parthenogenesis. Cytological analyses have shown that during parthenogenesis, egg diploidy is restored by fusion of the two central meiotic products. This peculiarity of the Cape bee preserves two products of a single meiosis in the daughters and can be used to map centromere positions using half-tetrad analysis. In this study, we use the thelytokous progenies of A. m. capensis workers and a sample of individuals from a naturally occurring A. m. capensis thelytokous clone to map centromere position for most of the linkage groups of the honeybee. We also show that the recombination rate is reduced by >10-fold during the meiosis of A. m. capensis workers. This reduction is restricted to thelytokous parthenogenesis of capensis workers and is not observed in the meiosis of queen within the same subspecies or in arrhenotokous workers of another subspecies. The reduced rate of recombination seems to be associated with negative crossover interference. These results are discussed in relation to evolution of thelytokous parthenogenesis and maintenance of heterozygosity and female sex after thelytoky.

Animals↗

A microsatellite-based linkage map of the honeybee, Apis mellifera L.

A linkage map for the honeybee (Apis mellifera) was constructed mainly from the progeny of two hybrid queens (A. m. ligustica x A. m. mellifera). A total of 541 loci were mapped; 474 were microsatellite loci; a few were additional bands produced during PCRs, one of the two rDNA loci (using ITS), the MDH locus, and three sex-linked markers (Q and FB loci and one RAPD band). Twenty-four linkage groups were estimated of which 5 were minute (between 7.1 and 22.8 cM) and 19 were major groups (>76.5 cM). The number of major linkage groups exceeded by three the number of chromosomes of the complement (n = 16). The sum of the lengths of all linkage groups amounts to 4061 cM to which must be added at least 320 cM to link groups in excess, making a total of at least 4381 cM. The length of the largest linkage group I was 630 cM. The average density of markers was 7.5 cM and the average resolution was about one marker every 300 kb. For most of the large groups, the centromeric region was determined genetically, as described in (accompanying article in this issue), using half-tetrad analysis of thelytokous parthenogens in which diploid restoration occurs through central fusion. Several cases of segregation distortion that appreared to result from deleterious recessives were discovered. A low positive interference was also detected.

Alleles↗

Antennal malformations in light ocelli drones of Apis mellifera (Hymenoptera, Apidae).

Malformed antennae of Apis mellifera light ocelli drones were drawn, dissected and mounted permanently on slides containing Canada balsam, in order to count the olfactory discs present in each segment, in comparison with the number of those structures in normal antennae of their brothers. Some drones presented morphological abnormalities in a single segment of the right or left antenna, but others had two or more malformed segments in a same antenna. Drones with malformations in both antennae were also observed. The 4th and 5th flagellum segments were the most frequently affected. In a low number of cases the frequency of olfactory discs in malformed segments did not differ from that one recorded for normal segments. However, in most cases studied, the antennal malformations brought about a significant reduction in the number of olfactory discs from malformed segments.

Animals↗

Morphometrics and adaptatives aspects in africanized honeybees (Apis mellifera).

The introduction of the African bees (Apis mellifera scutelata) in Brazil and their expansion in the American Continent created the opportunity to study the process of species adaptation in a new environment. In that process, within intra-specific variability, normalising selection can favour individuals that present a better adaptative morphology and they will constitute the most frequent type found in the population. To test that hypothesis morphometric analyses in samples of colonies of africanized bees and in samples of the populations were performed. The development of the colonies was also evaluated in terms of the amount of their brood, honey and pollen. Analysis of the data indicates that more developed colonies are formed by individuals closer to the population average with concerning morphological traits.

Journal Article↗

Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera: Apidae) promoted by topical application of juvenile hormone.

The present study analyzed, the influence of the treatment with juvenile hormone on the ultrastructure of Apis mellifera L. workers' venom glands. Newly emerged workers received topical application of 1 microl of juvenile hormone diluted in hexane, in the concentration of 2 microg/pl. Two controls were used; one control received no treatment (group C1) and other received topical application of 1 microl of hexane (group C2). The aspect of the glandular cells, in not treated newly emerged workers, showed that they are not yet secreting actively. Cellular modifications happened according to the worker age and to the glandular area considered. The most active phase of the gland happened from the emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic, being the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

Administration, Topical↗

Nectar robbery by bees Xylocopa virginica and Apis mellifera contributes to the pollination of rabbiteye blueberry.

Honey bees, Apis mellifera L., probe for nectar from robbery slits previously made by male carpenter bees, Xylocopa virginica (L.), at the flowers of rabbiteye blueberry, Vaccinium ashei Reade. This relationship between primary nectar robbers (carpenter bees) and secondary nectar thieves (honey bees) is poorly understood but seemingly unfavorable for V. ashei pollination. We designed two studies to measure the impact of nectar robbers on V. ashei pollination. First, counting the amount of pollen on stigmas (stigmatic pollen loading) showed that nectar robbers delivered fewer blueberry tetrads per stigma after single floral visits than did our benchmark pollinator, the southeastern blueberry bee, Habropoda laboriosa (F.), a recognized effective pollinator of blueberries. Increasing numbers of floral visits by carpenter bee and honey bee robbers yielded larger stigmatic loads. As few as three robbery visits were equivalent to one legitimate visit by a pollen-collecting H. laboriosa female. More than three robbery visits per flower slightly depressed stigmatic pollen loads. In our second study, a survey of 10 commercial blueberry farms demonstrated that corolla slitting by carpenter bees (i.e., robbery) has no appreciable affect on overall V. ashei fruit set. Our observations demonstrate male carpenter bees are benign or even potentially beneficial floral visitors of V ashei. Their robbery of blueberry flowers in the southeast may attract more honey bee pollinators to the crop.

Animals↗

Effective fall treatment of Varroa jacobsoni (Acari: Varroidae) with a new formulation of formic acid in colonies of Apis mellifera (Hymenoptera: Apidae) in the northeastern United States.

New formulations of formic acid and thymol, both individually and in combination with various essential oils, were compared with Apistan to determine their efficacy as fall treatments for control of Varroa jacobsoni (Oudemans), a parasitic mite of the honey bee, Apis mellifera L. Percent mite mortality in colonies treated with 300 ml of 65% formic acid averaged 94.2 +/- 1.41% (least square means +/- SE, n = 24), equivalent to those receiving four, 10% strips of Apistan (92.6 +/- 1.79%, n = 6). Treatment with thymol (n = 24) resulted in an average mite mortality of 75.4 +/- 5.79%, significantly less than that attained with Apistan or formic acid. The addition of essential oils did not affect treatment efficacy of either formic acid or thymol. The ratio of the coefficients of variation for percentage mortality for the formic acid (CVFA) and Apistan (CVA) groups was CVFA/CVA = 0.66. This indicates that the formic acid treatment was as consistent as the Apistan treatment. Thymol treatments did not provide as consistent results as Apistan or formic acid. Coefficient variation ratios for percentage mortality for the thymol group (CVT) with the Apistan and formic acid groups were CVT/CVA = 4.47 and CVT/CVFA = 6.76, respectively. In a second experiment, colonies received a 4-wk fall treatment of either 300 ml of 65% formic acid (n = 24) or four, 10% strips of Apistan (n = 6). The next spring, mite levels in the formic acid group (554.3 +/- 150.20 mites) were similar to those in the Apistan treatment group (571.3 +/- 145.05 mites) (P = 0.93). Additionally, the quantities of bees, brood, pollen, and nectar/honey in the two treatment groups were not significantly different (P > or = 0.50 each variable). These results suggest that formic acid is an effective alternative to Apistan as a fall treatment for varroa mites in temperate climates.

Animals↗

Bombus impatiens (Hymenoptera: Apidae): an alternative to Apis mellifera (Hymenoptera: Apidae) for lowbush blueberry pollination.

The pollination effectiveness of the commercially reared bumble bee Bombus impatiens Cresson, was compared in field studies to the honey bee, Apis mellifera L., for lowbush blueberry, Vaccinium angustifolium Ait. A preliminary study indicated that B. impatiens had potential as an alternative pollinator. In a 3-yr study, percentage fruit set, percentage harvested berries, berry weight, and seeds per berry were compared in blueberry fields stocked at 7.5 A. mellifera hives per hectare to 5, 7.5, or 10 B. impatiens colonies per hectare. Percentage of harvested berries (yield) was significantly higher in fields stocked with B. impatiens at 10 colonies per hectare. No other parameters measuring pollinator effectiveness were significantly different at 5, 7.5, or 10 colonies per hectare. Flower handling time was significantly faster for B. impatiens and it more frequently collected blueberry pollen. All parameters of pollinator effectiveness were similar for B. impatiens, A. mellifera, and native wild bees in a follow-up study. Overall, B. impatiens was a suitable alternative to A. mellifera.

Animals↗

Efficacy of modified hive entrances and a bottom screen device for controlling Aethina tumida (Coleoptera: Nitidulidae) infestations in Apis mellifera (Hymenoptera: Apidae) colonies.

This study was designed to test whether hive entrances reduced with polyvinyl chloride pipe reduce the ingress of Aethina tumida Murray into Apis mellifera L. colonies and whether screen-mesh bottom boards alleviate side effects associated with restricted entrances. Forty-eight colonies distributed equally between two locations each received one of six experimental treatments: 1) conventional solid bottom board and open entrance, 2) ventilated bottom board and open entrance, 3) conventional bottom and 1.9-cm-i.d. pipe entrance, 4) conventional bottom and 3.8-cm pipe entrance, 5) screen bottom and 1.9-cm pipe entrance, and 6) screen bottom and 3.8-cm pipe entrance. Results were inconsistent between apiaries. In apiary 1, colonies with 3.8-cm pipe entrances had fewer A. tuzmida than colonies with open entrances, but this benefit was not apparent in apiary 2. Pipe entrances tended to reduce colony and brood production in both apiaries, and these losses were only partly mitigated with the addition of screened bottom boards. Pipe entrances had no measurable liability concerning colony thermoregulation. There were significantly fewer frames of adult A. mellifera in colonies with 3.8- or 1.9-cm pipe entrances compared with open entrances but more in colonies with screens. There were more frames of pollen in colonies with open or 3.8-cm pipe entrances than 1.9-cm entrances. We conclude that the efficacy of reduced hive entrances in reducing ingress of A. tumida remains uncertain due to observed differences between apiaries. Furthermore, there were side effects associated with restricted entrances that could be only partly mitigated with screened bottom boards.

Animals↗

Acute contact toxicity of oxalic acid to Varroa destructor (Acari: Varroidae) and their Apis mellifera (Hymenoptera: Apidae) hosts in laboratory bioassays.

Laboratory bioassays were performed to characterize the acute contact toxicity of oxalic acid (OA) to Varroa destructor (Anderson and Trueman) and their honey bee hosts (Apis mellifera L.). Specifically, glass-vial residual bioassays were conducted to determine the lethal concentration of OA for V. destructor, and topical applications of OA in acetone were conducted to determine the lethal dose for honey bees. The results indicate that OA has a low acute toxicity to honey bees and a high acute toxicity to mites. The toxicity data will help guide scientists in delivering optimum dosages of OA to the parasite and its host, and will be useful in making treatment recommendations. The data will also facilitate future comparisons of toxicity if mite resistance to OA becomes evident.

Animals↗

Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera.

The caste-specific regulation of vitellogenin synthesis in the honeybee represents a problem with many yet unresolved details. We carried out experiments to determine when levels of vitellogenin are first detected in hemolymph of female castes of Apis mellifera, and whether juvenile hormone and ecdysteroids modulate this process. Vitellogenin levels were measured in hemolymph using immunological techniques. We show that in both castes the appearance of vitellogenin in the hemolymph occurs during the pupal period, but the timing was different in the queen and worker. Vitellogenin appears in queens during an early phase of cuticle pigmentation approximately 60h before eclosion, while in workers the appearance of vitellogenin is more delayed, initiating in the pharate adult stage, approximately 10h before eclosion. The timing of vitellogenin appearance in both castes coincides with a slight increase in endogenous levels of juvenile hormone that occurs at the end of pupal development. The correlation between these events was corroborated by topical application of juvenile hormone. Exogenous juvenile hormone advanced the timing of vitellogenin appearance in both castes, but caste-specific differences in timing were maintained. Injection of actinomycin D prevented the response to juvenile hormone. In contrast, queen and worker pupae that were treated with ecdysone showed a delay in the appearance of vitellogenin. These data suggest that queens and workers share a common control mechanism for the timing of vitellogenin synthesis, involving an increase in juvenile hormone titers in the presence of low levels of ecdysteroids.

Acetone↗

Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera.

Imidacloprid is a systemic nitroguanidine insecticide that belongs to the neonicotinoid family. As an agonist of the acetylcholine receptor, it attacks the insect nervous system and is extremely effective against various sucking and mining pests. Oral acute and chronic toxicity of imidacloprid and its main metabolites (5-hydroxyimidacloprid, 4,5-dihydroxyimidacloprid, desnitroimidacloprid, 6-chloronicotinic acid, olefin, and urea derivative) were investigated in Apis mellifera. Acute intoxication by imidacloprid or its metabolites resulted in the rapid appearance of neurotoxicity symptoms, such as hyperresponsiveness, hyperactivity, and trembling and led to hyporesponsiveness and hypoactivity. For acute toxicity tests, bees were treated with doses of toxic compounds ranging from 1 to 1,000 ng/bee (10-10,000 microg/kg). Acute toxicity (LD50) values of imidacloprid were about 60 ng/bee (600 microg/kg) at 48 h and about 40 ng/bee (400 microg/kg) at 72 and 96 h. Out of the six imidacloprid metabolites tested, only two (5-hydroxyimidacloprid and olefin) exhibited a toxicity close to that of imidacloprid. Olefin LD50 values were lower than those of imidacloprid. The 5-hydroxyimidacloprid showed a lower toxicity than imidacloprid with a LD50 four to six times higher than that of imidacloprid. Urea also appeared as a compound of nonnegligible toxicity by eliciting close to 40% mortality at 1,000 ng/bee (10,000 microg/kg). However, no significant toxicity was observed with 4,5-dihydroxyimidacloprid, 6-chloronicotinic acid, and desnitroimidacloprid in the range of doses tested. To test chronic toxicity, worker bees were fed sucrose solutions containing 0.1, 1, and 10 microg/L of imidacloprid and its metabolites for 10 d. Fifty percent mortality was reached at approximately 8 d. Hence, considering that sucrose syrup was consumed at the mean rate of 12 microl/d and per bee, after an 8-d period the cumulated doses were approximately 0.01, 0.1, and 1 ng/bee (0.1, 1, and 10 microg/kg). Thus, all tested compounds were toxic at doses 30 to 3,000 (olefin), 60 to 6,000 (imidacloprid), 200 to 20,000 (5-OH-imidacloprid), and >1,000 to 100,000 (remaining metabolites) times lower than those required to produce the same effect in acute intoxication studies. For all products tested, bee mortality was induced only 72 h after the onset of intoxication.

Administration, Oral↗

The changes of gene expression in honeybee (Apis mellifera) brains associated with ages.

Honeybee (Apis mellifera) worker bees (workers) are known to perform wide variety of tasks depending on their ages. The worker's brains also show the activity and behavior-dependent chemical and structural plasticity. To test if there are any changes of gene expression associated with different ages in the worker brains, we compared the gene expression patterns between the brains of newly emerged bees and old foraging workers (foragers) by macroarray analysis. The expression of genes encoding signal transduction pathway components, ion channels, and neurotransmitter transporters is elevated in the old forager brains, suggesting that the neuronal activities would be enhanced. The mRNA levels of cell adhesion protein, transcription related factors, and plasma membrane associated proteins are also increased in the old forager brains. Meanwhile, the mRNA level of one putative cell adhesion protein is decreased in the old forager brains. These results thus suggest that the dramatic changes of gene expression occur in honeybee brains associated with ages.

Age Factors↗

Ultrastructural and cytochemical aspects of metamorphosis in the midgut of Apis mellifera L. (Hymenoptera: Apidae: Apinae).

The midgut of Apis mellifera is remodeled during metamorphosis. The epithelium and, to a lesser extent, the muscular sheath degenerate between the end of the last larval instar and the onset of pupation (prepupa). The larval epithelium is shed to the midgut lumen and digested, while a new epithelium is reconstructed from larval regenerative cells. During pupation, some reorganization still occurs, mainly in brown-eyed pupae. In pharate adult, the midgut wall shows the characteristics of adult, although some cells have pycnotic nuclei. The localization of alkaline and acid phosphatases showed that these enzymes were not involved in the reabsorption of the midgut wall.

Animals↗

Modification of the AFLP protocol applied to honey bee (Apis mellifera L.) DNA.

The established amplified fragment-length polymorphism (AFLP) protocol was simplified and optimized for honey bee DNA (Apis mellifera L.). Compared to the original method, the following simplifications were made: (i) the digestion of DNA and ligation of the adapters are performed in one reaction vs. two, (ii) one restriction enzyme is used vs. two and (iii) amplification is accomplished in one reaction vs. two. PCR products are resolved in agarose-Synergel instead of polyacrylamide and are visualized by ethidium bromide staining rather than by autoradiography of labeled primers. Using the modified procedure for honey bee DNA, high reproducibility of the band patterns of PCR products and low sensitivity to the amplification conditions were seen. Analysis of honey bee DNA revealed considerable genetic variability within and between African and European bee samples. African- and European-specific fragments were found.

Animals↗

Classical conditioning of proboscis extension in harnessed Africanized honey bee queens (Apis mellifera L.).

Experiments are reported on learning in virgin Africanized honey bee queens (Apis mellifera L.). Queens restrained in a "Pavlovian harness" received a pairing of hexanal odor with a 1.8-M feeding of sucrose solution. Compared to explicitly unpaired controls, acquisition was rapid in reaching about 90%. Acquisition was also rapid in queens receiving an unconditioned stimulus of "bee candy" or an unconditioned stimulus administered by worker bees. During extinction the conditioned response declines. The steepest decline was observed in queens receiving an unconditioned stimulus of bee candy. These findings extend previous work on learning of Afrianized honey bee workers to a population of queen bees.

Animals↗

Isolation and characterization of major royal jelly cDNAs and proteins of the honey bee (Apis cerana).

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts > 500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of alpha-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.

Amino Acid Sequence↗

Cloning, expression and genomic organization of genes encoding major royal jelly protein 1 and 2 of the honey bee (Apis cerana).

Major Royal Jelly Protein cDNAs of Apis cerana (AcMRJP) were cloned and characterized. The open reading frames (ORFs) of the AcMRJP1 and AcMRJP2 genes were 1302 and 1392 nucleotides, encoding 433 and 463 amino acid residues, respectively. The sequence divergences between AcMRJP1 and AcMRJP2 and their corresponding protein families in A. mellifera were 0.0618 and 0.0934 at the nucleotide level and 0.0912 and 0.1438 at the protein level, respectively. Phylogenetic analysis supports the orthologous similarity between these proteins. The deduced amino acids indicated high essential amino acid contents of AcMRJP1 and AcMRJP2 (47.5 and 44.8 %, respectively). The genomic organization of both AcMRJP1 and AcMRJP2 was determined. Both the AcMRJP1 (3663 bp) and AcMRJP2 (3963 bp) genes contained six exons and five introns, where all boundaries conformed to the GT/AG rule. AcMRJP1 and AcMRJP2 cDNAs were cloned into pET17b, and both the recombinant (r) AcMRJP1 (47.9 kDa) and rAcMRJP2 (51.7 kDa) were expressed in the insoluble form. Western blot analysis and N-terminal sequencing of the solubilized proteins revealed successful expression of rAcMRJP1 and rAcMRJP2 in vitro. The yields of the purified rAcMRJP1 and rAcMRJP2 were approximately 20 and 8 mg protein per liter of the flask culture, respectively.

Amino Acid Sequence↗