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Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony.
Citation: Richard F-J, Tarpy DR, Grozinger CM (2007) Effects of Insemination Quantity on Honey Bee Queen Physiology. PLoS ONE 2(10): e980. doi:10.1371/journal.pone.0000980
Academic Editor: Robert Brooks, The University of New South Wales, Australia
Received: July 17, 2007; Accepted: September 12, 2007; Published: October 3, 2007
Copyright: © 2007 Richard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a USDA-NRI grant to CMG and DRT (2006-35607-16625) and the Eastern Apiculture Society.
Competing interests: The authors have declared that no competing interests exist.
While many previous studies have considered the effects of mating on the physiology and behavior of female insects (i.e., reference 1), the effects of mating number and semen quantity have not been broadly considered. Honey bees are an excellent system in which to study this process: colonies typically consist of a single reproductive queen and thousands of sterile worker bees, and the queen mates with several males (12 males on average; reviewed in reference 2) during a brief period in her early adult life. However, studies of the effects of mating number have focused almost exclusively on the consequences of worker genetic diversity on overall colony health [3]–[5] and social interactions [6]. Relatively little is known about how queen mating number may alter queen physiology and thereby alter intracolony social interactions. Understanding the physiological effects of multiple mating could offer insights into the mechanisms that govern mating behavior and how the process is regulated in honey bees and female insects in general, as well as what consequences this may have for colony organization in bees and other social insects.
Virgin honey bee queens initiate mating very early in their lives, when they are approximately 1–2 weeks old, by taking multiple mating flights and mating with numerous males (drones) on each flight. On average, queens are successfully inseminated by 12 males (based on molecular genotyping of workers [2]), but mating number is highly variable among queens (range from 1 to 28). The factors that determine mating frequency are not fully understood [7], [8]. Mating has profound and permanent effects on queen behavior, physiology, and resultant queen-worker interactions. Once they begin to oviposit, mated queens will never mate again and will remain in the colony for the rest of their lives (unless they depart during colony swarming). Mating stimulates vitellogenesis and oocyte-maturation in the ovaries [9], which prompts the initiation of egg-laying of up to 1500 eggs/day [10]. There are also profound changes in a queen's brain after mating, where levels of dopamine significantly decrease [11] and the ratio of the neuropil/cell body volume in the mushroom bodies significantly increases [12].
Finally, mated and virgin queens differ dramatically in their pheromone profiles, and these pheromones are important for regulating colony organization and worker behaviour [13], [14]. Queens produce pheromones from a variety of glands, and the complete "queen pheromone" has not been fully characterized [15]–[17]. However, five components in particular, termed "queen mandibular pheromone" or QMP, are produced by the mandibular gland and elicit many similar worker behavioral and physiological responses as a live queen. QMP consists of 9-oxo-(E)-2-decenoic acid (ODA), (R)- and (S)-9-hydroxy-(E)-2-decenoic acid (9-HDA), methyl p-hydroxybenzoate (HOB), and 4-hydroxy-3-methyoxyphenylethanol (HVA) [18]. QMP has both primer (long-term) and releaser (short-term) effects on worker behaviour and physiology. It elicits a "retinue response", in which workers are attracted from a distance (several cm) and then antennate and groom the queen [18]–[21]. QMP also inhibits worker ovary activation [22], [23], inhibits queen cell production by workers [24], [25], stimulates pollen foraging and brood rearing in small, newly founded colonies [26], increases nectar foraging [27], [28], and delays the age-at-onset of foraging and reduces juvenile hormone secretion [27]. Some components of QMP are involved in drone attraction and mating [29], [30]. However, while the five component QMP blend is a potent modulator of worker behaviour and physiology, it is still not as potent as a live queen, suggesting that additional pheromonal components may exist. Indeed, four additional compounds were identified that synergize with QMP to increase the retinue response, but even those nine compounds do not elicit as robust a response as a live queen [17].
Although it is clear that mandibular gland pheromone profiles differ dramatically between young virgins and naturally mated, laying queens, the short-term effects of mating on pheromone profiles are largely unknown. Plettner et al. [14] compared the quantities of the QMP components between 6-day-old virgins and 1-year-old mated laying queens, and found that mated queens had significantly higher levels of 9-ODA, 9-HDA, HOB and HVA than did virgins. However, a similar study by Slessor et al [13], which compared 6- and 12-day-old virgins with mated queens that had been laying for 1 day, 5 weeks, or 2 years, found different results. In that study, levels of 9-ODA were not significantly different between any of these groups, 9-HDA levels were significantly higher in mated versus virgin queens, HOB levels were significantly higher in the 2-year-old mated queens than in both the virgins and 1-day laying queens and intermediate in the 5-week laying queens, and HVA levels were significantly higher in the 2-year mated queens compared to all other groups. Thus, pheromone profiles are strongly affected by mating, age (or perhaps time since mating), and potentially by egg-laying. Finally, Apsegaite and Skirkevicius [31] found slight differences in the quantity of 9-ODA between different strains of honey bees, suggesting that genotypic differences might also alter pheromone profiles.
Given the highly variable mating number and the profound effects mating has on queens, we hypothesize that mating number could modulate queen physiology, behavior, and pheromone production. However, "mating number" comprises a host of factors, including the number and duration of mating flights, the physical act of insemination, the volume of ejaculate, the quantity and viability of sperm and seminal proteins in the ejaculate, and the genetic diversity of sperm and seminal proteins. Furthermore, given that queens mate during flights several meters above the ground, it is not possible to control natural mating behavior. However, the number of males that inseminate a queen can be precisely controlled by using instrumental insemination [32]. Previous studies have demonstrated that queens inseminated with lower quantities of semen (less than 8 µL) are more likely to continue to take mating flights [33], but the effects of insemination quantity on other aspects of queen physiology have not been considered. To begin to address these questions, we focused on single-drone inseminated (SDI, total semen from one drone) and multi-drone inseminated (MDI, total semen from 10 drones) queens. We monitored attraction of the workers to SDI or MDI queens in observation hive colonies, as well as the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts in cages. The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, since fully inseminated queens cease to be phototactic and no longer take mating flights, we measured brain expression levels in SDI and MDI queens of a gene that is associated with phototaxis in worker honey bees (Amfor, the foraging gene; [34].
Behavioral assays
Observation hives.
One of the most measurable effects of queen pheromone is the induced retinue response, in which workers are attracted to the queen from a short distance, and lick and antennate her. Following insemination, the retinue response to SDI and MDI queens was monitored in observation hives, twice a day for 5 days. MDI queens attracted significantly more worker bees in their retinue than the SDI queens (F = 6.73, p = 0.02; Figure 1). There was no significant effect of day (F = 0.46, p = 0.76) or day*treatment interaction (F = 0.68, p = 0.61).
Following insemination, the retinue response to SDI and MDI queens was monitored in observation hives, twice a day for 5 days. MDI queens attracted significantly more worker bees in their retinue than the SDI queens did (F = 6.73, p = 0.02). (SDI: 7 queens; MDI: 5 queens).
Cage assays.
Worker retinue responses to the mandibular gland extracts of virgin, SDI, and MDI queens were tested in cages containing 4-day-old bees. The retinue size was equivalent whether workers were exposed to virgin, SDI, or MDI queen mandibular gland extracts (H(2,18) = 0.615 p = 0.735; data not shown). However, worker bees exposed to two different mandibular gland extracts at the same time preferred gland extracts from SDI and MDI queens to virgins (respectively: t = 4.2, df = 36, p<0.001 and t = 3.1, df = 38, p<0.01, Figure 2), and preferred MDI extracts to SDI extracts (t = 2.7, df = 40, p<0.01). Data for the preference assay represent two trials which were not significantly different and were pooled for the subsequent analysis.
Caged workers were exposed to mandibular gland extract of two different queens simultaneously, and the retinue response to each extract was monitored. Workers were significantly more attracted to the extracts of inseminated versus virgin queens, and significantly more attracted to the extracts of MDI versus SDI queens (0.05 queen equivalents: Virgin vs SDI queens (n = 19); Virgin vs MDI (n = 20). MDI vs SDI (n = 21); all comparison with t-test: t<2.7, p<0.01). ** p<0.01 and *** p<0.001
Chemical analysis of queen mandibular gland profile
The chemical composition of the mandibular gland extract of virgin, SDI, and MDI queens were analysed using gas chromatography. A total of 27 compounds were apparent in the GC analysis and used in the subsequent comparisons. There were significant differences in the overall chemical profiles between the three groups of queens, as revealed by a discriminant analysis (F(33, 36) = 9.33, p<10−4; Figure 3). Two discriminant variables explained 100% of the variation. The chemical profiles of the virgin queen mandibular glands were most different from the two groups of mated queens (85% of the variation), and the other 15% of the variation could be attributed to insemination quantity.
Chemical composition of the mandibular gland extracts of Virgin, SDI and MDI queens were analysed using gas-chromatography. Discriminant analysis of mandibular extract of virgin, SDI, and MDI queens was based on the relative proportion of the chemical compounds (F(33, 36) = 9.33. p<10−4; All group distances <0.005). Ellipses have been drawn to emphasize the categories, but have no specific statistical meaning
Next, when the actual quantities of the compounds are analyzed (Table 1), mated queens had significantly lower levels of 18 compounds relative to virgins, and higher levels of one compound. MDI queens had significantly lower levels of seven compounds than SDI queens, and significantly higher levels of two compounds. Of the five QMP components, the quantities of 9-ODA, 9-HDA, and HVA were all significantly lower in mated queens compared to virgins. Levels of 9-ODA and 9-HDA were also significantly lower in the MDI queens than in the SDI queens.
Finally, we compared the relative proportions (compared to the total gland quantity) of the 27 individual compounds between virgin and mated queens, and between SDI and MDI queens (Table 2). The relative proportion of 13 compounds was significantly different between virgins and mated queens, and 10 of these compounds were significantly higher in mated queen mandibular glands. There were fewer compounds with significantly different relative proportions between SDI and MDI queens. Two compounds (8-hydroxyoctanoic acid and unidentified 5) were present in significantly higher proportions in SDI queens than in MDI queens (Table 2), while six compounds (4-hydroxybenzoic acid, unidentified 2, (E)-dec-2-enedioic acid, palmitic acid, alkane 2 and stearic acid) were significantly higher in MDI than in SDI queens mandibular glands (Table 2). Of the QMP components, relative levels of 9-ODA were significantly lower in mated queens compared to virgins, while HOB levels were significantly higher. None of the QMP components differed between SDI and MDI queens.
Brain gene expression levels of Amfor
We used quantitative real-time PCR to measure Amfor expression levels in the queens' brains to determine if insemination treatment altered brain gene expression and therefore presumably altered neuronal properties in the brain. Amfor expression was significantly higher in SDI queens than in MDI queens (U = 10, p = 0.02; Figure 4).
Expression levels of Amfor were monitored using quantitative real-time PCR using individual brains (SDI: N = 9; MDI: N = 9). SDI queens have significantly higher levels of Amfor than MDI queens (U = 10, p = 0.022). Data represent mean values, −/+ standard error (converted to the same arbitrary scale as the mean), relative to the SDI brains.
In this study, we instrumentally inseminated queen honey bees to determine if insemination quantity alters their physiology and social interactions under controlled environmental conditions. Our results clearly demonstrate that insemination quantity alters queen physiology, queen pheromone profiles, and queen-worker interactions. MDI queens elicited a stronger retinue than SDI queens in natural colony conditions, and their mandibular gland extracts were more attractive in preference assays with caged worker bees. Moreover, GC-MS analysis of the mandibular gland chemical profiles revealed significant differences between SDI and MDI queens. Finally, brain-expression levels of a behaviorally relevant gene were significantly different between the SDI and MDI queens. All of these results suggest that insemination quantity can have profound effects on queen physiology and behaviour.
Comparisons of the mandibular gland profiles between MDI and SDI queens may lead to the identification of new components of queen pheromone. Previously, studies focusing on compounds that elicited a retinue response identified the five-component QMP blend [18]. Another study identified four additional compounds that did not elicit a retinue on their own, but synergized with QMP to produce a stronger retinue response [17]. However, the retinue response was still not as strong as that elicited by the live queens, suggesting that additional active compounds may still exist. In this study, we found that MDI queens elicited a larger retinue than SDI queens in observation hive colonies. Since the mandibular glands are the main source of QMP, we tested the mandibular gland extracts of virgin, SDI, and MDI queens on worker retinue responses in cages. When tested individually, the glands were equally attractive, though all glands are qualitatively comparable and contained the main QMP compounds [18]. However, when the glands were compared in a choice test, there were clear differences. Indeed, the glands of the inseminated queens were more attractive than gland of virgins, while the gland extracts of MDI queens were more attractive than gland of SDI queens. This suggests that MDI queen mandibular gland extracts have additional compounds that synergize with 9-ODA and 9-HDA to make these extracts more attractive in a preference assay and that the relative proportions of the entire gland chemical profile affect the retinue response. Indeed, when the extracts were analyzed with GC-MS, the chemical profiles of the mandibular glands of the inseminated queens were significantly different from those of the virgins, and those of the MDI queens were significantly different from those of the SDI queens.
Our results suggest that these changes in pheromone profiles occur within days after insemination, and that additional changes occur over a long period of time. Previous studies have compared the queen mandibular gland compounds between 6-day-old virgin and 1-year-old mated laying queens, and mated queens queens had higher levels of 9-ODA, 9-HDA, HOB, HVA, and 10-HDAA, and lower levels of 10-HDA [14]. However, when virgins and mated queens of more similar ages were compared, the differences were less robust [13]. In particular, 12-day-old virgins and mated queens that were laying for only 1 day did not differ significantly between levels of 9-ODA, 9-HDA, HOB, or HVA. In our study, virgins and inseminated queens were age-matched, and all queens were collected 5 days after insemination and were not laying eggs. However, we still observed significant differences in the overall chemical profiles and in several individual compounds, suggesting that insemination quantity causes changes in pheromone production within days. Thus, our results suggest that changes in mandibular gland profiles occur immediately or shortly after insemination, but additional modifications occur over time that may be associated with age, egg laying, or both. Furthermore, the observation hives assays with live queens and the cage studies with queen mandibular gland extract demonstrate that workers can detect these differences in pheromone profiles and respond differently to virgins versus newly inseminated queens, and singly versus multiply inseminated queens.
Expression levels of a gene associated with phototaxis are also significantly altered by insemination quantity, suggesting that insemination quantity exerts effects of neuronal properties in the brain and possibly behavior. We focused our analysis on brain expression levels of the foraging gene (Amfor; GenBank Accession AF469010). Previous studies found that expression of Amfor is significantly higher in forager bees than in-hive bees [35]. Moreover, Amfor encodes a cGMP-dependent protein kinase (PKG) and treatment with a cGMP analog stimulates foraging behavior [35] and increases phototaxis [36]. Our results demonstrate that there are significant differences in gene expression levels in brains of SDI and MDI queens, and these differences may be associated with changes in flight and phototaxis, though behavioural studies are clearly necessary to determine if this is the case. However, previous work demonstrates that queens inseminated with lower quantities of semen (<8 µL) have an increased likelihood to attempt mating flights [37].
Clearly, insemination quantity has profound effects on queens, but what are the mechanisms by which insemination quantity triggers these changes in queen honey bees? As noted previously, a number of factors might play a role. The number of stored sperm may modulate queen post-mating physiology and behavior [38]. It is highly unlikely that queens can directly assess the number of sperm in their spermathecae since there is no nerve enervation into the spermathecae and the membranes of their spermathecae are rigid [39], [40], so there is likely to be some other mechanism—one that is possibly correlated with stored sperm number—that serves as the basis for these physiological and behavioral changes. One possible explanation is that insemination volume may be the cue that triggers these changes. As queens are inseminated with numerous drones, the large quantity of semen is temporarily stored in their lateral and median oviducts for several hours before a proportion (5–10%) of each male's sperm migrates into the spermathecae and the excess semen is excreted. Indeed, preliminary studies (Richard, Tarpy, Grozinger, unpublished data) suggest insemination volume may be a key contributor to the observed post-insemination changes. One possible mechanism to induce these changes is that queens may have stretch receptors in their oviducts that provide a negative stimulus for additional mating behavior. Similar mechanisms exist in other insect systems [41]–[43], and abdomen distension has been tested in honey bees [44], but it remains unclear if abdominal enlargement is a physiological mechanism for reproductive senescence in queen bees. Alternatively, queens may use a substance in their mates' ejaculate, such as a hormonal precursor or seminal protein that causes physiological changes and decreases their subsequent reproductive behavior. Male seminal proteins have shown to have significant inhibitory effects on mating in female Drosophila [45]–[52] and possibly bumble bees [53], [54], and several substances in drone honey bee ejaculate have been identified, including sugars, metal ions, and proteins [55]–[57]. Further studies will be necessary to determine which factor(s) is involved in causing the differences in queen pheromone profile and brain gene expression that we have observed between SDI and MDI queens.
Previous studies have demonstrated that there may be colony-level adaptive benefits for genetically diverse workers, in terms of increased resistance to disease [5], [58], homozygosity at the sex determination locus [59], or better regulation of colony division of labor [60], [61]. Here, we demonstrate that insemination quantity, and thereby potentially mating number, could also affect different aspects of queen physiology or quality that could affect colony fitness. Additional research will be necessary to see if the physiological and pheromonal differences of differentially inseminated or mated queens are used by the workers as honest signals of their insemination quality or fertility [62]–[64].
Materials and Methods
Bee rearing
Colonies headed by single-drone inseminated queens (Apis mellifera carnica, Glenn Apiaries, CA) were maintained at the NCSU Lake Wheeler Honey Bee Research Facility (Raleigh, NC). These colonies were used as genetic sources for producing supersister queens for the experiments. Due to the haplodiploid genome of the hymenoptera, the progeny of SDI-queens have a genetic relatedness of G = 0.75. Additionally, colonies headed by naturally mated Apis mellifera ligustica or Buckfast-SMR queens (B. Weaver, TX) were used to provide workers for the observation-hive and cage experiments. Note that while worker bees were derived from different source colonies for convenience, each individual experiment used workers from a single colony when testing differential responses to SDI or MDI queens and/or pheromone extract. Thus, though there might be differences between colonies in worker responses to queen pheromones (i.e., reference 65), this should not be a factor in our results.
Queen rearing
Supersister queens were produced by grafting young larvae (<24 h) from a single source colony and reared as queens in a queenless colony [32]. Once all queen cells were capped, they were transferred to a dark incubator at 33°C and ∼40% relative humidity. One to two days prior to emergence, each capped queen cell was placed into an individual Plexiglas cage (10×10×7 cm). Frames of brood were removed from a hive and incubated at 33°C. Day-old workers were collected from the brood frames and 100 adult worker bees were placed in the cages with the capped queen cell. Bees were provided with water, food (45% honey/45% pollen/10% water), and a solution of 50% sucrose, and all cages were maintained in an incubator (at 33°C and ∼40% RH). The food was changed every two days. Five days after emergence, queens destined for insemination were treated with CO2 for 4.0 minutes [66], while virgins were handled but left untreated, since we wanted a baseline nonreproductive control group and CO2 treatment accelerates the transition to egg-laying [66]. Seven days after emergence, queens were again treated with CO2 for 4.0 minutes, during which time they were inseminated with semen from either one drone (single-drone inseminated treatment group, or SDI) or 10 brother drones (multi-drone inseminated treatment group, or MDI) by following standard insemination protocols which readily produce laying queens [32], though in our experiments queens were reared in cages without honeycomb, so there was no opportunities for them to lay eggs. The average drone produces approximately 1 µl of semen, thus SDI queens were inseminated with approximately 1 µl and MDI queens were inseminated with approximately 10 µl. Queens were then returned to their respective cages and collected 5 days after insemination onto dry ice and stored at 80°C. A total of 12 virgin, 15 SDI and 11 MDI queens were collected. Queens were reared in three separate cohorts (Cohorts 1∶ 5 SDI and 4 MDI; Cohorts 2∶ 5 SDI and 2 MDI and Cohorts 3∶ 12 virgin, 5 SDI and 5 MDI), but queens from different cohorts were combined for the subsequent chemical analysis and behavioural assays.
Prior to dissection, queen heads were removed and partially lyophilized to facilitate dissection [67], after which their mandibular glands and brains were dissected out on dry ice and stored for future processing. In order to verify insemination success, we counted the number of sperm in each spermatheca (SDI, N = 15 and MDI, N = 8). As would be expected, spermathecae of MDI queens (65*104±25*104 sperm) contained significantly more sperm than those of SDI queens (15*104±8*104 sperm; U = 20.0, p = 0.0098). Note that these quantities of sperm are lower than typical for inseminated queens, but the lack of comb, the presence of a small number of workers, and the small size of the cages may have minimize sperm storage [38].
Behavioral assays
Observation hives.
Supersister queens were produced as described above. Capped queen cells were removed from the queen-rearing colony and individually placed into 1.5 frame observation hives (34″×5″×21.5″ LxWxH). The entrance to each hive was blocked with queen excluder material so the queens could not exit the hives to take mating flights. Observation hives were stocked with ∼1000 one-day-old bees and a half-frame of honey/pollen. The observation hives were stored in a 33°C incubator for 3 days (to ensure that workers were old enough to properly thermoregulate) prior to being placed in a room with access to the outside via a window. As the queens emerged, individual plastic numbered tags (Thorne Ltd, UK) were glued to their thoraxes. Approximately five days after emergence, queens were captured and treated with carbon dioxide for 4.0 minutes. Two days later, queens were captured again and treated with carbon dioxide for 4.0 minutes during their insemination procedure. As outlined above, queens were inseminated with semen from either a single drone (single-drone inseminated, SDI) or 10 brother drones (multi-drone inseminated, MDI). Queens were then released back into their respective hives. One hour later, the number of worker bees in the queen's retinue (surrounding the queen, licking and antennating her) was counted every minute over a period of 5.0 minutes [17], [18]. One hour later, the number of workers contacting the queen was counted again. The retinue size was calculated by summing across all of the observations, as in previous studies [17], [68]. The retinue was monitored every day, twice a day, for five days. In total, 2 SDI and 2 MDI queens were assayed in 2005 and 5 SDI and 3 MDI in 2006.
Differences in the retinue size for SDI and MDI queens in observation hives were analyzed using a mixed-model ANOVA (PROC mixed in SAS, Cary, NC), with treatment and day viewed as fixed factors, while trial, trial*treatment and replicate (trial*treatment) were random effects. Thus 'day' was treated as a split plot factor.
Cage assays.
We next compared worker responses to mandibular gland extracts of virgin, SDI, and MDI queens. The glands were extracted from each supersister queen produced as outlined above.
Frames of brood were removed from a hive and incubated at 33°C. Day-old workers were collected from the brood frames and 35 bees were placed in Plexiglas cages (10×10×7 cm). Bees were provisioned as described above. The cages were kept in a 33°C incubator with ∼40% relative humidity, and manipulations and observations were performed under red light to negate any potential behavioural effects. Cages were maintained for five days.
Experiment 1: Retinue response
The retinue response was monitored as described previously [17], [21], [68], [69]. Extracts of mandibular glands of individual queens were produced as described below (see chemical analysis section). 0.05 queen equivalents (Qeq) of queen mandibular gland extract from individual queens (Virgin: n = 3; SDI: n = 8; MDI: n = 7) was placed onto a glass coverslip, the solvent was allowed to evaporate, and the coverslip was then placed inside the cage at the same time every day for five days. The number of bees antennating and licking the coverslip was counted in each cage ("retinue"). The retinue was recorded 5, 10, and 15 minutes after pheromone introduction each day of the 5-day time course. A single trial was performed.
Experiment 2: Preference assay
This experiment was repeated twice (Trial 1: Fall 2005 and Trial 2: Spring 2006). Bees were exposed to 0.1 Qeq of synthetic QMP (Pherotech, Canada). Every day, 10 µl of QMP was placed on a microscope slide and allowed to evaporate before being placed in the cage. This amount of QMP mimics a live queen in assays of worker behaviour and physiology [70]. Since worker maturation is altered in the absence of queen pheromone [67], [68], [71], we reared these workers with QMP to mimic natural colony conditions. On the fifth day of the experiment, workers were presented with two slides containing equal quantities of extract (0.05 Qeq) from either a virgin and SDI queen (Trial 1: n = 9 and Trial 2: n = 10), a virgin and MDI queen (Trial 1: n = 10 and Trial 2: n = 10), or a SDI and MDI queen (Trial 1: n = 11 and Trial 2: n = 10). The number of workers contacting each slide was counted every 5.0 minutes for 15 minutes after slide presentation. Note that a total of 6 virgin, 6 SDI, and 6 MDI queens were used in this analysis. Queens were derived from 2 cohorts. Trial 1 and trial 2 were not significantly different, so the data were pooled for the subsequent analysis.
Data are presented as mean±SEM. The effect of the mated number was evaluated with a non-parametric Kruskall-Wallis ANOVA on ranks for global comparison. The worker preferences between two slides were evaluated with a parametric t-test.
Chemical analysis
The mandibular glands were dissected and immersed in 50 µl of diethyl ether containing 0.4 µg/µl of undec-10-enoic acid (as an internal standard) for minimum of 24 hours. A 5 µl portion (approx. 0.1 bee equivalents) of an extract was placed in a small glass insert and the solvent gently evaporated. The residue was sylylated overnight at room temperature in the insert with 10 µl of neat bistrimethylsilyltrifluoroacetamide (BSTFA) [17]. The derivatized sample was diluted with hexane (100 µl) and a 2 µl portion was analyzed using gas chromatography on a HP 5890 equipped with capillary column (30 m×0.25 mm ID. 0.5 µm film thickness) DB-5 (5% diphenyl-95% dimethylsiloxane) column (J&W scientific, Folsom, CA) in splitless mode. Helium was used as the carrier gas at a head pressure of 18 psi (flow rate = 1.3 ml/min). The GC temperature was held at 100°C for 1 min and then increased at 5°C/min to 200°C (5 min), followed by an increase of 10°C/min to 250°C (15 min). Injector and FID temperatures were both set at 250°C. We extracted the mandibular gland of 12 virgin queens, 15 SDI queens and 10 MDI queens. All queens used for chemical analysis were raised from the same grafting source to reduce any genetic variation in their pheromone profiles. However, the queens were reared in three different cohorts.
Compound identification was achieved by splitless capillary gas-chromatography-mass spectrometry using a Hewlett-Packard 6890 GC and a model 5973A msd with an electron impact ion source and a HP-5ms capillary column (30 m×0.25 mm ID×0.25 µm film thickness).
To examine differences in profiles related to number of inseminations based on the relative proportion of the chemical compounds, a stepwise discriminant analysis was employed, using all the chemical compounds (Statistica 6.0. StatSoft® Inc.). A MANOVA test was also used to compare treatment effects (queen insemination quantity). As the MANOVA test was significant, we compared each compound using a univariate analysis on the relative proportion for each compound. Both parametric and non-parametric tests were used for individual component comparisons, and both tests revealed comparable statistic p value. Here, we present the results from the Mann-Whitney test.
The following abbreviations are used for chemicals found in the analysis: methyl p-hydroxybenzoate (HOB), 8-hydroxyoctanoitic acid (8-HOAA), 4-hydroxy-3-methoxyphenylethanol (HVA), (E)-9-oxodec-2-enoic acid (ODA), (E)-9-hydroxydec-2-enoic acid (9-HDA), 10-hydroxydecanoic acid (10-HDAA), (E)-10-hydroxydec-2-enoic acid (10-HDA), decanedioic acid (C10∶0 DA) and (E)-dec-2-enedioic acid (C10∶1 DA)
Brain gene expression levels of Amfor
Total RNA was isolated from dissected brains using a RNeasy RNA extraction kit (Qiagen, Valencia CA), yielding 0.6-1.5 µg/brain. cDNA was synthesized from 150 ng RNA using Arrayscript reverse transcriptase (Ambion, CA). Expression levels of Amfor were measured using quantitative real-time RT-PCR (qRT-PCR) with an ABI Prism 7900 sequence detector and the SYBR green detection method (Applied Biosystems, Foster City, CA). eIF3-S8, a housekeeping gene that did not vary in expression levels in previous bee brain microarray studies [67], [72], was used as a loading control. For each sample, triplicate qRT-PCR reactions were performed and averaged. A standard curve was generated for each primer using dilutions of genomic DNA to calculate the relative quantities of mRNA levels for each sample. A dissociation curve and negative control (cDNA reaction without RT-enzyme) were used to ensure primer specificity and lack of contamination.
The sequences for the primers (5′ to 3′) used are as follows:
Amfor -F: AATATAACTTCCGGTGCAACGTATT;
Amfor -R: CGTTTGGATCACGGAAGAAAG;
eIFS8-F: TGAGTGTCTGCTATGGATTGCAA;
eIFS8-R: TCGCGGCTCGTGGTAAA;
We evaluated the brain gene-expression levels of 9 SDI queens and 9 MDI queens (derived from two cohorts). For each individual brain sample, the ratio of the expression level of Amfor to that of the control gene (eIF3-S8) was calculated.
We would like to thank Coby Schal and Michael Roe for use of their GC and GC/MS instruments, Cavell Brownie for her advice on the statistical analyses, Josh Summers, Jennifer Keller, and Joe Flowers for their beekeeping assistance, Kelly Hutcherson and Cynthia Rouf for their assistance in the field. This experiment complies with the laws of the USA.
Author Contributions
Conceived and designed the experiments: CG FR DT. Performed the experiments: FR DT. Analyzed the data: CG FR. Contributed reagents/materials/analysis tools: CG DT. Wrote the paper: CG FR DT.
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text:
By Pontotoc Progress
PONTOTOC – Mayor Jeff Stafford has ordered a two-hour parking limit downtown.
The signs should be in the city and posted within the next week to 10 days.
Parking in the lot on the south side of the library has been especially hard because it has been used by students, rather than them using the lot provided behind the school.
The mayor said another handicapped parking space added in the lot for those who can't walk very far.
Once the signs are installed, officers will mark the tires and if a car is still in a two-hour parking zone, the owner will be fined. Fine amounts are yet to be determined.
Click here for more news in Pontotoc at Pontotoc360.com.

subset: commoncrawl


text: Nursing Excellence At Providence Health & Services, nursing excellence reflects cutting edge quality, innovation and compassion in all that we do. It means not only do our patients receive outstanding care, but also that nurses are actively engaged in professional development and advancing the profession itself to improve the health of all that we serve. Our nurses work in clinical teams to develop innovative care improvement strategies such as: Leading interdisciplinary care teams to partner with patients through their care journey Striving to protect our vulnerable patients through eliminating health care-acquired conditions such as falls, pressure ulcers and infections Developing early warning systems that alert care teams to intervene rapidly with subtle changes in a patient's condition indicating decline. Our nurses are leading and building a nationally-recognized Providence palliative care program available to every patient who needs it, wherever they are. Active nursing research programs are in place in many of our ministries and regions, in pursuit of scientific evidence to support better patient care outcomes Unique to our system, Providence nurses can advance their nursing education by participating in an innovative, distance-based RN-to-bachelor of science in nursing program offered through the University of Great Falls, our Providence-sponsored university. Our nurses live the Providence mission in service to their communities and through international outreach, contributing their knowledge, time and skill in many countries around the world. This past year, Providence has forged a deep and sustaining partnership with nurses and nurse leaders in Guatemala to improve the health of the country itself. Our system-wide plan for nursing excellence is reflected in our commitment to support all eligible ministries to achieve national Magnet® or Pathway to Excellence® recognition. These rigorous, nationally-accredited programs are the gold standard for recognizing outstanding highest quality in both nursing care and nursing workforce. Being a nurse requires a special calling. Being a Providence nurse is the greatest calling of all. I am so very proud of each of our 20,000-plus nurses, working around the clock in every community we serve. We are here for you, your family, each other and our communities. I am honored to serve with this talented and dedicated Providence caring community. What a privilege to declare, "I am a Providence nurse." Deborah A. Burton, Ph.D., RN, CNAA Vice President, Chief Nursing Officer


text: NATIONAL JOINT COMMITTEE FOR SPANISH RELIEF BASQUE CHILDREN'S COMMITTEE Minutes of Joint Meeting of the two Committees held on Tuesday, November 23rd, 1937, in Room 16, House of Commons, at 5.0 p.m. PRESENT: The Duchess of Atholl, M.P. (in the Chair) Miss Eleanor Rathbone, M.P. The Earl of Listowel Mr. Tewson Mr. Wilfrid Roberts, M.P. ) Capt. J.R.J. Macnamara, M.P.) Mr. D.R. Grenfell M.P. ) Mr. G.T. Garratt (Hon. Administrator) Miss B.L. Arne (Organising Secretary) Bilbao Delegation: Dame Janet Campbell Dr. Norman White Mrs. M. Gee Miss M. Denner Representatives of the following organisations: British Youth Peace Assembly, Committee against Malnutrition, Friends Service Council, League of Nations Union (County Hall Branch), International Committee for Relief of Victimised Teachers, Peace Pledge Union, Salvation Army, Save the Children Fund, Spanish Medical Aid Committee, Spanish Women's Committee for Help to Spain, Trades Union Congress Individual Members: Mr. Henry Brinton Lady Layton Mrs. Wilfrid Roberts Dr. Audrey Russell Miss P. Smith Miss Ellen Wilkinson Mrs. Wood Representative of the Basque Delegation in London: Senor Don Miguel Uranga


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The drunk-driving case of Darrell Williams, the vice president of business development for The Roane Alliance, was resolved Monday in General Sessions Court.
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Public Officials Warned About Taking Former L.A. Mayor's Advice
by Jon Melegrito and Allison Padgett | December 04, 2012
Los Angeles millionaire and former Mayor Richard Riordan got a lot of things wrong when he was running the city from 1993 to 2001 and his threat to put a draconian pension-cutting initiative on the ballot is another wrong idea that doesn't deserve a second chance.
In a recent blog posted by Madeline Janis, the former L.A. Community Redevelopment Agency commissioner said that Riordan's threat prompted the City Council last month to hastily adopt its own pension-cutting plan, "a plan that almost certainly will be thrown out by the courts as a violation of existing collective bargaining agreements." Riordan's ballot initiative would move newly hired workers into 401(k)-style plans and freeze retirement benefits for current workers.
But a funny thing happened the other day after trying to gather the required 300,000 signatures. Unable to meet a Dec. 28 deadline, the whole ballot measure abruptly collapsed. Apparently, there aren't enough people willing to support Riordan's initiative. In recent weeks, city workers and community supporters have attacked the proposal with public protests and e-mail blasts.
During his term of office, Riordan blamed public sector unions for every woe facing the region. "Even worse, Riordan vigorously opposed several city laws designed to give workers and communities the benefit of city investment in economic development," adds Janis. "Richard Riordan got a lot of things wrong when he was mayor. Current elected officials should be wary about taking the former mayor's advice today."
Or any day for that matter.
Previous: Illinois Governor Terminates State Contract, AFSCME Responds


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Current and new Last Minute Travel Club members enjoy 5,000 complementary TripleDip points as of a few days ago. If you are not a member of the Last Minute Travel Club yet, head over to www.LMTClub.com and sign up for an account. The membership fee is $49.95 per year and you will enjoy savings up to 65% on over 13,000 hotels worldwide.
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text:
Valuing Women's Unwaged Work
by Phoebe Jones Schellenberg Chair of Philadelphia Women Count Working Group
The International Women Count Network (IWCN) grassroots delegation to the UN Fourth World Conference on Women in Beijing succeeded in getting governments to agree to measure and value unwaged work. This issue is important to all women because for the first time there would be a true measure of what it really takes for a society to function. It would also give everyone, policy makers and men and women themselves, a measure of how much inequality has to be eliminated. It would allow for realistic evaluations of how much cutbacks in the U.S. and other industrialized countries - and "structural adjustment programs*" in non-industrialized countries - are costing women and communities.
To find out more and to get involved, contact:
IWCN P.O. Box 11795 Philadelphia, PA 19101 Voice: (610) 668-9886; Fax: (610) 664-8556 E-mail: [email protected]
(International and other addresses in the U.S. are available on request).
*Belt tightening imposed by the International Monetary Fund and World Bank on debtor nations seeking loans.
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Infuse your wardrobe with personality with this 'Thea' Tunic/Dress from White Mark. This shift dress features beautiful crochet lace hemline and natural waist.
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text:
Last week, World Food Day emphasized 'sustainable' in agriculture and food systems. One interesting element of the day was how the discussion spread beyond the development community, in many cases demonstrating how necessary breaking down silos really is. Case in point, Landscapes for People, Food and Nature Co-Organizer Conservation International (CI) took this opportunity to highlight some of its ongoing work in Madagascar. By bringing in a spatial element to examine how land uses are influencing the provision of ecosystem services in a landscape, CI expects its systems approach to conservation, agriculture, and sustainable development to provide tools and stimulate discussion in the policy realm. The following is an excerpt from a post by Rachel Neugarten, manager of conservation priority setting, on the HumanNature Blog.
Madagascar is a land of rice. To understand the relationship between people and nature here, that's the first thing you need to know.
Malagasy people eat more rice per capita than most countries in the world. In Malagasy, friendship is referred to as "rice and water"; perfection is "rice with milk and honey." I once saw a map that showed how agricultural wages varied across the country, and the units were kilos of rice, not money.
Everything about this place — land use, water use, income and poverty, nutrition and food security, deforestation and habitat loss — can be measured in terms of rice.
The second thing you have to know is that Madagascar is hungry … and growing. Over 90% of people live on less than the international poverty rate of US$ 2 per day. Rates of chronic malnutrition are so high that half the children under five have stunted growth. And the population is growing at an astounding 2.8% per year, twice as fast as India.
The people are literally eating their country — stripping the land bare, sifting every last fish from the surrounding sea. Madagascar has lost 90% of its native vegetation cover already, making it one of the most threatened landscapes on Earth.
The most common way to grow rice is tavy — slash-and-burn agriculture. Tavy leaves the land naked. When it rains, the red soil leaches into the rivers, and people say that Madagascar is bleeding. When a cyclone hits — and they hit several times a year — massive flooding drowns entire valleys, destroys rice paddies and homes, and takes lives. Survivors retreat to higher ground and wait for the waters to recede, so they can rebuild and replant.
Climate change is making the cyclones more intense, while also making rainfall more unpredictable. So people may face catastrophic floods one year and crippling droughts the next.
The final thing you have to know about Madagascar is, of course, that it is like no other place on Earth. There are chameleons the size of beetles and bats the size of housecats. The islands off its shores were once home to the dodo, and "fossil" fish known as coelacanth still lurk in the deepest waters. It seems new species — even new primates — are discovered every time a biologist stumbles into the forest.
It has been estimated that over 90% of the species of plants, amphibians and terrestrial mammals found here are found nowhere else. If they disappear, they will be gone from the face of the planet. One local biologist told me, "We aren't saving these species; we are archiving them."
It is against this backdrop of rice, hunger and cyclones that CI is attempting to map out — literally — the links between nature and people in Madagascar. It is one of many efforts to remind people at all levels — local communities, civil society, public officers, corporations — that in order to save their own skin, they need to save the living "skin" of this country. After all, it is Madagascar's vegetation and biota that filters their water, makes their air breathable and provides refuge from the storm.
Scientists have already done much of the work to map Key Biodiversity Areas (KBAs) — the most important areas for species' survival — in Madagascar. But CI's mission now encompasses people, too. So to these existing maps, we are seeking to add new ones. Maps depicting ecosystems that are critical for providing fresh water, for food, for climate resilience and for recreational and cultural values.
Click here to read the full post.


text:
- Member: BrennaCeDria
- Title: Final Fantasy X-2 - Cutie Yuna Flash!
- Premiered: 2006-05-15
- Categories:
- Koda Kumi Cutie Honey
- Anime:
Just a quick video I've made to have something to do this weekend... Based solely on the idea that "well, all Honey does is change clothes and save the world, and that's about all Yuna does anymore!" while I was half asleep.
Song: Koda Kumi's version of Cutie Honey
Video: Final Fantasy X-2
Software: DVDdecrypter, Nova extractor, PSS Plex, DVD2AVI, Windows Movie Maker
Now, for some self-critism:
Things I don't like
Windows Movie Maker
Too much Lip Flap
Multiple spots that just wouldn't sync
Windows Movie Maker
Not enough source footage
Couldn't get Rikku's fight from the concert to fit in to go along with Paine's fight at the concert
Windows Movie Maker
Easily the most difficult thing in this video was getting the footage; even when WMM hated me and refused to do anything else for the night the editing was as easy as it can be for so little source footage. Anyway, the video's not meant to be taken seriously, and while it's far from my best at least I can honestly say it's better than my first. (Trust me, you don't want to go looking for it!)
Opinions (7)
- Orig
- Visual
- Sound
- Synch
- Lip
- Effects
- Effort
- Re-View
- Overall
- 7.80
- 9.40
- 9.80
- 8.00
- 8.60
- 6.60
- 8.20
- Link Format Bitrate Codec Duration Filesize Link Check Information Comments
WMV3/Windows Media Audio
45.3 MiB
Local File Final Fantasy X-2 - Cutie Yuna Flash.wmv Duration 185 seconds Video Track 0 kb/s WMV3 [WMV3] 720 x 480 @ 1000 fps Audio Track Windows Media Audio @ 160.04 kbps 44.1 kHz, stereophonic sound


text:
Kenneth Feinberg
Founder, Feinberg Rozen Llp
Kenneth R. Feinberg has been key to resolving many of our nation's most challenging and widely known disputes. He is best known for serving as the Special Master of the Federal September 11th Victim Compensation Fund of 2001, in which he reached out to all who qualified to file a claim, evaluated applications, determined appropriate compensation, and disseminated awards. Mr. Feinberg shared his extraordinary experience in his book What Is Life Worth?, published in 2005 by Public Affairs Press. Just a few years later, Mr. Feinberg became Fund Administrator for the Hokie Spirit Memorial Fund following the tragic shootings at Virginia Tech. Mr. Feinberg also has served as Special Master in Agent Orange, asbestos personal injury, wrongful death claims, Dalkon shield, and DES (pregnancy medication) cases.


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What's New
Added a French translation.
App Description
School 26 is a unique game of empathy, strategy and relationship building.
School 26 follows Kate, a student whose nomadic family has made it difficult for her to maintain long-term friendships. As she enrolls at her 26th new school, she and her parents strike a deal: if Kate makes good friends here, the family will stay put. Now the player must help Kate use intuition, empathy, and strategy to build friendships and navigate the often-treacherous social hierarchies of high school in this unique game of social mastery.
School 26 Hands-On Preview from SlideToPlay.com
"We've seen a lot of quality games at GDC this week, including shooters, racers, and puzzle games. But none of them quite has the same creative spark as School 26, an upcoming iPhone and iPad game geared towards teenage and pre-teen girls. But really, School 26 is for anyone who wants to become a better communicator. In this game, empathy is your main tool, resulting in an incredibly unique storytelling experience..."
App Changes
- June 12, 2014 Initial release
- December 12, 2014 New version 1.7


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Sunday, February 7, 2010
Happy Valentine's Day - A Corny Valentine
5 comments:
That is really cute.
And the winner is…
Are you the winner? See if you can guess.
~ Just Joany
Red Wagon FlightsWeren't those older cards just the sweetest things you ever saw? Why can't we have cards like that, now?
~ Yaya
Yaya's Changing World
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Gwenn Dress
Can't find your size? Special Order
- Self: 100% silk
Lining: 100% poly - Fully lined
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- Side cut outs
- Center back zipper closure
- Revolve Style No. PKER-WD368
- Manufacturer Style No. PA1266LAR
- Modeled in size S.
- Shoulder seam to hem measures approx 33" in length
- Model Measurements: Height 5'9", Waist 24", Bust 32", Hips 34"
- Size Guide
- Need size help? Contact our Style Expert!
The New York-based design team behind Parker knows that the modern woman loves sophistication and sexy in the same look. Parker's collection of dresses and tops feature flirty details and girly prints perfect for day or night, work or play.


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In it, but not of it. TPM DC
Dead Dems Walking: TPM's List Of Candidates Who've Been Written Off By Their Party
- 1. Allen Boyd (FL-02): One of the most conservative Democrats in the House has held on to his seat since 1997, along the way flirting with Social Security privatization and other GOP ideas. That didn't do him any favors in a year when he needed strong Dem support, and his overtures to the base -- Boyd surprised everybody by voting for the final health care bill, and for Wall Street reform -- haven't been enough to keep him above water.
- 2. Kathy Dahlkemper (PA-03): Support from unions hasn't helped Dahlkemper overcome high unemployment in her district. The pro-life freshman Democrat helped broker a compromise on the abortion provisions in the health care bill.
- 3. Steve Driehaus (OH-01): Dreihaus skirmished with House Minority Leader John Boehner earlier this year after the top Republican -- who represents a neighboring district -- said Driehaus would be a "dead man" if he voted for the health care bill. Driehaus, a freshman, was receiving death threats at the time. The DCCC recently pulled financial support for him.
- 4. Chet Edwards (TX-17): The ten-term Democrat had a genial enough relationship with his party leadership that after Barack Obama won his presidential nomination, Speaker Nancy Pelosi floated his name as a potential Vice Presidential candidate. Edwards represents one of the most conservative districts in the country and has a voting record to match. It hasn't helped.
- 5. Debbie Halvorson (IL-11): Halvorson's seat has been in Republican hands for more than a decade before she won in 2008 with a solid 58 percent of the vote. But that support hasn't held against Republican challenger, and Iraq war veteran, Adam Kinzinger.
- 6. Steve Kagen (WI-08): Not every insider has written off Kagen completely, but most say he won't be around for a third term -- a victim of a very tough year in Wisconsin.
- 7. Mary Jo Kilroy (OH-15): Freshman Kilroy only narrowly won her seat in 2008 in a Democratic wave year, so it's no surprise that the man she narrowly defeated in 2008 is poised to defeat her this year -- this time, with the backing of the Tea Party.
- 8. Ann Kirkpatrick (AZ-01): Kirkpatrick won an open seat in a Republican district by a significant margin in 2008. She's one of many freshmen members in tough districts who watched as a conservative opponent emerged and shot ahead in the polls.
- 9. Suzanne Kosmas (FL-24): Yet another freshman who charged to the center once the political environment this year was set in stone. Kosmas supported the extension of all the Bush tax cuts -- after she agonized over health care reform and voted against the House bill, but for the final legislation.
- 10. Betsy Markey (CO-04): Markey also moved to the center after winning her seat in 2008. Most handicappers and party faithful count her out, but the DCCC did dump some money in her district in recent days.
- 11. Glenn Nye (VA-02): As much as Nye -- another conservative freshman -- has a claim to independence from his party leaders, and as much as he's tried to run away from them, he can't shake the Democratic label.
- 12. Carol Shea-Porter (NH-01): This one's heartbreaking for progressives. Shea-Porter, a sophomore member and a social worker by trade, supported the Democratic agenda in a mixed district (it went for Obama in '08 but for George W. Bush in '00 and '04). Her opponent is a Republican former mayor of Manchester.
- 13. Harry Teague (NM-02): Yet another freshman, Teague won an open seat in a Republican district in 2008, but now runs far behind the man who vacated that seat, former congressman Steve Pearce.
There are several other Democrats who have a high mountain to climb going into Tuesday's elections. They include: John Boccieri (OH-16), Alan Grayson (FL-08), Paul Kanjorski (PA-11), Frank Kratovil (MD-01), Jim Marshall (GA-08), Jerry McNerney (CA-11), Scott Murphy (NY-20) John Spratt (SC-05), and Dina Titus (NV-03).
Likewise, a bunch of Dems from seats in conservative districts are retiring. Most of those will be picked up by Republicans. They include: Reps. Brad Ellsworth (IN-08), Bart Gordon (TN-06), Charlie Melancon (LA-03), Dennis Moore (KS-03), Vic Snyder (AR-02), and John Tanner (TN-08).
Republicans are also almost certain to win the vacant seat in New York's 29th district.
Got all that?


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Geoff Keighley
YouTube Gaming's E3 show to return with Geoff Keighley as host
For the second year in a row, YouTube will provide its own coverage of E3.
Geoff Keighley headlines
Ten world premieres at The Game Awards 2015, confirms Keighley
This year's Game Awards ceremony will feature ten world premieres.
Geoff Keighley returns to E3 with YouTube's expo hub
YouTube and Geoff Keighley have teamed up to produce a live E3 show.
The Game Awards to debut in December as Keighley bows out of Spike VGX
Spike VGX host Geoff Keighley will head a new video game awards show next month, with the backing of Microsoft, Nintendo and Sony.
Microsoft's Spencer feels fanboys have "unhealthy dislike" for other formats, exec praises Sony
Xbox One and PS4 format debates are here to stay unfortunately, but Microsoft's Phil Spencer has called out the "unhealthy dislike" of rival formats in a new interview, while taking time to applaud Sony's efforts in the new generation so far.
Geoff Keighley to host an hour-long interview with Hideo Kojima on March 13
Metal Gear Solid creator Hideo Kojima will be sitting down for an interview with Spike's Geoff Keighley this Thursday.
The Final Hours of Tomb Raider app now available
If you're a fan of in-depth behind-the-scenes looks at games, then Geoff Keighley's newest Final Hours app on Tomb Raider might be right up your alley. You can get it now on iOS, Kindle Fire and Steam for £1.99/€2.99/$2.99.
UE4 platforms "beyond PC" TBA, UE3 supports Wii U
Geoff Keighley has said in a tweet Unreal Engine 4 won't be compatible with Wii U, referring to a GDC comment from Epic CEO Mike Capps.
A standalone Mass Effect FPS almost happened
The Final Hours of Mass Effect 3 just went live but already there are some very interesting stories coming to light about the development of the series' third installment.
Keighley launches The Final Hours of Mass Effect 3 for iPad
GTTV host Geoff Keighley's confirmed The Final Hours of Mass Effect 3, announced on Monday, is now available. You can pick it up for £1.99/$2.99 for iPad, with Keighley saying in a tweet PC and Mac versions are coming soon.
Keighley announces Final Hours of Mass Effect 3
GameTrailers host Geoff Keighley's confirmed that his next personal project will be The Final Hours of Mass Effect 3.
Car Crash: Why VGAs are in a serious need of change
The 2011 Spike VGAs were meant to celebrate the year's best games and hype future releases. Instead, Johnny Cullen argues, they played to stereotypes in the worst possible way.
Keighley: The Last of Us VGAs unveil to be "in-game"
Geoff Keighley's promised the unveil of the massive Sony exclusive "you won't believe," called The Last of Us, will be an in-game affair, not a CG effort.
Keighley: Alan Wake XBLA is "effectively" Alan Wake 2
Alan Wake's debut on Xbox Live Arcade is more than you think, according to VGA boss and GameTrailers TV host Geoff Keighley, who's said it's "effectively" Alan Wake 2.
Behind-the-scenes book of Portal 2 now available on Steam
We all know that Valve makes great games – but now the company has dabbled in making great books, too. Portal 2 – The Final Hours is a digital tome that will give you an "unvarnished look" behind the scenes on Valve's most recent puzzler.
Keighley launches premium Portal 2 iPad feature
US games journalist Geoff Keighley launched The Final Hours of Portal 2 last night, a $1.99 iPad app detailing the creative process behind Valve's puzzler. The feature itself comes in at 15,000 words and includes interactive diagrams, magazine-style production and such. "The Final Hours of Portal 2 is a gripping and dramatic story brought to […]
Batman: Arkham City VGAs trailer will see new villain revealed
Geoff Keighley's confirmed the new trailer for Batman: Akrham City at tonight's VGAs will see a new baddie for the game being announced for the first time.
10 VGA premieres promised by Keighley
Geoff Keighley has confirmed there will be at least ten premieres at next month's Video Game Awards.
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Geoff Keighley latest
YouTube Gaming's E3 show to return with Geoff Keighley as host
Ten world premieres at The Game Awards 2015, confirms Keighley
The Game Awards to debut in December as Keighley bows out of Spike VGX
Microsoft's Spencer feels fanboys have "unhealthy dislike" for other formats, exec praises Sony
Geoff Keighley to host an hour-long interview with Hideo Kojima on March 13
Batman: Arkham City VGAs trailer will see new villain revealed
Keighley: Date for Halo: Reach open beta "coming very soon" [Update]
Spike's Video Game Awards is tonight, in case you forgot [UPDATE]
Keighley – Halo Reach is "huge leap forward," will be 2010's "biggest game by far"


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About Our Practice
Preventive Cardiology, Inc. utilizes an old practice model to deliver current and cutting-edge clinical care. Dr. Baum runs the only LDL-Apheresis center in Florida that offers both FDA-approved systems – Kaneka's Liposorber® and B. Braun's H.E.L.P.® Futura – enabling his Comprehensive Lipid Center to treat even the highest risk hyperlipidemic patients.
What We Do
As this is a cardiology practice, echocardiograms, nuclear stress tests, and carotid ultrasounds are performed on site when necessary. The overarching theme of the practice is to prevent heart attacks and strokes through appropriate lifestyle changes and the management of all cardiovascular risks.
About Dr. Baum
Dr. Seth J. Baum, a graduate of Columbia College of Physicians and Surgeons, completed training in Internal Medicine, Cardiology, Interventional Cardiology, and Electrophysiology. Dr. Baum's practice in Boca Raton, Florida is devoted predominantly to Preventive Cardiology and Lipidology.


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Dr. Moku has helped over 250,000 people learn Hiragana and Katakana
No matter your age or experience you will be amazed at your ability to learn and recall Japanese writing.
"I looked around and came across Dr. Moku,
30 minutes later I had memorized all 46 Hiragana"
"After a half hour, I was blazing through each exercise: highly recommended for beginners."
Read more reviews >Learn to write Hiragana and
We have added animated stroke guides so you can
learn how to write Japanese calligraphy!
Learn to speak the essentials
Includes Japanese words and phrases with audio.
There are different categories in both Hiragana &
Katakana versions. (iOS only for now)
Master tricky Modified Kana
with all new quiz modes!
After you learn Hiragana and Katakana you need to cement it further into your mind with different quiz types
"I love you Dr. Moku, you are a lifesaver! I struggled for weeks with Japanese Hiragana until I found your app. "
- Amanda W. from USA
Read more reviews >Try Dr.Moku
In one hour you could be reading and
writing Hiragana and Katakana.
How cool is that?
View all Products


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Photo Gallery: Chargers vs. Titans | Top Photos | More Photos
"It was a bit like the Brady Bunch out there,"
Offensively, on the team's final possession, they had only two of their projected starting five offensive linemen on the field. And between those two,
"All of a sudden you go from being healthy with the group you saw what it was going to be all in training camp, who was going to be the five guys, then you look up and before you know it there are only a couple of them that are out there," said
It would be easy to point the finger at having to plug so many holes as a reason why the Chargers dropped their second heartbreaking decision of the season, a 20-17 last second loss to the Tennessee Titans. But the Bolts weren't buying any of that.
"We've got a good football team here and that's why you've got 53 on the roster," said Head Coach Mike McCoy. "I think the players who had an opportunity to step in today did a nice job. They really did. They came in on short notice because of what happened during the week. That's all part of the game. (We) had every opportunity to win the football game with the guys that were playing. We just didn't get it done at the end."
The players did not hesitate to agree with their head coach.
"Our mentality is always the next man up," said
Instead, it was too many mistakes that shot the Chargers in the foot as the Titans ended up making one more play than San Diego did. As a result, the Bolts fell to 1-2 to start the season.
"In a football game like this, (we had) too many mistakes at critical times during the game," McCoy said. "It comes down to the end of the game where we've got to make a play, and they made one more play than we did. So give them the credit where it's deserved. We put them in a situation, and we didn't finish the game. So we've got to improve there…Little mistakes killed us in this game today. (We had) a number of them. We'll go home and analyze the film, look at it and correct it as soon as we can."
Offensively, the first of those mistakes came in the first quarter with the Chargers in a 3rd-and-1 around the Tennessee 39-yard line. While they have been so good up to this point converting in those situations, they failed to do so and were forced to punt. Later in the first half, San Diego had 1st-and-goal at the five yard line. It appeared that
"There were just little things," said Rivers. "We played good for the most part. We had a 3rd-and-1 when we were up 7-0 (and) we could have taken a 14-0 or 10-0 lead. Then we had that one down in the 1st-and-goal at the five and we had the touchdown taken off the board there. We got it going, we just didn't finish a couple of those drives. And I thought there at the end, if we don't get the holding penalty, we're in a 3rd-and-3, 3rd-and-4, which is realistic to go get it and finish the game. It became 3rd-and-10."
Defensively, the Chargers allowed Jake Locker to make plays with his legs, picking up first downs and chunks of yards on broken plays. The Titans converted 6-of-14 third downs as well as one fourth-down conversion, and many of those plays were in long to-go situations. On the final drive of the game,
"We've been through it already against Houston, and we rebounded," he said. "Obviously Dallas is as talented a team as they come, so there's no feeling sorry for ourselves. We've got to correct it, look at the film and see what could have been done better. There were some things out there where when you get a team on the ropes you've got to take advantage of it. Convert third downs and get off the field on 3rd-and-10. So there's stuff that we can improve on. We fought. It showed good character in us that we fought until the end. We've just got to make the plays when they come our way."


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marketing plan Archives
Inbound Marketing Machine How To Build A Better One
Inbound marketing can be an extremely powerful tool in any business' marketing plan. In order to see results, just like any other strategy, you must pair these with other efforts to see a direct effect on your bottom-line. Marketo partnered with Column Five to take a visual approach to explaining how to build a well-oiled… Read More →
How Can You Make Your Business More Appealing To Potential Investors?
The reality is more and more companies are competing for a limited supply of funding, so much like in the dating scene you want to appear attractive and engaging. Whether your business is seeking financial support from a bank, a private investor or a venture capitalist group, it is crucial that you make the right… Read More →
Why Creating A Marketing Plan Matters
A lot has been said about the importance of creating a marketing plan, but the task at hand often appears so daunting that many business owners and executives never get down to the business of tackling this challenge. Instead, they choose to simply approach things on a day-to-day basis, functioning in reactive mode rather than… Read More →


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The iPhone is the most anticipated new product launch in recent years, and the most important for
Except research indicates the opposite. A recent study by Compete Inc. asked 680 potential iPhone shoppers this question: "How important would each of the following be on your decision to purchase an iPhone?" The results were revealing:
Price of the device: 81%
Performance of the phone: 77 %
Battery life: 76%
Overall ease of use: 75%
Design/look of device: 46%
Ease of using touch screen: 45%
Ability to synch device with music collection: 44%
Wi-fi: 37%
Ease of accessing e-mail: 37%
And, in last place: The fact that Apple makes the device: 32%
You mean, all the anticipation is about the actual, physical product and its features–and the Apple brand is at the bottom of the list, less than half as important as something as banal as "battery life"? After all, conventional marketing wisdom says that some time around 1980, consumers stopped buying products and started buying brands instead. And no brand has been more vaunted than Apple.
Could it be that great brands are the product (no pun intended) of something other than branding? In fact, they are! What's more, branding and great brands are made of altogether different stuff. Branding seeks to transcend tangible benefits, or even compensate for their absence. But great brands are built from the inside out, on tangible benefits. Branding seeks to create reality through perceptions, engendered by compelling communications.
Great brands, meanwhile, are the result of compelling products and services. The exception–a few image-driven categories such as beer, liquor or fashion–only proves the rule.
For those with a stake in selling marketing communications, branding is a convenient confusion of cause and effect. But brands such as Apple didn't become great because of good ads. Rather, the unique and differentiated propositions of the products made for good ads. Macintosh computers really are more beautiful inside and out: easier to use, and better to look at.
A brand is neither a goal nor a means, but a result of consistent delivery against a differentiating, relevant benefit. A brand is not an end in itself, and it doesn't even provide a hedge against future bets.
Case in point: Apple's flops, such as its early 1980s Lisa computer, its highly-hyped Newton PDA and the Motorola/Apple RokR iTunes phone. The Lisa was done in by a high price, the Newton by weak handwriting technology. Most recently, and most to the point, the RokR flopped because of mediocre software and hardware design. In each instance, the Apple brand was only as good as the product.
Great products speak for themselves. Not by coincidence, the iPhone ads are as straightforward as a product demo can be: a hand in front of a black background operating the device. Could
Those features better work–and wow–because a brand, even one with Apple's vaunted reputation, won't carry the day by itself.
Marc E. Babej and Tim Pollak are partners at Reason Inc., a marketing-strategy consulting firm that works with clients in a range of categories, including media and entertainment, financial and professional services, packaged goods and the public sector.


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The Liberty Bowl has reached a six-year deal with the Big 12 that will take effect in the 2014 season.
Under the agreement announced Friday, the Liberty Bowl will match a Big 12 team against a representative from the Southeastern Conference. In a release announcing the deal, Liberty Bowl officials say they will have the fourth selection of Big 12 teams after that league's college football playoff representatives have been chosen.
The Liberty Bowl currently matches the Conference USA champion against an SEC representative, assuming the SEC has enough bowl-eligible teams to fill its Liberty partnership. The bowl's contract with Conference USA expires after this season.


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Mouth Diseases |
Can`t swallow01/09/2009 |
hi i am asking on hehalf of my mother. she as problem swalloing food she alwys complain that it gets stuck she hardly eats any solid food for this reason and for her age is under wait. she went to many doctors in south africa but no one seems to no whats wrong pls help thanks
She should be evaluated by an otolaryngologist (Ears, Nose and Throat doctor) or a gastroenterologist (digestive system specialist).
Carl M Allen, DDS, MSD Professor Emeritus of Oral Pathology College of Dentistry The Ohio State University |


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GM Retirees Face an Uncertain Future
Rebecca ReisnerRobert and Joan Allen, 83 and 79 respectively, have what is known in Detroit as a mixed marriage. Both spent their careers with General Motors, but he worked a union job as a shipping clerk in a GM warehousing facility, while she held a salaried position as a senior analyst for the Chevrolet division. Now with GM having filed for bankruptcy, the Allens worry that neither will retain benefits or pension payments adequate to sustain them in their condominium in South Lyon, Mich.
"I don't think my husband's union benefits are safe. Maybe they'll take away all of it, or maybe just dental and eye," says Joan. "And I don't know about our pensions either. You hear this, and you hear that, but you don't know what to believe. I retired from GM in 1985, paying nothing for my benefits. Then they started pecking away at them." The Allens receive a combined monthly pension of $1,900 from GM.
Worrying about the future isn't new to GM's 493,000 retirees and surviving spouses, who have watched the automaker lose market share for years. But with the company's Chapter 11 bankruptcy filing on June 1, retirees have entered a new, uncertain zone. GM's restructuring will affect salaried retiree health care, some executive pensions, and retiree life insurance, the company says. But details are incomplete.
salaried retirees are more at risk
On June 2, GM spokesman Tom Wilkinson acknowledged that there's no solid answer for retirees yet, although GM filed a first-day motion to continue employee benefits, including the pension plan. "But that's not certain until the judge approves the sale of GM," Wilkinson explained. "I believe the assumption everyone has is that those [employee benefits] will be moved to the new company, but there probably will be some reduction in those benefits." He added that those reductions are more likely to involve health-care and life insurance benefits—rather than pension payments—for salaried retirees.
That won't reassure the Allens, who have already seen one change this year. In January, GM revoked all health benefits for salaried retirees older than 65, replacing them with a $300-a-month payment to cover the costs of private-market insurance to supplement Medicare. Joan Allen was paying $138 for the combined medical, dental, and vision insurance GM once promised would be free for life. Now she's on Medicare and has purchased a $192-per-month health insurance policy from AARP to cover what Medicare doesn't; she dropped dental insurance altogether because it stretched her budget too far. Plus, she's had to figure out which specialists are on her new medical plan, and which aren't.
While both salaried and union retirees are likely to face changes, benefits offered to salaried retirees—who are not covered by collective bargaining agreements—appear to be most at risk under the transition. For the moment, pensions appear safe.
"We were assured that GM would move our Salary Retirement Plan—which is the formal name of what we call 'pension'—into the new GM and there will be no disruption," says Jack Dickinson, president of Over the Hill Car People, a Hoover (Ala.) membership organization that looks out for the interests of salaried GM retirees. "It appears that GM has taken the proper steps to protect it. The fund is in excellent shape. And they've indicated it will take care of retirees for years and years to come."
More pressed into early retirement
However, according to GM's figures, pensions for hourly workers and salaried employees were both underfunded at the end of 2008, by a total of more than $12 billion.
Dean Gloster, a lawyer hired by the GM Retirees Assn., doubts GM's ability to replenish the pension fund within the next few years. "I'm deeply worried because, while statements from the White House and GM indicate it will be a leaner, meaner company, that is in part because more employees will be pushed into retirement early," says Gloster, a partner in the San Francisco law firm Farella Braun & Martel. "There is the possibility that the business will not be capable of contributing enough to the pension fund. One thing we as baby boomers are poor at is math. We refuse to realize that fewer people are putting money in than are taking it out."
David Certner, AARP's legislative policy director, feels more optimistic about the pension prospects. "GM's pension plan is in relatively good shape, decently funded. Every pension fund in the U.S. is underfunded because of the market's being bad," Certner says.
As a last resort, pensions are somewhat guaranteed by the Pension Benefit Guaranty Corp., a federal outfit. Pensions for retirees 65 and older are guaranteed for up to $54,000 a year. Coverage is lower for younger retirees.
"weasel words" inserted
Of more concern is the future of health benefits. The United Auto Workers, which represents nonsalaried employees, will administer health-care benefits through the new Voluntary Employees' Beneficiary Assn. trust that is being established as part of the restructuring. For hourly workers, their union—not their former employer—will determine what health-care benefits they receive. Union retirees are already scheduled to lose vision and dental coverage and will have prescription-drug benefits cut under an agreement reached last month between GM and the union.
For salaried retirees, GM possibly could cancel health benefits for those who are under 65, according to Gloster. "The precedent has been set," he says. "It's a matter of the 'weasel words' contained in 'summary plan descriptions' that employees receive every year. The weasel words give the company the option to 'amend, modify, and terminate' the benefits employees have been promised. Back in the 1970s and 1980s, those weasel words weren't even in the summary plan description booklets. Yet in 1998, the Sixth Circuit Court of Appeals gave GM the right to enforce them."
However the scenarios play out, Kathleen Buczko, the Allens' niece, says she and her husband are preparing for the worst. "We're worried my aunt's white collar pension will disappear now that GM has gone bankrupt, and they won't be able to afford to stay in their home," says Buczko, a Los Angeles consultant who says the Allens acted as parents to her after her own mother and father died. "We're trying to figure out whether we can keep them in their condo in Michigan or move them here to California. So we'll take care of my aunt and uncle, but what about the other retirees who don't have anyone to help them? They're going to be out there, competing with teenagers for jobs to stock the shelves at Wal-Mart (WMT)."


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OEBB Insurance open enrollment is now open online and will be closed on September 8th.
Carriers will be available for quetsions at convocation on 8/30 or at the district office on 8/25 from 1pm - 3pm
OEBB Insurance Information from the GBEA
The district is offering Medic First Aid courses at their office during the weeks of 8/22 and 8/29. Classes are 4 hours each and cost $34
You can search for ODS providers by going here, we are under the ODS Plus network
Bargaining on 8/13 at CAL has been CANCELED
The next scheduled Bargaining date is 8/30 at CAL 4:30pm - 8:30pm
What happens on the political front directly affects us as teachers and our students!
Stay informed! Sign up to receive Education Votes updates on http://educationvotes.nea.org/
It's easy! All you need is your email address and zip code
The GBEA would like to give special thanks to two of our retiring members for their years and years of tireless contributions to the association.
Nancy Chase has been Treasurer for many years, more than anyone can remember, and has done an outstanding job. Thank you, Nancy, for all your hard work and dedication!
Jan Speciale has been our long-time Grievance Chair and assisted countless members with problems and questions in regards to our contract. In addition, she has been a member of our Bargaining Team for years as well. Her experience and wisdom has benefited us all so many ways. Thank you, Jan, for your many many hours of work with our members!
Thanks to everyone who attended the GBEA Appreciation Party!!
OEA SUMMER CONFERENCE open to all members Aug 1-3 in Bend
At the 2011 OEA Summer Conference, discover the power you hold as an OEA member to engage in collective action. Learn to use your own skills to advocate for our schools and students. Youíll network, youíll enhance your organizing and leadership skills, and youíll walk away with some expert professional development tools.
See link for more information and registration:


text: If you're really running out of words to create with the board you have, try using all of the words so that you can flip it and be given a new set. Got a nice little stack of Coins built? Splurge on a freeze time or a 4x multiplier, as that wad is only going to waste otherwise. Remember: you can add an 'S' to loads of words, so look out for those plurals.