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Behavioral genomics of honeybee foraging and nest defense
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The honeybee has been the most important insect species for study of social behavior. The recently released draft genomic sequence for the bee will accelerate honeybee behavioral genetics. Although we lack sufficient tools to manipulate this genome easily, quantitative trait loci (QTLs) that influence natural variation in behavior have been identified and tested for their effects on correlated behavioral traits. We review what is known about the genetics and physiology of two behavioral traits in honeybees, foraging specialization (pollen versus nectar), and defensive behavior, and present evidence that map-based cloning of genes is more feasible in the bee than in other metazoans. We also present bioinformatic analyses of candidate genes within QTL confidence intervals (CIs). The high recombination rate of the bee made it possible to narrow the search to regions containing only 17–61 predicted peptides for each QTL, although CIs covered large genetic distances. Knowledge of correlated behavioral traits, comparative bioinformatics, and expression assays facilitated evaluation of candidate genes. An overrepresentation of genes involved in ovarian development and insulin-like signaling components within pollen foraging QTL regions suggests that an ancestral reproductive gene network was co-opted during the evolution of foraging specialization. The major QTL influencing defensive/aggressive behavior contains orthologs of genes involved in central nervous system activity and neurogenesis. Candidates at the other two defensive-behavior QTLs include modulators of sensory signaling (Am5HT 7 serotonin receptor, AmArr4 arrestin, and GABA-B-R1 receptor). These studies are the first step in linking natural variation in honeybee social behavior to the identification of underlying genes.
KeywordsApis mellifera Recombination rate Insulin-like signaling Foraging behavior Aggressive behavior Candidate genes Behavior genetics
Mapping genes for natural variation in behavior
The need to understand within- and between-population variation in behavior, especially social behavior and its evolution, remains a central theme in biology. Honeybee societies have facultatively sterile female workers and a specialized workforce. Honeybee social behavior is dramatically demonstrated by the efforts of worker bees collecting and hoarding pollen and nectar, and mass stinging responses in defense of the nest. Division of labor within a honeybee nest is a consequence of age-related changes in physiology plus genetic variation within the colony for tendency to perform specific tasks (Page and Robinson 1991). A number of studies have demonstrated genetic variation within and between honeybee populations in specialization for water, nectar, and pollen collecting (reviewed in Page et al. 2000) and variation in defensive responses (reviewed in Breed et al. 2004). The genetic architectures of these two task sets are more thoroughly characterized than other behavioral traits in the honeybee. For these reasons, we sought to determine whether it is possible to define, within a manageable number of candidate genes, regions influencing these behaviors.
Quantitative trait locus (QTL)-mapping studies provide information about the genetic architecture of a trait that cannot be determined by other methods. This information includes estimates for the number and location of loci influencing population variation in the trait, the mode of inheritance of these loci (dominance, epistasis, or imprinting effects), and amount of phenotypic variance each locus accounts for (Lander and Botstein 1989). But QTL studies do not have a good track record for isolation of causal genes. For example, genes for only 20 of 2,000-rodent QTLs have been cloned (Flint et al. 2005). Identifying genes responsible for naturally occurring phenotypic variation is especially challenging for behavioral traits where complex interactions of genes and the environment are expected (Flint 2003; Plomin and McGuffin 2003; Arnholdt and Mackay 2004; Goldman et al. 2005).
The endgame in determining nucleotide sequence variation responsible for natural variation in honeybee behavior will require identification of QTLs, confirmation in independent crosses, fine-scale mapping, expression assays, and finally, experimental modulation of gene expression or complementation tests (Mackay 2004). The biggest problem in taking a map-based approach to isolation of genes is that confidence intervals (CIs) for QTL location are usually quite large. The size of the CI primarily depends on the number of individuals in the mapping population, the number of genetic markers scored, the magnitude of effect that the QTL has on the phenotype, and the recombination rate (numbers of crossovers per unit of chromosomal distance). The first two factors, sample size and marker density, are important for reducing CIs, but they are subject to diminishing returns as more individuals or more markers are added to the experiment. The effect of the QTL is a major concern because QTLs often account for only a small proportion (often <5%) of phenotypic variance. The QTL effect can only be increased up to a certain level by improving the phenotypic assay and using the most appropriate cross (Darvasi 1998; Arnholdt and Mackay 2004). On the other hand, recombination rate can have a large effect on the size of chromosomal regions covered by CIs because the physical size scales inversely with recombination rate. For this reason, fine-scale mapping studies are often designed to effectively increase recombination by taking advantage of historical recombination events through linkage-association studies in populations or by developing multigeneration, recombinant inbred lines (Darvasi 1998). In the honeybee, map resolution is enhanced by a meiotic recombination rate that currently ranks highest among metazoans (Hunt and Page 1995; Solignac et al. 2004), so that large genetic distances correspond to relatively small physical regions containing few genes.
In this study, we review what is currently known about the genetics of honeybee foraging and defensive behaviors, and how a map-based approach leads us to a manageable number of candidate genes that seem to fit what is known of the behavioral patterns. Information from the draft honeybee genome sequence (Honeybee Genome Sequencing Consortium, HGSC 2006) was used to delimit sets of candidate genes flanked by marker sequences that were identified in prior QTL studies, and expression data from candidate genes provided additional information on likely candidates.
Behavioral genetics of foraging
Preferential foraging for either a nectar or protein source such as pollen are sequential parts of the gonotrophic cycle of many insect females. When nonreproductive, females tend to forage for nectar as a carbohydrate source for maintenance. When reproductively active, insects such as solitary bees and mosquitoes seek protein that is incorporated into eggs (Amdam et al. 2004). Although worker honeybees are facultatively sterile, they can produce eggs if their ovaries develop sufficiently as happens in the absence of a queen. Amdam et al. (2004, 2006) hypothesized that remnants of the ancestral gonotrophic cycle and the correlated foraging behavior remain and "drive" foraging behavior (Fig. 1). In support of this hypothesis, it was established that workers from the high pollen-hoarding strain are characterized by elevated titers of the conserved yolk precursor vitellogenin and have larger and more active ovaries than low strain bees. It was also found that bees that were unselected for pollen hoarding but had enlarged ovaries foraged earlier in life showed a preference for pollen foraging and collected nectar of lower concentration than bees with fewer ovarioles. Thereby, "wild type" bees show the same correlated phenotypes that differ between high and low pollen-hoarding strains (Amdam et al. 2006).
In honeybees, ovariole number is determined during the third larval instar through a nutrient-dependent endocrine signaling cascade. The endocrine factors, juvenile hormone and ecdysteroids, are involved in the initiation of vitellogenin expression at adult emergence, and vitellogenin and juvenile hormone interact during adult life to affect sensory responsiveness and onset of foraging behavior (Guidugli et al. 2005). In solitary insects, endocrine cascades involving juvenile hormone and ecdysteroids have pleiotropic effects on sensory tuning, yolk protein production, ovarian physiology, and life span (Amdam et al. 2004; Flatt et al. 2005; Guidugli et al. 2005). This hormonal pleiotropy appears to be regulated by upstream signaling through the insulin/insulin-like signaling (IIS) pathway (Claeys et al. 2001; Flatt et al. 2005; Tu et al. 2005). The association between traits in high and low pollen-hoarding bees, therefore, suggests that honeybee foraging division of labor has evolved from an ancestral reproductive regulatory network involving IIS. With knowledge of the association between components of physiology and foraging behavior, we expected an overrepresentation of genes involved in IIS and ovarian development within the CIs for "pollen" QTLs.
Behavioral genetics of defensive behavior
Honeybee defensive behavior is not as thoroughly characterized as foraging behavior in terms of correlated physiological and sensory traits. Honeybees exhibit defensive behavior near the nest, but highly defensive bees may pursue for considerable distances away from the nest. Defensive behavior involves at least two tasks: guarding behavior at the hive entrance and flying out and stinging. Guards specialize in exploratory behavior in the nest entrance. They learn to recognize the hydrocarbon blend in the cuticles of their nestmates by olfaction, and they reject non-nestmates by biting or stinging. Only 10–15% of workers have been observed to guard the entrance during their lifetime (Moore et al. 1987). Both the number of days that individuals in a colony persist at guarding and the number of bees guarding the nest entrance correlate with the intensity of the stinging response (Arechavaleta-Velasco and Hunt 2003; Breed et al. 2004).
Multiple sensory modalities influence stinging behavior. A moving visual stimulus usually is necessary to release stinging behavior (Free 1961). Substrate vibrations also increase the chance of a mass stinging response. Alarm pheromone also is an important component of colony defense. This pheromone blend is released from the sting apparatus during the act of stinging and as guards extrude their stings at the colony entrance in response to relevant stimuli. A transient increase in metabolic rate occurs after exposure of bees to alarm pheromone, and this increased rate genetically correlates with the defensiveness of colonies (Southwick and Moritz 1985; Moritz and Southwick 1987; Andere et al. 2002). Although the alarm pheromone components vary with strains of bees, QTLs influencing this variation were distinct from QTLs influencing stinging behavior (Hunt et al. 1999, 2003). Defensive strains of bees respond more quickly to all of these stimuli.
Materials and methods
QTL mapping and confirmation
QTLs' influencing traits related to foraging behavior or defensive behavior were mapped and confirmed previously (Hunt et al. 1995, 1998; Page et al. 2000; Rueppell et al. 2004, 2006), but new analyses were performed on the data to include additional markers, and in addition, "sting" QTLs were reanalyzed by combining two traits as stated below. Maps were based on crosses that are appropriate for a haplodiploid, colonial species. For QTL detection, haploid drone progeny of an F1 queen were each backcrossed to sister queens (that were daughters of a single haploid drone). Colony phenotypes were correlated with inheritance of paternal marker alleles. Whole-colony phenotypes resulted from the behavior of many individuals, such as the number of stings in a leather patch in 1 min or the area of wax combs containing stored pollen. Confirmation studies all involved the use of F1 queens backcrossed to single drones and analyses of worker progeny for individual behavior and genotypes. Linkage maps that were used to identify candidate genes were constructed with JoinMap (3.0) and MapManager QTX software using the Kosambi mapping function (Van Ooijen and Voorrips 2001; Manly et al. 2001), and interval mapping was performed with MapQTL (v. 4.0; Van Ooijen et al. 2002). Interval mapping was performed as previously described, except that new analyses were used to map defensive-behavior QTL that combined two sets of phenotypic data. Individual z scores for the two correlated traits, the number of stings in 1 min, and ratings for the degree to which bees flew up at the beekeeper during colony manipulations were averaged to produce composite z scores used for interval mapping. The z scores were calculated using the formula z = (y−μ)/δ, in which y is the phenotypic value, μ is the mean, and δ is the standard deviation. This provides a new trait value with a mean of zero and standard deviation of one. The colonies were rated on a relative scale of one to five based on the researchers' experience for the tendency of bees to fly up during colony manipulations. Ratings from two observation periods were averaged. This analysis resulted in somewhat reduced CIs and higher LOD scores for the three QTLs (3.84, 2.25, and 2.39, respectively for sting1, sting2, and sting3). A map with 1,154 markers was used to locate candidate genes for "sting" QTLs, and two maps, each with about 400 markers, were used to locate "pln" QTLs. Markers in common between the dense map and other maps were used to interpolate the location of pln QTL and align QTL CIs with the physical map because more cloned markers were available from the dense-map population.
Simple interval mapping was used rather than multiple factor interval mapping to determine CIs to have a more conservative (inclusive) search for candidate genes. The genomic region within 1.5 LOD value of the LOD-score peak was used to define each CI for QTL location. This corresponds to an approximate 97% CI for QTL location in a dense-marker map (Dupuis and Siegmund 1999). The LOD score is a log-likelihood estimator, for the probability that a QTL influencing the trait is present at a given map position (Lander and Botstein 1989). The honeybee genome assembly (v. 3.0) and positions of sequenced markers were used to identify predicted peptides that were evaluated for likely gene function.
Cloning and sequencing marker fragments
Sequences derived from DNA markers were obtained to integrate physical and genetic maps. More than 300 marker fragments not only from (primarily) amplified fragment length polymorphic markers (AFLPs), but also from random amplified polymorphic DNAs (RAPDs) and microsatellites linked to behavioral QTLs and throughout the genome, were cloned and sequenced. The first step in cloning was the excision of fragments from gels. For AFLPs from polyacrylamide gels, products were re-amplified and resolved on agarose gels to verify correct size before cloning with the TOPO-TA cloning kit and the pCR4-TOPO vector (Invitrogen, Carlsbad CA). RAPD marker fragments were excised from agarose gels and cloned into the same vector. Multiple sequences were obtained from each clone, and the consensus sequence was aligned with the genomic sequence scaffolds (HGSC 2006) using the nucleotide–nucleotide basic local alignment search tool (blastn) algorithm with Pymood BLAST software (Allometra, Davis CA).
RACE and cDNA cloning
Before expression analyses by quantitative real-time polymerase chain reaction (qRT-PCR), cDNA cloning was performed to confirm sequences and the gene prediction (location of introns and exons) and to provide information on sequence variation within some of the candidate genes. In the case of the serotonin receptor, this process provided complete sequence of the gene by making primers based on sequence of a putative G protein-coupled receptor (GPCR). Rapid amplification of cDNA ends (RACE) and cloning was performed using kits and manufacturers' instructions. Total RNA was extracted from individual bees using the RNAqueous kit (Ambion, Austin TX). The cDNA was synthesized using the SMART PCR cDNA Synthesis kit (SMART, simple modular architecture research tool, BD Biosciences, Palo Alto CA). The cDNA clones were obtained using the TOPO-TA kit and the pCR4-TOPO vector (Invitrogen). Clones were sequenced from multiple worker bees. Several sequence reads were obtained from each clone.
Comparative bioinformatics
This study is based on predicted peptides from the draft sequence (HGSC 2006). However, gene ontology (GO) terms and functional annotation of many of the homologous genes are incomplete. For this reason, further analyses were performed. Predicted peptides from the HGSC "Glean3" dataset that were found within QTL CIs by first using blastn to determine which sequence scaffolds from the genome assembly contained sequences corresponding to markers within the CI. Then, scaffolds were searched for presence of predicted peptides using protein–nucleotide 6-frame translation (tblastn). Predicted peptides from the QTL CIs were used to search the nonredundant database (using protein–protein BLAST [blastp]) at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). Protein domain information, gene similarities, and GO terms of homologs or orthologs were recorded. Literature and website searches were performed to assign putative functions based on reports from homologous genes of various species.
Quantitative real-time PCR
Some candidate genes for defensive behavior QTLs were tested for differential expression to better evaluate their potential for influencing behavior. These were chosen from a list of genes we were initially interested in based on functional studies in other species. Two families of worker bees were used: a low-defensive source and high-defensive source, each having a queen naturally mated to about 12–17 drones. The source families differed in the number of stings per minute deposited in a leather target (0 for low line and more than 100 for the high-defensive source) using a standard assay (Hunt et al. 1998). Although it is not known at which life stage a gene might exert its influence on defensive behavior, bees that are 10- to 20-days old are much more likely to sting than younger bees. Workers were collected within 12 h of emergence from brood combs placed in an incubator or marked and co-fostered in an unrelated hive and then collected 20 days later. Co-fostering was performed to eliminate potential environmental effects between hives. All bees were frozen in liquid nitrogen and kept at −80°C before RNA extraction. Heads of eight to nine bees from each of the two families were removed, and RNA was extracted using the RNAqueous® kit (Ambion). RNA yield was quantified using Ribogreen™ (Molecular Probes) dye on a fluorometer (Turner Biosystems, Sunnyvale CA). An aliquot of RNA was treated with DNAse (DNAfree® kit from Ambion) to remove any genomic DNA contamination before cDNA synthesis.
The cDNA template for qRT-PCR was generated according to Puthoff et al. (2005). For first-strand cDNA synthesis, the SuperScript First Strand cDNA Synthesis kit (Invitrogen) was used as per manufacturers protocol. The cDNA synthesis was monitored in a parallel tracer reaction as follows. A 5-μl aliquot was removed from each sample and mixed with 1 μl of a 1:5 dilution of 32P–dCTP (dCTP, 2′-deoxycytidine 5′-triphosphate, Amersham, Piscataway, NJ) in water. The remaining 15 μl of each reverse transcriptase reaction and the corresponding 5-μl tracer reaction were incubated at 42°C for 2 h. Reactions were terminated at 70°C for 15 min then chilled on ice. The 32P-tracer reactions were used to quantify the amount of cDNA synthesized in the larger experimental samples. The 32P-tracer reactions were spotted onto DE-81 filters (Fisher, Fairlawn, NJ), dried for 10 min, and then washed four times for 4 min each in 0.5 M sodium phosphate buffer (1 M of monobasic and 1 M of dibasic in 4 l of water). After two 1-min rinses in water, filters were washed in 95% EtOH and allowed to dry. Each filter was placed in a scintillation vial containing 5 ml of ScintiVerse (Fisher), and radiation from the newly synthesized cDNA was quantified in a scintillation counter. Resulting counts were used to normalize the cDNA from the corresponding reverse transcriptase reactions to a final concentration of 10 ng per μl of sample.
qRT-PCR was conducted on an ABI 7000 using the following mixture: 2 μl of normalized cDNA, 10 μl of 2X SYBR Green Mix (ABI, Foster City CA), and 0.25 μM of each primer in a 20-μl reaction. Reactions were carried out using the following cycling parameters: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 10 s and 60°C for 1 min. At the end of each run, a melt curve analysis was conducted to ensure primer specificity and purity of the PCR product. Relative mRNA levels were calculated by the standard curve method (User Bulletin 2: ABI PRISM 7700 Sequence Detection System) as described here. An aliquot was taken from each cDNA to construct a pooled sample. This pooled sample was serially diluted and subjected to qRT-PCR. The threshold cycle (Ct) for each dilution was plotted against its cDNA concentration (with an arbitrary starting quantity for the undiluted pooled sample assigned the value of 1) and used as the standard curve regression equation to generate the arbitrary expression values (AEVs). A standard curve was generated for each target gene on the same PCR plate that held the experimental samples. Linear standard curves, with a slope between −3.5 and −3.2 and R 2 value of at least 0.98, were required for all primers used in this study. The AEVs were then normalized to the expression values of the eukaryotic initiation factor EIF-S8 for each bee. Two technical replicates for each bee were used, and results averaged before obtaining the family average. Normalized, average AEVs for each bee were analyzed by two-factor analysis of variance (ANOVA) without replication to compare transcript levels of the high-defensive family to the low-defensive family.
Results and discussion
Bioinformatic analyses of putative gene functions and results of qRT-PCR allowed us to form hypotheses concerning gene networks involved in either foraging or defensive behaviors. Results of analyses and hypotheses concerning genes with potential to influence behavior are presented in the following two sections.
Candidate genes for honeybee foraging behavior
Annotation of peptides within 97% confidence intervals for foraging-behavior QTLs
The pln-2 region contains two LOD-score peaks that were resolved into two CIs, suggesting separate but linked QTLs (Fig. 2b). The CI with the higher LOD score contains a bee ortholog of a nuclear hormone receptor (Dmel/HR46). Nuclear hormone receptors bind ligands (such as ecdysteroids) and enter the nucleus as transcription factors (Simonet et al. 2004). The bee ortholog of HR46 was differentially regulated in microarray experiments that compared bees with and without application of queen mandibular pheromone treatment, which inhibits worker ovarian development and delays onset of foraging behavior (Grozinger et al. 2003). The pln-2 region also contains the tyramine receptor AmTyr1, which has a higher expression level in young bees selected for high levels of pollen collection and storage (M. H. Humphries, unpublished data). A Drosophila mutant for the tyramine receptor (hono) is deficient in its behavioral response to olfactory stimuli, an observation that is relevant because honeybee foragers respond to floral scents and brood pheromone. In addition, tyramine increases responsiveness to sucrose and is found at elevated levels in the brains of egg-laying worker bees, a pattern that fits the behavioral and reproductive state of pollen foragers (Scheiner et al. 2004). The second CI associated with pln-2 contains an ortholog of Dmel/skittles, encoding 1-phosphatydylinositol-4-phosphate 5-kinase (PIP5K), one of two phospholipid kinases known to produce phosphoinositol 4,5 phosphate (PI4,5P2), which is a key metabolite involved in IIS and the substrate of class I PI3K activity (Fig. 4; Carricaburu et al. 2003). The Drosophila skittles mutant is deficient in oocyte polarity and nurse cell development, and the gene is essential for germ line development (Hassan et al. 1998; Table 1).
At the most likely position for pln-3 is the honeybee ortholog of the fly gene for a class II PI3K (PI3K 68D). Class II PI3Ks have been shown to respond to insulin signals in mammals and use phosphoinositol to produce phosphoinositol phosphate, which influences glucose transport, a common effect of IIS (MacDougall et al. 2004; Shepherd 2005). In addition, three predicted peptides with glucose transport domains were found in the pln-3 CI. It is unknown whether class II PI3Ks can act directly on PI4,5P2 to produce PI3,4,5P3, thereby stimulating the primary downstream kinase in the IIS pathway 3-phosphoinositide-dependent kinase 1 (PDK1). However, the honeybee gene encoding PDK1 also lies within the pln-3 CI. PDK1 is a positive regulator of cell growth and size through its action on downstream protein kinase B (PKB; Fig. 4; Rintelen et al. 2001), which, in Drosophila, is required for egg chamber development and influences egg follicle cell size (Cavaleire et al. 2005). The presence of these genes at pln QTLs suggests a possible network of genes influencing ovarian development and foraging (Fig. 4). Linkage of a PI3K and PDK1 at pln-3 is intriguing, especially given the presence of PIG-P and the bazooka ortholog at pln-1 and the PIP5K ortholog near pln-2. PIG-P has the potential to activate PI3K, and thus PDK1, whereas bazooka has been shown to bind the protein tyrosine phosphatase PTEN. This phosphatase is a negative regulator of PI3K because it dephosphorylates PI3,4,5P3, and binding of dPTEN by the product of the bazooka modulates this process (Von Stein et al. 2005). The convergence of these pathways also suggests the potential for interaction between pln-3 and both the pln-1 and pln-2 regions, which has been observed experimentally (Rueppell et al. 2004, 2006).
A search of the 10 cM window surrounding AmFOR (pln-4) revealed only three predicted peptides, one of which was the insulin receptor substrate (IRS). Although AmFOR was chosen as a candidate because of its influence on foraging behavior in Drosophila and association with foraging-related behavioral states in bees (Ben-Shahar 2005), the IRS could be partly or wholly responsible for the behavioral effects of this QTL. In Drosophila, ovarian expression of this gene is necessary for vitellogenesis, independent of the action of juvenile hormone and ecdysteroids (Richard et al. 2006).
When the phenotypic architecture of foraging behavior is taken into consideration (Fig. 1), the identification of genes encoding class II PI3K and PDK1 at pln-3 and other key components of IIS at pln-1, pln-2, and pln-4, supports the hypothesis that IIS is the upstream mediator of foraging division of labor (Fig. 4). These IIS components do not constitute random distributions of genes. We obtained a rough estimate of the likelihood that a gene lies within the CIs for foraging behavior QTLs by comparing the genetic size of these four QTL regions relative to the genome (about 145:4600 cM, or 0.03). There are 12 Drosophila genes in Flybase with the GO term for insulin receptor signaling pathway (GO: 0008286), so the expected number of these genes within the CIs is 0.36. However, bee orthologs for two of these genes occur within foraging behavior QTL CIs, at least 5.5 times the expected number assuming independence of gene distributions. In addition, orthologs of four of these 12 genes were not in genome sequence assigned to chromosomes and so could not be sampled in our analyses. We also found at least four other genes known to interact with IIS, but not annotated as such with GO terms, and one nuclear hormone receptor within the QTL regions, which are characterized by epistatic interactions suggesting the presence of components of a common pathway. In contrast, we could not find any genes influencing IIS within CIs for QTLs that influence defensive behavior, although they represent a region of comparable size.
Candidate genes for defensive behavior
Annotation of peptides within 97% confidence intervals for defensive-behavior QTLs
Expression of candidate genes in high-defensive bees relative to low-defensive worker bees
The primary stimuli that elicit stinging behavior are moving visual targets and alarm pheromone. The sting-2 region contains two obvious candidates for modulation of response to these stimuli. At the most-likely position of the QTL is the bee ortholog of Drosophila arr1 (AmArr4, Fig. 5b), an arrestin that binds metarhodopsin, the light-activated form of rhodopsin in the eye. Arrestins are involved in the desensitization of specific GPCRs and their recycling through clathrin-mediated endocytosis. But the so-called visual arrestins are also expressed in the antennae of Drosophila and are involved in olfaction. Fly arr1 mutants are insensitive to classes I and II odorants (Merrill et al. 2005). Near the edge of the CI is an ortholog of the fly gene encoding the metabotropic gamma-aminobutyric acid receptor (GABA-B-R1). GABA serves as the primary inhibitor of neuronal excitability in the CNS of both insects and mammals (Bettler et al. 2004).
The sting-3 CI contains only 17 predicted peptides in 0.96 Mb of DNA. Like sting-2, it has genes with the potential to modulate sensitivity to visual and olfactory stimuli. Results of cDNA sequencing of a putative GPCR revealed a 5-HT7 serotonin receptor (Am5HT 7, Fig. 5c). Serotonin influences associative learning and circadian rhythm in mollusks and insects, and both serotonin and GABA influence mood disorders in mammals (Hayley et al. 2005). Application of serotonin to the optic lobe of the bee brain reduced behavioral and neural responses to moving visual stimuli (Erber and Kloppenburg 1995). However, pharmacological experiments likely target all types of serotonergic neurons. The 5-HT7 receptor is just one of four serotonin receptor types known in insects and is unique in that it activates adenylate cyclase, resulting in increased levels of cyclic adenosine monophosphate (AMP) and activation of PKA. Therefore, activation of 5-HT7 receptors would likely cause a stimulatory response. Total serotonin levels have sometimes been reported to influence mammalian, insect, and crustacean aggressive interactions (Nelson and Chiavegatto 2001; Panksepp et al. 2003). Recent evidence suggests that 5-HT7 receptors modulate exploratory behavior and anxiety in mice (Takeda et al. 2005). The sting-3 region also contains one of the three catalytic subunits of cyclic AMP-dependent PKA, AmPKA-C1, a gene known to affect behavior in flies and mammals, including behavioral responses to alcohol, learning, and locomotor rhythm (Eisenhardt et al. 2001). Finally, the ortholog of homer, a dendritic gene involved in calcium signaling and synaptic plasticity, is closely linked to AmPKA-C1 (Diagana et al. 2002; Szumlinski et al. 2004).
Measurement of gene expression by qRT-PCR for defensive-behavior candidate genes showed several interesting trends. There were few statistically significant differences, because in this first screen, families were chosen that had divergent behavior but were genotypically diverse. Variation in expression between individuals was high, but numerical differences indicated a trend towards higher gene expression in older defensive bees. Only 14-3-3 Epsilon showed significantly higher transcript levels in the defensive family of bees. In contrast, the CG8165 ortholog, a putative jumonji-domain transcription factor, had numerically higher levels in newly emerged defensive adults, yet significantly lower levels in older defensive bees (Table 3). This suggests an earlier peak in expression of this transcription factor in defensive bees. It was interesting that the mRNA levels of the GABA-B-R1 receptor, part of the major inhibitory pathway of neural signaling, were significantly lower in newly emerged defensive bees. Transcript levels of seven other genes showed trends for higher expression in defensive bees at the >1.3-fold level. At sting1, an unknown carboxylesterase showed the greatest numerical expression difference of all, but its levels also showed the highest interindividual variability, resulting in no significant difference between families. As previously stated, this gene also showed the highest allelic variability in cDNA sequence. At sting2, both the ortholog of Dmel/discs lost and AmArr4 (arrestin) trended towards higher expression in defensive bees. Three other genes showed high numerical but not significantly different levels in defensive bees: oxysterol binding protein, the Dmel/tango ortholog, and the Am5HT 7 serotonin receptor (1.7-fold higher). Eight of the 19 genes tested appear to be more highly expressed in older defensive bees at 1.3-fold or higher although only one is significantly so. The fact that so many of these linked genes trended towards higher mRNA levels in defensive bees may mean that they are regionally regulated. More sampling of other bees and genomic regions is necessary. Some genes may be expressed at higher levels in high-defensive bees because of elevated metabolic rates (Harrison et al. 2005). Presumably, genes that influence behavior will show differences in expression levels of protein at some stage between low- and high-defensive alleles. However, demonstration of differences in transcript levels does not prove a causal connection to the phenotype. Conversely, failure to find a difference in transcript level does not disprove a causal connection to behavior and could be a consequence of not sampling the most relevant developmental stage, or that small differences in expression are still relevant to the phenotype.
The advantage of high recombination rates
Comparison of number of candidate genes in QTL confidence intervals from Apis and Drosophila a
Interval Size (cM)b
Number of QTL
Predicted no. of genes based on genetic or physical sizec
Actual no. of genes
Pollen foraging
Defensive behavior
Ovariole number
Our findings lead us to propose that foraging division of labor (Fig. 1) is influenced by a gene network involving IIS (Fig. 4). This is just a hypothesis, but a testable one. We also suggest that the genetically variable defensive responses of bees may be explained by allelic differences in neuronal transcription factors and genes involved in G protein-coupled signaling pathways. The potential involvement of the Am5HT 7 serotonin receptor in defensive/aggressive behavior implies that the bee may be used to elucidate a role of serotonin in novelty-seeking (guarding) behavior and that this behavior could be modified by specific agonists and antagonists of this receptor subtype. Linkage mapping at a finer scale using many single nucleotide polymorphisms combined with genome-wide expression assays could be the next step in finding the sequences responsible for behavioral variation. The bee is likely to become an important species in this process and may become the first invertebrate model for understanding how gene-regulation of life histories are remodeled by social evolution.
We thank T. F. C. Mackay and T. Flatt for valuable discussions and comments. This work was supported by the following grants: National Science Foundation, IBN 0110842, 0090482, 0076811, National Institute of Health grant nos. R29 GM548580, NIA PO1 AG22500, and US Department of Agriculture (USDA) 93-37302-8880, 35302-10137, and 2003-01620. This was a joint effort with USDA/Agricultural Research Service and was supported in part by USDA Current Research Information System number 3602-22000-020-00D.
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text:
Let's start with Lana Rain.
Mirubro edition #2.
Hello and welcome once again to Sunday /cara/, I'm back from the advent market in my old hometown and it's time to begin.
Make yourself a cup of tea and get comfy with us, will you?
Giving a FAQ:
>who is she and what does she do? i would google her but i doubt it would be worth the effort.
Cara Delevingne is primarily occupied with being the #1 qtpie waifu and stealing hearts of men and women. Other her hobbies include changing the world of fashion forever and being a pretty good actress.
Comment too long. Click here to view the full text.
I'm just about to hop in the shower and then I'll be back to funpost. My IT prof started teaching again on friday so i'll have to get up early tomorrow.
JP bro reporting in at 1 am. Gotta love stress induced all nighters.
Maybe I just sperged out but I made us an IRC channel to communicate thread timings or to talk about anything when the thread isn't up. I was expecting a midweek thread but apparently we agreed not to make one for example.
here's the link:
channel and password name is cara4life
el bumpo
My current favorite Japanese girl.
Post any sets you have!
Sexy sport bras
I'm trying to complete this set/pics of this girl and I know I'm missing some. Any help would be greatly appreciated!
I've just finished reorganising a few of my /s/ folders:
>Over saturated pictures
>British slags
Could post one of these, you decide.
Folder 1 example first
Folder 2
Folder 3
Folder 1 mate, no doubt!
Here's a few of mine, eh?
A vid of her
anyone interested in seeing this chicks nude i got of her?! hooked up with her this past weekend!
what do you think of her guys??! come on no one wants to see her nude pic?!
go on then show us
Dumping some pics, enjoy!

subset: commoncrawl


text:
Students at Lanigan Elementary School have full buckets and a reason to celebrate.
The Positive Behavioral Interventions and Supports (PBIS) committee at the school introduced in September the idea of promoting a positive learning environment through "bucket filling."
Students fill their metaphoric buckets each school day by demonstrating kindness, appreciation and acknowledging the school's following four behavioral expectations: Be Respectful, Be Responsible, Be Safe, and Be a Problem Solver.
With the first quarter of the school year behind them and their buckets filled to the brim, students at the school were rewarded with a PBIS celebration and an opportunity to watch a movie or participate in a fun activity of their choice.


text:
Comments Posted By Robert
Displaying 61 To 90 Of 219 Comments
the white collar criminal hit the little girl, who was his daughter, for steling candy.
he would not raise another crook.
» Posted By robert On 01.26.2012 @ 3:30 pm
I was in the sandy desert it was really hot through the day but cold at night. I wished I was somewhere with more water as I was really thirsty.
» Posted By Robert On 12.29.2011 @ 10:30 am
Patents have become completely obsolete in the modern age. As companies scoop up patents on idea after idea in order to simply levy law suits when someone gets around to inventing it, it discourages the innovation that made this country great.
» Posted By Robert On 11.08.2011 @ 6:59 pm
leaves color my world again. cold cuts at my neck and i'm sick of these buildings. i'd rather be somewhere i could enjoy this, but for now i'm stuck.
» Posted By robert On 11.07.2011 @ 12:29 pm
Downpour was his name, and he was ashamed of it. I mean, how could he not be? His parents were deaf, and they didnt know how anything sounded really, so when they said it, it just sounded stupid. To him it sounded stupid, maybe it wouldve been better if he wouldve known what it actually sounded like.
» Posted By Robert On 11.04.2011 @ 5:14 pm
I had a profound moment a day and half ago. I realized my true dream may not be to just finish college. Quite frankly, I just wanted to finish high school. I never had dreams about life after it. But now, almost three years in, I'm feeling some sort guilt.
» Posted By Robert On 11.02.2011 @ 8:39 pm
I hover. When I feel your touch, all I can do is hover. Hover over the land, the sky, the moon.
I hover when I feel your touch.
» Posted By Robert On 09.11.2011 @ 10:04 pm
Usually a female person. Most common in big firms and porn movies.
She can also be very helpfull around the office.
» Posted By Robert On 08.27.2011 @ 1:55 pm
I was walking through the streets of Madrid. I exited the Metro at "Ciudad Universitaria" and it started to rain. Of course, I didn't have my umbrella.
But I'm glad I didn't. I let the rain fall on my head and wash me clean.
» Posted By Robert On 08.24.2011 @ 9:59 pm
Today, I had to drop my dad off at the airport. At 4:55 in the morning.
As I went downstairs, I walked by the refrigerator and I remembered that I have some soy milk in there. Then I went to the car. And drove my dad. Then went back home. And fell back asleep. I didn't drink any Silk Soy Milk.
» Posted By Robert On 08.23.2011 @ 1:10 pm
sex pain naughty hurting crushed lonely heartbroken dead inside alone
» Posted By Robert On 08.14.2011 @ 3:39 pm
Lock, to keep out? Or to keep in? What are you locking? A room? A box? Something you cant see or hear? Why would you want to lock it? Is it dangerous?
» Posted By Robert On 08.05.2011 @ 8:42 pm
crashes. lightning crashes: its a song that penetrated my family's ears for years. That and dolphin's cry. Really it the band "Live" that got to us; the spirituality, the sheer beauty of the words.
» Posted By robert On 07.25.2011 @ 1:54 pm
When I was in high school, I broke my right arm. It happened during PE class while playing "touch" football. The strange thing is that I didn't realize it was broken until the next day when I drove myself to the doctor.
» Posted By Robert On 07.07.2011 @ 7:10 am
I am given a word. I find that ironic, I really do. As if to say, 'here, write this'. It's a gift, or something like that.
» Posted By Robert On 07.02.2011 @ 1:10 pm
cells… cells are what make us exist, they're pretty important. Plants have cells, humans have cells. I'm having a mindblank but did you know that cellphones do not contain the same kind of cells as plants? :O
Yeah, fact maestro. Ermm. hurry
» Posted By Robert On 06.21.2011 @ 3:28 pm
Man. I wish I could actually predict the future. Not for any real personal gain persay, just for personal easement-of-mind. I'd be so relaxed. But maybe i'd become super lazy, too. Hard to say. Hard to predict.
» Posted By Robert On 06.20.2011 @ 9:07 pm
Every day, a new series of memories is made. Blips and clips of families and friends, all wrapped up into some tiny little brain cells that you can keep around for as long as you want. I can't imagine going without such a power.
» Posted By Robert On 05.31.2011 @ 7:51 pm
Joe Mauer has the best sideburns ever. David Tennant's are awesome too. They're named after that Civil War guy, Burnside. I'd like to grow great sideburns also, but I don't know how well I'd be able to pull it off. Yeah.
» Posted By Robert On 05.29.2011 @ 8:25 pm
I hate thieves. Scum of the earth. They never think about the people they're stealing from, only themselves. And does it really help them to steal? I think not. Even if the thief doesn't feel guilty about what they do, which most of them do not, instead rationalizing their actions by saying things like "I deserve this." or, "That person doesn't need it anyway." I'd tell that person to try and obtain whatever they're stealing the honest, hard working way like everyone else. Then I'd take it from them, and say "See how that feels douchebag? Not cool is it?"
» Posted By Robert On 05.23.2011 @ 1:36 pm
sun earth people man women computer house houme dog cat mouse ,hamster,mom,dad
» Posted By Robert On 05.13.2011 @ 9:59 am
not much to write on this, consoles are hard to find
» Posted By robert On 05.11.2011 @ 8:01 am
and how descriptive it is. keeps one locked in, locked out. impenetrable. isolated. dead.
» Posted By Robert On 04.29.2011 @ 8:39 am
The changes came in waves that made is body shake. He didn't know what it was, and he didn't care. He wasn't intrigued, but felt that he should have been. This, in fact, was what in intrigued him. Not that he had no control over his body, but that he wasn't concerned that he had no control over his body. This, he knew, was a sign that he no longer wanted to live.
» Posted By Robert On 04.24.2011 @ 10:14 pm
a longing for something that you don't have. Water is only one kind of thing that fixes a single physiological thirst. People ache for things all of the time. Buddhists try and separate from feelings of thirst for anything.
» Posted By Robert On 04.10.2011 @ 11:42 pm
policies are the things that you have to abide by in order to do things on certain websites just like this one. why not follow them so that you can enjoy the fruits of some internet site? eh sound good?
» Posted By robert On 03.22.2011 @ 2:20 am
I was wandering through the store. I didn't know what I wanted to buy. Or even if I wanted to buy anything. I just knew that I had to be anywhere but home. For some reason, I felt better walking among the aisles of canned peas and thirteen brands of ketchup. I dont know what it is that made me come here.
» Posted By Robert On 03.15.2011 @ 10:06 am
The lssexpensie things are the less we ppreciate them. It seems that weys consider value along side expense.
» Posted By robert On 02.28.2011 @ 7:41 pm
Items that may get the job done… once!
» Posted By Robert On 02.28.2011 @ 4:03 pm
Back To Stats Page
strong… muscular i guess, though strong can mean emotionally, mentally strong as well. strong is the ability to deal with whatever comes your way, be it a heavy sofa, a puzzle with too many unknowns, or just a hard day at work.
» Posted By robert On 02.27.2011 @ 4:11 am


text:
About Me
Robert completed his Part II studies in Architecture at the University of Kent in 2018. He chose to specialise his time in his Masters exploring computational design as a method to generate form and space in different ways. He has previously had Architectural experience woking in the residential sector on new build and existing dwellings with a focus on technical design. During his time there he was also involved in commercial and masterplan projects. Previous to working in practice, he has spent time on site including assisting stonemasons in the restoration of a London townhouse. This was beneficial in gaining an understanding of basic construction processes. His architectural interests include the integration of new technologies and construction techniques to create more innovative and intuitive architectural solutions. Creating bespoke design design outcomes to benefit and enhance as the needs of environments continually develop.


text:
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New NIC-USB Loytec Network LonMark RS FT LPT TP TP | eBay
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text:
Cherokee (CHS) Class of 1963 Alumni List
Help us Update the Cherokee 1963 Class List by adding any names missing from the list below:
Registered Alumni
1963 Class List
Know Cherokee Class of 1963 graduates that are NOT on this List? Help us Update the 1963 Class List by adding missing names.
The most popular movie of 1963 was Cleopatra which has grossed over $48,000,000 since its release. The TV series The Beverly Hillbillies was the most popular show of 1963.
Hit Songs From 1963
Candy GirlFour Seasons
Tie Me Kangaroo Down, SportRoll Harris
From A Jack To A KingNed Miller
Walking The DogRufus Thomas
Little Red RoosterSam Cooke
The Night Has A Thousand EyesBobby Vee
Take These Chains From My HeartRay Charles
Shut DownBeach Boys
Fools Rush InRick Nelson
That Sunday, That SummerNat King Cole
Hit Movies From 1963
Reunions and Cherokee High School News
More 1963 alumni from Cherokee HS Cherokee, AL have posted profiles on Classmates.com®. Click here to register for free at Classmates.com® and view other 1963 alumni.
Alumni from the Cherokee High School class of 1963 that have been added to this alumni directory are shown on this page. All of the people on this page graduated in '63 from Cherokee . You can register for free to add your name to the CHS alumni directory.
If you are a Cherokee alumn, we encourage you to register and be sure to sign up for the alumni newsletter.


text:
...was born in Allentown, PA. Adam is a multi-instrumentalist (keyboards, guitar, vocals, drums) who writes & performs a variety of styles of music incorporating jazz, rock, funk, electronic, and hip-hop. Adam pursues music with the intent of inspiring hearts and fostering improvement in communication and concentration.
Adam has studied music with teachers Paul Amrod, Alex Gergar amongst others and has completed jazz program courses at Bucknell University, Moravian College, and Lebanon Valley College. Adam's musical adventures began with Vanilla Ice dance contests at the neighbor's house and followed with listening to classical music and learning the piano. Adam learned to play rock guitar and drums as a teen and eventually entered the jazz and progressive landscape. Over the years, Adam has had the opportunity to perform projects opening for musical artists such as Jurassic 5, Jaguar Wright, and Keller Williams and record on Meatloaf's Hang Cool Teddybear.
In 2010 in New York City, Adam became acquainted with Project K-Paz through common friends. After a few performances Adam joined the group, as there was a common path found through Spontaneous Music. Adam seeks to approach music with the fluidity that one may speak a language, seeing music similar to a river - at ease in it's natural flow. Adam's on-going project in New York, The Flowdown, is similarly an integrative band that bridges together various musical styles and personalities to convey peace through musical potency.


text:
STATEN ISLAND, N.Y. -- Wagner College has announced the appointment of Dr. Lily McNair as provost and vice president for academic affairs.
Dr. McNair is currently associate provost of research and professor of psychology at Spelman College in Atlanta, Ga. She will begin her new responsibilities at Wagner July 1.
She replaces Dr. Devorah Leiberman, who had announced she would be leaving at the end of the academic year to return to her home state of California to lead the University of La Verne, a comprehensive, independent university located 35 miles outside Los Angeles. She would be the first woman in the school's 119-year history to hold the position.
"Here at Wagner are delighted that Lily will be joining us as our next provost. She has a strong track record of working collaboratively with colleagues across academic departments to help them develop research opportunities and initiatives," Wagner College President Richard Guarasci said.
"Her superb leadership will continue Wagner's strong focus on student learning, civic engagement and global education."
Wagner College has become known across the country for its innovative curriculum, the Wagner Plan, which combines a foundation of classes in the liberal arts and science with hands-on learning in the community.
The Grymes Hill school has been ranked on U.S. News' list of Up and Coming colleges for each of the past two years, and has been cited for its curriculum plan by numerous publications and organizations.
A New Jersey native, Dr. McNair has served as professor of psychology on the faculties of the University of Georgia, Spelman College and the State University of New York College at New Paltz. She also spent two years at Vassar College as a clinical psychologist.
"I look forward to the opportunities my new role will offer. These are challenging times for higher education, but such times can serve as fertile ground for the emergence of creative, innovative and immensely promising ideas," she said. "I am happy to have this opportunity to contribute."
A clinical psychologist by training, Dr. McNair's research focuses on the development and evaluation of community-based interventions targeting substance use and early sexual behavior in African-American youth.
Her work has been funded by the National Institute of Mental Health as well as the Centers for Disease Control and Prevention. She also is the co-editor of "Women: Images and Realities, A Multicultural Anthology," an introductory women's studies textbook in its fourth edition.
Dr. McNair received her undergraduate degree in psychology at Princeton University, and her doctorate in psychology at the State University of New York at Stony Brook.
She has served as mentor professor and advisor to nearly a dozen doctoral students in clinical psychology at the University of Georgia, where she was actively involved in both undergraduate and graduate training.


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According to the Centers for Medicare and Medicaid Services, "dual eligible" beneficiaries refers to those qualifying for both Medicaid and Medicare.
A "dual eligible" demonstration program is also called an Integrated Care Delivery System (ICDS), which is a system of managed care plans selected to coordinate physical, behavioral, and long-term care services for individuals over the age of 18 who are eligible for both Medicaid and Medicare. "Demonstration" simply means that the program is in its pilot testing phase. MNS currently handles dual eligible demonstration programs for Ohio (MyCare) and Texas (StarPlus MMP). MNS is contracted with Molina, CareSource, and Buckeye for the MyCare Ohio program and Amerigroup Texas MMP for the Texas duals demonstration. We are looking to expand our network with other states in the future.
While both Medicaid and Medicare programs provide individuals with access to critical services and care, there has always been a lack of coordination between these programs. MyCare Ohio provides this coordination. Plus, MyCare Ohio plans may include additional services to their members. There is no additional cost to participate in this program.
For more information:
The second phase of the MyCare Ohio demonstration was effective January 1, 2015.
What does this mean?
Right now, people who live in the demonstration counties―and are eligible for Medicare and Medicaid―receive their Medicaid benefits through a MyCare Ohio managed care plan. On January 1, 2015, their Medicare coverage changed to the same managed care plan that provides their Medicaid benefits. This plan also covers prescription drugs. Members have the option to opt-out of the Medicare portion of the plan.
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If members want to receive their Medicare benefits and their Medicaid coverage through the same plan, you don't have to do anything.
If members want to change how they receive their Medicare benefits, you must call the Ohio Medicaid Consumer Hotline at 800-324-8680 and tell them you do not want to change your Medicare plan.
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Gringo Cover
Days after the self proclaimed Dancehall King and Zylofon's newest signee drops his best and most expensive video yet for Gringo, which got him trending for days, IWAN jumps on it to show he is still the best lyricist in the game.
Check out the original verson here; Shatta Wale – Gringo (Directed by Sesan)
Produced by Body beatz and recorded By Kv Bangers in RMG studios, IWAN talks about choosing lyrics over gimmicks.
Check out the this new cover from IWAN and don't forget to leave your comment below.


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About shannonsteeves
Boudoir slideshow of jackie & Ryan
shannonsteeves replied to JUANCARLOST's topic in Destination Wedding in CaboGorgeous Juan! Congrats.
Wedding Planning
shannonsteeves replied to dms8447's topic in Newbies!!Hi Dana, Congrats on your engagement! I know flights on Delta are all non-stop, even to Cancun so you've got a lot of options. Enjoy the forum, I know you'll get a lot of info! Shannon
Engaged in Paris
shannonsteeves replied to amandalovesryan's topic in Share your Wedding & Engagement Stories!Congratulations! Paris is a fantastic city. Shannon Steeves Photography firstname.lastname@example.com 941.375.3519
Quote: Originally Posted by march1508 hello ladies.. can anyone help? does anyone live in nyc within the 5 boroughs? My fh and I didnt take engagement photos, but I am thinking that now I wanna take them.. our wedding is march 08.I know its kind of late, but I really wanna use the photos and have a guest book created.. on each page there will be pictures of him and I so that they can view while signing the book..i saw it at a wedding that we attended and loved the idea.. can u recommend any place that u might have gone to or a friend? thanks so much.. octavia We'll be in NYC in early Feb. doing an engagement session with another couple. We're doing their shoot in Soho since they want to get street life/cafes in there. But the park is always beautiful for engagement shoots. Shannon Steeves Steeves Photography email@example.com
Newbie planning for Bahia Principe Coba
shannonsteeves replied to CarrieRN's topic in Newbies!!Quote: Originally Posted by CarrieRN Hi everyone, My fh and just started planning for our dw in Riviera Maya. I live in Pittsburgh and just discovered this forum. Tons of great info here. Dave and I are planning on Nov 6-13, 2008 with our wedding being on Nov 7, 2008. I'm trying to get everything done early so I have less to worry about. Carrie Hi Carrie, Congrats! Have fun planning. Shannon
St. Croix - Carambola Beach Resort?
shannonsteeves replied to Alyssa's topic in The Islands WeddingsQuote: Originally Posted by Mrs.B 2008 We are considering Carambola Beach Resort - Home - St. Croix, United States Virgin Islands for our wedding - has anyone been there or heard anything about it? so far - i know that John Rockefeller built Carambola and Caneel Bay (St. John) as family retreats and that Caneel Bay was bought by Rosewood Hotels and maintained and is 5 Star. Carambola apparently fell to the wasteside a bit and a new owner came in last year with $20Million and started extensive renovations. It looks pretty great and sounds even better from my conversations with them but if any of you ladies have any info it is greatly appreciated!!! Thanks, Alyssa I know Carambola is a nice resort. We were on St. Croix several months ago and they were completing renovations. We were doing a wedding at The Buccaneer Resort St Croix Hotels, Virgin Island Hotels- Luxury Rooms and Suites at The Buccaneer Resort Hotel and they did a great job for our couple. We were very impressed how nice the property was & how nice the staff was to everyone. Good luck deciding!
shannonsteeves replied to USMC_Wife2Be's topic in Florida WeddingsQuote: Originally Posted by USMC_Wife2Be Anyone know of any good and reasonably priced photographers in the Florida /Keys area? I currently have one booked that is traveling from Ohio. But there is a family illness that just came up and it may force him to back out. He's a great friend so I understand the situation, but now I'm looking around like nuts. Does anyone have any ideas? We're in Florida & would be able to help you out if you still need it.
any suggestions?
shannonsteeves replied to crys17tal's topic in Florida WeddingsQuote: Originally Posted by robindepaula Key West weddings, receptions & honeymoons - Ocean Key Wedding Resort, FL Key West Audubon House and Gardens, Weddings, Tour, Shop, Audubon Prints, Group Tours - key west florida These are two that caught my eye . . . The Keys seems like a great location for a state-side wedding!!! Happy planning! Robin If you're still looking for a location, I also recommend checking out the Hyatt. http://keywest.hyatt.com/hyatt/hotel...ings/index.jsp butI agree with Robin regarding the Ocean Key Resort. They are right at Duvall St., Old Town & the pier. Key West weddings, receptions & honeymoons - Ocean Key Wedding Resort, FL Good Luck! Shannon Steeves Photography firstname.lastname@example.com 941.375.3519
Newbie - Steeves Photography
shannonsteeves replied to shannonsteeves's topic in Newbies!!Thanks everybody!
Newbie - Steeves Photography
shannonsteeves posted a topic in Newbies!!Hi everybody! Just wanted to introduce myself and say congrats to all the brides. We're destination wedding photographers based out of Florida. We've shot weddings internationally for the past year and love working with brides. Please check us out & if you have any questions on certain venues feel free to ask. We've worked with some great resorts & private estates. Have fun!


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Ruth Bader Ginsburg Alive? Or Not?
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Published: 3 months ago
A concert was held in honor of Ruth Bader Ginsberg, but no photos of her attending have been released leading many to ask, "Where's Ruth?". Joe & Doug Hagmann discuss the possibility of a cover up.
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bettyboob4you , 58 years old
Raleigh, United States
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Tabitha turns 15 years old today. She is an amazing young woman. I liked to call her my little dynamo when she was one year old because she was this tiny little person, with a blonde afro, who never quit moving. She is still my dynamo. She likes to play and ref soccer, swim, ski and has recently taken up snowboarding. She adores nature and her dogs and often takes herself on a mountain hike with one of her dog brothers. She was the glue that held the Okanagan Lake Dip Challenge together. Not once did she complain; she was always the first one into the cold lake and always exited the water with a smile on her face.
She often has a mischievous grin and I know she is plotting some sort of adventure or fascinating project. Her mind is seldom at rest. She actually enjoys hunkering down at her desk to solve math problems. She is constantly creating some type of art work, design or baking a fabulous dessert. She loves to sing, play the piano and will begin cello lessons this month (she begged me to let her study the cello and I caved). Tabby spends hours devouring novels and taking photographs. I created the poster above by using several pictures from one of Tabs' self portrait photography series.
However, when Tabby does rest, she rests hard. She's always been one to sleep suddenly and deeply. She is very much Tabby the cat with whiskers.
The truth is, Tabitha is magic and I am honoured to be her mom.