Honey Bee Nutrition.

Optimal nutrition is crucial for honey bee colony growth and robust immune systems. Honey bee nutrition is complex and depends on the floral composition of the landscape. Foraging behavior of honey bees depends on both colony environment and external environment. There are significant gaps in knowledge regarding honey bee nutrition, and hence no optimal diet is available for honey bees, as there is for other livestock. In this review, we discuss (1) foraging behavior of honey bees, (2) nutritional needs, (3) nutritional supplements used by beekeepers, (4) probiotics, and (5) supplemental forage and efforts integrating floral diversity into cropping systems.

Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites.

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic mite Varroa destructor. This research was conducted to study associations between grooming behavior and the expression of selected immune, neural, detoxification, developmental and health-related genes. Individual bees tested in a laboratory assay for various levels of grooming behavior in response to V. destructor were also analyzed for gene expression. Intense groomers (IG) were most efficient in that they needed significantly less time to start grooming and fewer grooming attempts to successfully remove mites from their bodies than did light groomers (LG). In addition, the relative abundance of the neurexin-1 mRNA, was significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than in NG, but it was not different from that of control bees. The abundance of vitellogenin mRNA was not changed by grooming activity. However, the abundance of blue cheese mRNA was significantly reduced in IG compared to LG or NG, but not to control bees. Efficient removal of mites by IG correlated with different gene expression patterns in bees. These results suggest that the level of grooming behavior may be related to the expression pattern of vital honey bee genes. Neurexin-1, in particular, might be useful as a bio-marker for behavioral traits in bees.

Biased Allele Expression and Aggression in Hybrid Honeybees may be Influenced by Inappropriate Nuclear-Cytoplasmic Signaling.

Hybrid effects are often exhibited asymmetrically between reciprocal families. One way this could happen is if silencing of one parent's allele occurs in one lineage but not the other, which could affect the phenotypes of the hybrids asymmetrically by silencing that allele in only one of the hybrid families. We have previously tested for allele-specific expression biases in hybrids of European and Africanized honeybees and we found that there was an asymmetric overabundance of genes showing a maternal bias in the family with a European mother. Here, we further analyze allelic bias in these hybrids to ascertain whether they may underlie previously described asymmetries in metabolism and aggression in similar hybrid families and we speculate on what mechanisms may produce this biased allele usage. We find that there are over 500 genes that have some form of biased allele usage and over 200 of these are biased toward the maternal allele but only in the family with European maternity, mirroring the pattern observed for aggression and metabolic rate. This asymmetrically biased set is enriched for genes in loci associated with aggressive behavior and also for mitochondrial-localizing proteins. It contains many genes that play important roles in metabolic regulation. Moreover we find genes relating to the piwi-interacting RNA (piRNA) pathway, which is involved in chromatin modifications and epigenetic regulation and may help explain the mechanism underlying this asymmetric allele use. Based on these findings and previous work investigating aggression and metabolism in bees, we propose a novel hypothesis; that the asymmetric pattern of biased allele usage in these hybrids is a result of inappropriate use of piRNA-mediated nuclear-cytoplasmic signaling that is normally used to modulate aggression in honeybees. This is the first report of widespread asymmetric effects on allelic expression in hybrids and may represent a novel mechanism for gene regulation.

A Search for Parent-of-Origin Effects on Honey Bee Gene Expression.

Parent-specific gene expression (PSGE) is little known outside of mammals and plants. PSGE occurs when the expression level of a gene depends on whether an allele was inherited from the mother or the father. Kin selection theory predicts that there should be extensive PSGE in social insects because social insect parents can gain inclusive fitness benefits by silencing parental alleles in female offspring. We searched for evidence of PSGE in honey bees using transcriptomes from reciprocal crosses between European and Africanized strains. We found 46 transcripts with significant parent-of-origin effects on gene expression, many of which overexpressed the maternal allele. Interestingly, we also found a large proportion of genes showing a bias toward maternal alleles in only one of the reciprocal crosses. These results indicate that PSGE may occur in social insects. The nonreciprocal effects could be largely driven by hybrid incompatibility between these strains. Future work will help to determine if these are indeed parent-of-origin effects that can modulate inclusive fitness benefits.

Finding the missing honey bee genes: lessons learned from a genome upgrade.

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Fine-scale linkage mapping reveals a small set of candidate genes influencing honey bee grooming behavior in response to Varroa mites.

Populations of honey bees in North America have been experiencing high annual colony mortality for 15-20 years. Many apicultural researchers believe that introduced parasites called Varroa mites (V. destructor) are the most important factor in colony deaths. One important resistance mechanism that limits mite population growth in colonies is the ability of some lines of honey bees to groom mites from their bodies. To search for genes influencing this trait, we used an Illumina Bead Station genotyping array to determine the genotypes of several hundred worker bees at over a thousand single-nucleotide polymorphisms in a family that was apparently segregating for alleles influencing this behavior. Linkage analyses provided a genetic map with 1,313 markers anchored to genome sequence. Genotypes were analyzed for association with grooming behavior, measured as the time that individual bees took to initiate grooming after mites were placed on their thoraces. Quantitative-trait-locus interval mapping identified a single chromosomal region that was significant at the chromosome-wide level (p<0.05) on chromosome 5 with a LOD score of 2.72. The 95% confidence interval for quantitative trait locus location contained only 27 genes (honey bee official gene annotation set 2) including Atlastin, Ataxin and Neurexin-1 (AmNrx1), which have potential neurodevelopmental and behavioral effects. Atlastin and Ataxin homologs are associated with neurological diseases in humans. AmNrx1 codes for a presynaptic protein with many alternatively spliced isoforms. Neurexin-1 influences the growth, maintenance and maturation of synapses in the brain, as well as the type of receptors most prominent within synapses. Neurexin-1 has also been associated with autism spectrum disorder and schizophrenia in humans, and self-grooming behavior in mice.

High-resolution linkage analyses to identify genes that influence Varroa sensitive hygiene behavior in honey bees.

Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene 'no receptor potential A' and a dopamine receptor. 'No receptor potential A' is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection.

The effects of young brood on the foraging behavior of two strains of honey bees (Apis mellifera).

Honey bee foragers specialize on collecting pollen and nectar. Pollen foraging behavior is modulated by at least two stimuli within the nest: the presence of brood pheromone and young larvae and the quantity of stored pollen. Genetic variation in pollen foraging behavior has been demonstrated repeatedly. We used selected high and low pollen-hoarding strains of bees that differ dramatically in the quantity of pollen collected to determine if the observed differences in foraging could be explained by differential responses to brood stimuli. Workers from the high and low pollen-hoarding strains and wild-type bees were co-fostered in colonies with either brood or no brood. As expected based on previous studies, returning high pollen-hoarding foragers collected heavier pollen loads and lighter nectar loads than low pollen-hoarding bees. Effects of brood treatment were also observed; bees exposed to brood collected heavier pollen loads and initiated foraging earlier than those from broodless colonies. More specifically, brood treatment resulted in increased pollen foraging in high pollen-hoarding bees but did not affect pollen foraging in low pollen-hoarding bees, suggesting that high pollen-hoarding bees are more sensitive to the presence of brood. However, response to brood stimuli does not sufficiently explain the differences in foraging behavior between the strains since these differences persisted even in the absence of brood.

The effects of foraging role and genotype on light and sucrose responsiveness in honey bees (Apis mellifera L.).

In honey bees, the sensory system can be measured by touching sugar water to the antennae, eliciting the extension of the proboscis. The proboscis extension response (PER) [6,13] is closely associated with complex behavioral traits involving foraging and learning [30-32,34-36,43-49]. Bees specializing in pollen foraging are more responsive to low concentrations of sucrose solution and, as a consequence, perform better in associative learning assays [4,43,46-48]. An important unanswered question is whether sensory-motor differences between pollen and nectar specialists are restricted to the gustatory modality or whether pollen foragers are in general more sensitive to sensory stimuli associated with foraging. We used an assay designed to test responsiveness to varying intensities of light [11] and tested responsiveness to varying concentrations of sucrose in wild-type pollen and non-pollen foragers and bees artificially-selected for differences in pollen-hoarding behavior [27]. Workers of the high pollen-hoarding strain are more likely to specialize on collecting pollen. In wild-type bees, pollen foragers were more responsive to sucrose and light than non-pollen foragers. In the selected strains, high pollen-hoarding pre-foragers were more responsive to sucrose and light than low pollen-hoarding pre-foragers. These PER and light assays demonstrate a positive relationship between the gustatory and visual sensory modalities with respect to foraging behavior and genotype. We propose that light responsiveness, in addition to sucrose responsiveness, is a component of a pollen-hoarding behavioral syndrome - a suite of traits that covary with hoarding behavior [51,52] - previously described for honey bees [14,37,41]. We suggest that the modulation of the sensory system may be partially constrained by the interdependent modulation of multiple sensory modalities associated with hoarding and foraging.

Sensory response system of social behavior tied to female reproductive traits.

BACKGROUND: Honey bees display a complex set of anatomical, physiological, and behavioral traits that correlate with the colony storage of surplus pollen (pollen hoarding). We hypothesize that the association of these traits is a result of pleiotropy in a gene signaling network that was co-opted by natural selection to function in worker division of labor and foraging specialization. By acting on the gene network, selection can change a suite of traits, including stimulus/response relationships that affect individual foraging behavior and alter the colony level trait of pollen hoarding. The 'pollen-hoarding syndrome' of honey bees is the best documented syndrome of insect social organization. It can be exemplified as a link between reproductive anatomy (ovary size), physiology (yolk protein level), and foraging behavior in honey bee strains selected for pollen hoarding, a colony level trait. The syndrome gave rise to the forager-Reproductive Ground Plan Hypothesis (RGPH), which proposes that the regulatory control of foraging onset and foraging preference toward nectar or pollen was derived from a reproductive signaling network. This view was recently challenged. To resolve the controversy, we tested the associations between reproductive anatomy, physiology, and stimulus/response relationships of behavior in wild-type honey bees. METHODOLOGY/PRINCIPAL FINDINGS: Central to the stimulus/response relationships of honey bee foraging behavior and pollen hoarding is the behavioral trait of sensory sensitivity to sucrose (an important sugar in nectar). To test the linkage of reproductive traits and sensory response systems of social behavior, we measured sucrose responsiveness with the proboscis extension response (PER) assay and quantified ovary size and vitellogenin (yolk precursor) gene expression in 6-7-day-old bees by counting ovarioles (ovary filaments) and by using semiquantitative real time RT-PCR. We show that bees with larger ovaries (more ovarioles) are characterized by higher levels of vitellogenin mRNA expression and are more responsive to sucrose solutions, a trait that is central to division of labor and foraging specialization. CONCLUSIONS/SIGNIFICANCE: Our results establish that in wild-type honey bees, ovary size and vitellogenin mRNA level covary with the sucrose sensory response system, an important component of foraging behavior. This finding validates links between reproductive physiology and behavioral-trait associations of the pollen-hoarding syndrome of honey bees, and supports the forager-RGPH. Our data address a current evolutionary debate, and represent the first direct demonstration of the links between reproductive anatomy, physiology, and behavioral response systems that are central to the control of complex social behavior in insects.

Synthesis of an immobilized Bombyx mori pheromone-binding protein liquid chromatography stationary phase.

The pheromone-binding protein from the silkworm moth, Bombyx mori (BmorPBP) has been covalently bonded to a liquid chromatographic stationary phase. The resulting column was evaluated using radiolabeled bombykol and the immobilized protein retained its ability to bind this ligand. The data also demonstrate that the BmorPBP column was able to distinguish between four compounds, and rank them in their relative order of affinity for the protein from highest to lowest: bombykol>bombykal>1-hexadecanol>(Z,E)-5,7-dodecadien-1-ol, and that the immobilized BmorPBP retained its pH-dependent conformational mobility. The results of this study demonstrate that pheromone-binding protein from the silkworm moth, Bombyx mori and an odorant binding protein (OBP) obtained from the female mosquito Culex quinquefasciatoes have been immobilized on a silica support with retention of ligand-binding activity. The data indicate that proteins from non-mammalian organisms can be used to create liquid chromatography affinity columns.

Kinetics and molecular properties of pheromone binding and release.

Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an "on" rate of 0.068 +/- 0.01 microM(-1).s(-1). With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is approximately equal to 1 ms. A pH-dependent conformational change (BmorPBP(B) --> BmorPBP(A)), associated with the release of pheromone, is a first-order reaction (k = 74.1 +/- 0.32 s(-1); t(1/2), 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K(D) = 105 nM) at pH 7 but low affinity (K(D) = 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBPDeltaP129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.

Intriguing olfactory proteins from the yellow fever mosquito, Aedes aegypti.

Four antennae-specific proteins (AaegOBP1, AaegOBP2, AaegOBP3, and AaegASP1) were isolated from the yellow fever mosquito, Aedes aegypti and their full-length cDNAs were cloned. RT-PCR indicated that they are expressed in female and, to a lesser extent, in male antennae, but not in control tissues (legs). AaegOBP1 and AaegOBP3 showed significant similarity to previously identified mosquito odorant-binding proteins (OBPs) in cysteine spacing pattern and sequence. Two of the isolated proteins have a total of eight cysteine residues. The similarity of the spacing pattern of the cysteine residues and amino acid sequence to those of previously identified olfactory proteins suggests that one of the cysteine-rich proteins (AaegOBP2) is an OBP. The other (AaegASP1) did not belong to any group of known OBPs. Structural analyses indicate that six of the cysteine residues in AaegOBP2 are linked in a similar pattern to the previously known cysteine pairing in OBPs, i.e., Cys-24-Cys-55, Cys-51-Cys-104, Cys-95-Cys-113. The additional disulfide bridge, Cys-38-Cys-125, knits the extended C-terminal segment of the protein to a predicted alpha2-helix. As indicated by circular dichroism (CD) spectra, the extra rigidity seems to prevent the predicted formation of a C-terminal alpha-helix at low pH.