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This study was conducted to evaluate the Indiana “mite-biter” honey bee stock, which has been selected for increased mutilation of Varroa destructor mites (“mite biting” behavior). A comparison between colonies of the selected stock and colonies of unselected Italian bees showed that the proportion of mutilated mites, the severity of mutilations, and winter colony survival were higher in Indiana mite-biter colonies. Additionally, the number of fallen mites and the rate of mite population growth were lower in the colonies of the selected genotype than in those of the unselected genotype. The expression of a gene associated with grooming behavior, AmNrx-1 (neurexin), was significantly higher in the selected stock. Moreover, AmNrx-1 expression was positively correlated with the proportion of mutilated mites but not with mite population growth. AmNrx-1 may have the potential to be used for marker-assisted selection. This study provides evidence that selection for mite-biting behavior reduces V. destructor infestations, increases colony survival and increases the expression of a grooming behavior–associated gene.

Little is known about the combined effects of stressors on social immunity of honey bees (Apis mellifera) and related gene expression. The interaction between sublethal doses of a neurotoxin, clothianidin, and the ectoparasite, Varroa destructor, was examined by measuring differentially expressed genes (DEGs) in brains, deformed wing virus (DWV) and the proportion and intensity of self-grooming. Evidence for an interaction was observed between the stressors in a reduction in the proportion of intense groomers. Only the lowest dose of clothianidin alone reduced the proportion of self-groomers and increased DWV levels. V. destructor shared a higher proportion of DEGs with the combined stressors compared to clothianidin, indicating that the effects of V. destructor were more pervasive than those of clothianidin when they were combined. The number of up-regulated DEGs were reduced with the combined stressors compared to clothianidin alone, suggesting an interference with the impacts of clothianidin. Clothianidin and V. destructor affected DEGs from different biological pathways but shared impacts on pathways related to neurodegenerative disorders, like Alzheimer’s, which could be related to neurological dysfunction and may explain their negative impacts on grooming. This study shows that the combination of clothianidin and V. destructor resulted in a complex and non-additive interaction.

Sexual reproduction brings genes from two parents (matrigenes and patrigenes) together into one individual. These genes, despite being unrelated, should show nearly perfect cooperation because each gains equally through the production of offspring. However, an individual's matrigenes and patrigenes can have different probabilities of being present in other relatives, so kin selection could act on them differently. Such intragenomic conflict could be implemented by partial or complete silencing (imprinting) of an allele by one of the parents. Evidence supporting this theory is seen in offspring-mother interactions, with patrigenes favoring acquisition of more of the mother's resources if some of the costs fall on half-siblings who do not share the patrigene. The kinship theory of intragenomic conflict is little tested in other contexts, but it predicts that matrigene-patrigene conflict may be rife in social insects. We tested the hypothesis that honey bee worker reproduction is promoted more by patrigenes than matrigenes by comparing across nine reciprocal crosses of two distinct genetic stocks. As predicted, hybrid workers show reproductive trait characteristics of their paternal stock, (indicating enhanced activity of the patrigenes on these traits), greater patrigenic than matrigenic expression, and significantly increased patrigenic-biased expression in reproductive workers. These results support both the general prediction that matrigene-patrigene conflict occurs in social insects and the specific prediction that honey bee worker reproduction is driven more by patrigenes. The success of these predictions suggests that intragenomic conflict may occur in many contexts where matrigenes and patrigenes have different relatednesses to affected kin.

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.

The adaptive advantage of the eusocial lifestyle is evident from the fact that social insects represent more than half of the world's arthropod biomass. This topic explores how the recent advances in genomics and epigenomics are helping researchers to ask and answer questions concerning the evolution of social behavior and the genetic and epigenetic mechanisms behind phenotypic plasticity, i.e., how environmental signals can morph the same genome in a reproductive or non-reproductive individual resulting in dramatically different phenotypes. The articles in this research topic deal broadly with the evolution of reproductive and sterile castes (workers), mechanisms of caste determination, and the role of epigenetic processes for division of labor. The termites were the first group of insects to evolve eusociality and a thorough review describes what is known about the development of subcastes from a mechanistic perspective (nymphs, workers, soldiers) and the genomic contributions of gut symbionts and their hosts in digestion of wood, and the role of symbionts in host fitness (Scharf). Korb et al. compares the genomes of two termites with contrasting social complexities and symbioses. One of the interesting findings was that gene families involved in chemical communication in other social insects are not expanded in termites with more complex social organization. But transposable elements are, suggesting a role for transposition in social evolution but perhaps also pointing toward other mechanisms.

Fusarium graminearum Schwabe (Fusarium head blight, FHB) and Puccinia triticina Eriks (leaf rust) are two major fungal pathogens posing a continuous threat to the wheat crop; consequently, identifying resistance genes from various sources is always of importance to wheat breeders. We identified tightly linked single nucleotide polymorphism (SNP) markers for the FHB resistance quantitative trait locus (QTL) Qfhs.pur-7EL and the leaf rust resistance locus Lr19 using genotyping-by-sequencing (GBS) in a wheat–tall wheatgrass introgression-derived recombinant inbred line (RIL) population. One thousand and seven hundred high-confidence SNPs were used to conduct the linkage and QTL analysis. Qfhs.pur-7EL was mapped to a 2.9 cM region containing four markers within a 43.6 cM segment of wheatgrass chromosome 7el2 that was translocated onto wheat chromosome 7DL. Lr19 from 7el1 was mapped to a 1.21 cM region containing two markers in the same area, in repulsion. Five lines were identified with the resistance-associated SNP alleles for Qfhs.pur-7EL and Lr19 in coupling. Two SNP markers in the Qfhs.pur-7EL region were converted into PCR-based KASP markers. Investigation of the genetic characteristics of the parental lines of this RIL population indicated that they are translocation lines in two different wheat cultivar genetic backgrounds instead of 7E–7D substitution lines in Thatcher wheat background, as previously reported in the literature.

In order to identify genes that are influencing defensive behaviors, we have taken a new approach by dissecting colony-level defensive behavior into individual behavioral measurements using two families containing backcross workers from matings involving European and Africanized bees. We removed the social context from stinging behavior by using a laboratory assay to measure the stinging response of individual bees. A mild shock was given to bees using a constant-current stimulator. The time it took bees to sting in response to this stimulus was recorded. In addition, bees that were seen performing guard behaviors at the hive entrance were collected. We performed QTL mapping in two backcross families with SNP probes within genes and identified two new QTL regions for stinging behavior and another QTL region for guarding behavior. We also identified several candidate genes involved in neural signaling, neural development and muscle development that may be influencing stinging and guarding behaviors. The lack of overlap between these regions and previous defensive behavior QTL underscores the complexity of this behavior and increases our understanding of its genetic architecture.

Invertebrate models have greatly furthered our understanding of ethanol sensitivity and alcohol addiction. The honey bee (Apis mellifera), a widely used behavioral model, is valuable for comparative studies. A quantitative trait locus (QTL) mapping experiment was designed to identify QTL and genes influencing ethanol vapor sensitivity. A backcross mating between ethanol-sensitive and resistant lines resulted in worker offspring that were tested for sensitivity to the sedative effects of alcohol. A linkage map was constructed with over 500 amplified fragment length polymorphism (AFLP) and sequence-tagged site (STS) markers. Four QTL were identified from three linkage groups with log of odds ratio (LOD) scores of 2.28, 2.26, 2.23, and 2.02. DNA from markers within and near QTL were cloned and sequenced, and this data was utilized to integrate our map with the physical honey bee genome. Many candidate genes were identified that influence synaptic transmission, neuronal growth, and detoxification. Others affect lipid synthesis, apoptosis, alcohol metabolism, cAMP signaling, and electron transport. These results are relevant because they present the first search for QTL that affect resistance to acute ethanol exposure in an invertebrate, could be useful for comparative genomic purposes, and lend credence to the use of honey bees as biomedical models of alcohol metabolism and sensitivity.

Several candidate genes identified from quantitative trait loci (QTL) for defensive behavior in honey bees (Apis mellifera L.) are homologous to genes known to influence ethanol sensitivity in other organisms. To investigate this possible link between aggression/defense and ethanol sensitivity, assays were developed to evaluate ethanol vapor responses in worker bees from a low-defensive (gentle) colony and a high-defensive colony. Defensive workers exhibited characteristic signs of ethanol-induced sedation significantly faster than gentle workers upon exposure to ethanol vapor. Backcross workers displayed ethanol sensitivity intermediate to the parental defensive and gentle lines, suggesting a genetic basis for the trait. Workers were screened with sequence-tagged site markers linked to three defensive-behavior QTL and their genotypes were tested for associations with ethanol sensitivity. There were no significant associations, indicating that the defensive QTL were not having a pleiotropic effect on ethanol sensitivity. It is possible that gentle-source alleles at these QTL are dominant with respect to sensitivity, one or more of these QTL were not segregating in the backcross family, or unidentified QTL are influencing alcohol sensitivity.

SummaryQueen honey bees were fed Nosema apis spores in sucrose solution, returned to their hives, and examined later for N. apis infection by a polymerase chain reaction test. Eggs, larvae and pupae from the hives were also examined for infection on three observation dates during a 39 day period following the inoculation of the queens. Six of seven surviving queens developed N. apis infections in their ventriculi, but none had detectable N. apis in their ovaries. No eggs, larvae or pupae taken from the hives of Nosema inoculated queens contained detectable N. apis. These results suggest that N. apis is not transmitted vertically, unlike many other Microsporidia in other invertebrate hosts.In an effort to determine if the stress of shipping increased the susceptibility of Nosema exposed bees, another set of mated, queen honey bees was fed N. apis spores in sucrose, and the queens were then either mailed in small shipping cages, from Kentucky to Indiana and back, or kept caged for the same period in the lab...

Background Varroa mites are widely considered the biggest honey bee health problem worldwide. Until recently, Varroa jacobsoni has been found to live and reproduce only in Asian honey bee (Apis cerana) colonies, while V. destructor successfully reproduces in both A. cerana and A. mellifera colonies. However, we have identified an island population of V. jacobsoni that is highly destructive to A. mellifera, the primary species used for pollination and honey production. The ability of these populations of mites to cross the host species boundary potentially represents an enormous threat to apiculture, and is presumably due to genetic variation that exists among populations of V. jacobsoni that influences gene expression and reproductive status. In this work, we investigate differences in gene expression between populations of V. jacobsoni reproducing on A. cerana and those either reproducing or not capable of reproducing on A. mellifera, in order to gain insight into differences that allow V. jacobsoni to overcome its normal species tropism. Results We sequenced and assembled a de novo transcriptome of V. jacobsoni. We also performed a differential gene expression analysis contrasting biological replicates of V. jacobsoni populations that differ in their ability to reproduce on A. mellifera. Using the edgeR, EBSeq and DESeq R packages for differential gene expression analysis, we found 287 differentially expressed genes (FDR ≤ 0.05), of which 91% were up regulated in mites reproducing on A. mellifera. In addition, mites found reproducing on A. mellifera showed substantially more variation in expression among replicates. We searched for orthologous genes in public databases and were able to associate 100 of these 287 differentially expressed genes with a functional description. Conclusions There is differential gene expression between the two mite groups, with more variation in gene expression among mites that were able to reproduce on A. mellifera. A small set of genes showed reduced expression in mites on the A. mellifera host, including putative transcription factors and digestive tract developmental genes. The vast majority of differentially expressed genes were up-regulated in this host. This gene set showed enrichment for genes associated with mitochondrial respiratory function and apoptosis, suggesting that mites on this host may be experiencing higher stress, and may be less optimally adapted to parasitize it. Some genes involved in reproduction and oogenesis were also overexpressed, which should be further studied in regards to this host shift. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3130-3) contains supplementary material, which is available to authorized users.

This study was conducted to analyze the stinging response thresholds of individual European and Africanized worker honeybees (Apis mellifera L.) to electrical stimulation. Newly emerged workers were identified, and either were placed into an incubator, into their natal colonies, or cross-fostered in common colonies of European or Africanized ancestry. Nest and guard bees of each type were collected and exposed to an electric stimulus of 0.5 mA, and the time they took to sting a leather substrate was recorded. Africanized bees consistently had significant lower thresholds of defensive response than European bees across all of the environments tested. Guards were faster to sting than nest bees only for the Africanized genotype, suggesting that alleles of African origin have pleiotropic effects on guarding and stinging. This is the first study that shows that single individuals specialized in guarding also may have a lower response threshold for stinging. Environmental effects were also evident. In all cases, bees responded faster to the electrical stimulation after being kept in environments other than their natal nest. Moreover, significant genotype by environment and genotype by task specialization interactions were found. Our results fit a model of division of labor based on differences in response thresholds to stimuli among workers of different genotypes and task groups that result in non-additive effects on colony behavior.

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.

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.

The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.

This study was conducted to detect binary trait loci (BTLs) that insuence guarding behavior of individual honey bees, Apis mellifera L., andto locate genetic markers that are associated with these BTLs on genetic maps d erivedfrom guardbees from two reciprocal backcross colonies. Samples of guards and control bees were taken from two backcross colonies derived from a defensive colony anda gentle colony. AmpliÞedfragment length polymorphism (AFLP) markers were produced from DNA samples of guards. Two genetic maps were generated, one for each type of colony. A chi-square goodness-of-Þt test was performed for each marker in the map to look for deviations from the 1:1 segregation of the markers. For those markers that signiÞcantly deviated from the 1:1 ratio in the guards, AFLPs were generated from two samples of control bees, one for each type of backcross. Those markers that showeda skewedsegregation pattern in the guards but not in the controls were analyzedwith a 2 2 chi-square to test for associations between the markers andthe expression of the trait. Ten markers were associatedwith guarding behavior (Þve in each backcross). The 10 markers representedseven putative BTLs that insuence honey bee guard ing behavior. One of the BTLs represents a QTL that was previously detected in analysis of colony-level stinging response, others represent new loci speciÞc to the behavior of individuals guarding the colony entrance.

Honeybees have an age-based division of labour that is influenced by genetic variability for the tendency to perform specific tasks. Individuals in a honeybee colony comprise diverse genotypes and their interactions can influence task allocation. Colonies from an African race (Africanized honeybees, AHB, Apis mellifera scutellata Ruttner) usually produce a much stronger defensive response than do European races of honeybees (EHB), and these races may differ in how individuals are allocated to the tasks of guarding and stinging. We observed guarding behaviour in colony environments that varied in proportions of genotypes (AHB, EHB) and population size. In large colonies, AHB showed much greater guarding persistence (number of days guarding) than EHB; hybrids were intermediate. In another series of experiments, three families each of AHB and EHB were cofostered in colonies with different AHB: EHB ratios, then tested in large and small colonies. In colonies of both sizes, colony environment interacted with both famly and type (AHB or EHB) for propensity to guard. Individuals of both types guarded more persistently in large colonies, but family and type both interacted with environment. EHB were more likely to initiate guarding bouts in low-AHB colonies, but persistence did not change with environment. AHB were insensitive to effects of environment for the tendency to initiate guarding behaviour, but were more persistent in high-AHB environments. EHB and AHB may differ in how they allocate individuals to guarding. The positive reinforcement of behaviour that occurs in high-defensive environments and in large populations could cause a stronger stinging response through alarm pheromone recruitment.  2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.

SUMMARYMany assays for measuring defensive behaviour of honey bee (Apis mellifera) colonies are reported in the literature, but their reliability in most cases has not been assessed. This study was conducted to determine the reliability and convenience of four field assays: (1) a test to score behavioural traits that differ between defensive and gentle bees (ratings assay), (2) two stinging tests in which a leather patch is presented to the bees above the brood chamber (flag assay), or (3) in a confined space (box assay), and (4) a test to measure the numbers of bees recruited at the hive entrance in response to a chemical stimulus (pheromone assay). Six colonies each of three different bee types known to vary in their defensive responses (European, Africanized, and hybrid) were established and tested with the assays. To determine the reliability of the assays, coefficients of variation (CV) among trials for single colonies were calculated. Additionally, colonies were ranked according to their degree of d...

This study was conducted to test for the effect of three stinging behaviors QTLs (sting-1, sting-2 and sting-3) on the expression of guarding and stinging behavior of individual honey bees, and to determine if results of defensive behavior QTLs found in studies with Africanized honey bees could be extended to other populations of bees. Samples of guards, stingers, foragers and nurse bees were taken from two backcross colonies derived from a defensive colony and a gentle colony. The genotype of each bee for both types of colonies was determined for two sequence tagged site (STS) markers linked to sting-1 and for another two STSs, one linked to sting-2 and one linked to sting-3. Results showed that sting-1 had an effect on the expression of both stinging and guarding behaviors, sting-2 and sting-3 influenced the expression of guarding behavior. These results indicate that division of labor is influenced by specific QTLs. Results also show that QTLs mapped in a population of Africanized honey bees using colony level phenotypes also influenced the expression of guarding and stinging behavior of individual bees of other populations.

We analyzed the alarm pheromone components from five colonies of Africanized honeybees and three colonies of European honeybees collected in Mexico. Analyses revealed a novel alarm pheromone component that was only present in appreciable quantities in the Africanized bee samples. Analysis of the mass spectrum and subsequent synthesis confirmed that this compound is 3-methyl-2-buten-1-yl acetate (3M2BA), an unsaturated derivative of IPA. In Africanized honeybees, sampling from stings of guards showed that 3M2BA was present at levels of 0–38% the amount of isoamyl acetate (IPA). Behavioral assays from three colonies each of Africanized and European bees showed that 3M2BA recruited worker bees from hives of both Africanized bees and European bees at least as efficiently as isopentyl acetate IPA, a compound widely reported to have the highest activity for releasing alarm and stinging behavior in honeybees. However, a mixture of of 3M2BA and IPA (1:2) recruited bees more efficiently than either of the compounds alone. None of the compounds differed in their efficacy for inducing bees to pursue the observers.

Background Meiotic recombination has traditionally been explained based on the structural requirement to stabilize homologous chromosome pairs to ensure their proper meiotic segregation. Competing hypotheses seek to explain the emerging findings of significant heterogeneity in recombination rates within and between genomes, but intraspecific comparisons of genome-wide recombination patterns are rare. The honey bee (Apis mellifera) exhibits the highest rate of genomic recombination among multicellular animals with about five cross-over events per chromatid. Results Here, we present a comparative analysis of recombination rates across eight genetic linkage maps of the honey bee genome to investigate which genomic sequence features are correlated with recombination rate and with its variation across the eight data sets, ranging in average marker spacing ranging from 1 Mbp to 120 kbp. Overall, we found that GC content explained best the variation in local recombination rate along chromosomes at the analyzed 100 kbp scale. In contrast, variation among the different maps was correlated to the abundance of microsatellites and several specific tri- and tetra-nucleotides. Conclusions The combined evidence from eight medium-scale recombination maps of the honey bee genome suggests that recombination rate variation in this highly recombining genome might be due to the DNA configuration instead of distinct sequence motifs. However, more fine-scale analyses are needed. The empirical basis of eight differing genetic maps allowed for robust conclusions about the correlates of the local recombination rates and enabled the study of the relation between DNA features and variability in local recombination rates, which is particularly relevant in the honey bee genome with its exceptionally high recombination rate. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1281-2) contains supplementary material, which is available to authorized users.

Genetic correlations for behavioral characteristics and forewing length of worker honey bees, Apis mellifera L., were estimated. All characteristics associated with defensive behavior were correlated with each other. The tendency of bees to fly off the combs was correlated with their tendency to run on the combs, and with stinging behavior. Tendency to run was positively correlated with tendency to hang from combs, and with hygienic behavior, but was negatively correlated with forewing length. Forewing length was negatively correlated with hygienic behavior, but it was not significantly correlated with stinging behavior. The correlations obtained suggest that smaller bees have higher activity levels than larger bees. The implications of these results on the adaptive success of Africanized honey bee populations to tropical environments and on selective breeding are discussed.

The stinging and guarding components of the defensive behavior of European, Africanized, hybrid, and backcross honeybees (Apis mellifera L.) were compared and analyzed at both colony and individual levels. Hybrid and Africanized backcross colonies stung as many times as Africanized ones. European backcross colonies stung more than European bees but not as many times as Africanized or Africanized backcross colonies. The degree of dominance for the number of times that worker bees stung a leather patch was estimated to be 84.3%, 200.8%, and 145.8% for hybrid, backcross European, and backcross Africanized colonies, respectively. Additionally, both guards at the colony entrance and fast-stinging workers of one European backcross colony had a significantly higher frequency of an Africanized DNA marker allele, located near "sting1," a QTL previously implicated in stinging behavior at the colony level. However, guards and fast-stinging bees from a backcross to the Africanized parental colony did not differ from control bees in their frequency for the Africanized and European markers, as would be expected if large genetic dominance effects for sting1 exist. These results support the hypothesis that genetic dominance influences the defensive behavior of honeybees and confirm the effect of sting1 on the defensiveness of individual worker bees.

Increasingly, studies demonstrate significant intra-specific variation in genome-wide recombination rates, supporting the notion that local rates of meiotic recombination are evolving. The honey bee, Apis mellifera, exhibits the highest rate of recombination across the genome among multicellular animals. Multiple linkage maps have been constructed and agree on the overall high recombination rate but local rates have not been compared in detail. Here, we compared eight genome-wide recombination maps at different scales to assess how they relate to each other. We found that variation from a 50 to 1,000 kbp scale does not systematically affect the moderate correlations among the recombination maps. Individual chromosomes differed from each other in how much recombination rates were conserved but no apparent relation to chromosome size or average recombination existed. Finally, the overall similarities among maps related more to methodological than to mapping population coalescence. Therefore, recombination maps that were constructed using different methods should be compared with caution and results that are derived from such multiple data sets should prove more robust than analyses of single maps, irrespective of intra-specific variation in recombination rates.

The complete mitochondrial genome from an Africanized honey bee population (AHB, derived from Apis mellifera scutellata) was assembled and analyzed. The mitogenome is 16,411 bp long and contains the same gene repertoire and gene order as the European honey bee (13 protein coding genes, 22 tRNA genes and 2 rRNA genes). ND4 appears to use an alternate start codon and the long rRNA gene is 48 bp shorter in AHB due to a deletion in a terminal AT dinucleotide repeat. The dihydrouracil arm is missing from tRNA-Ser (AGN) and tRNA-Glu is missing the TV loop. The A + T content is comparable to the European honey bee (84.7%), which increases to 95% for the 3rd position in the protein coding genes.

We demonstrate the effects of a new quantitative trait locus (QTL), designated pln3, that was mapped in a backcross population derived from strains of bees selected for the amount of pollen they store in combs. We independently confirmed pln3 by demonstrating its effects on individual foraging behavior, as we did previously for QTLs pln1 and pln2 (Hunt et al. 1995). QTL pln2 is very robust in its effects on foraging behavior. In this study, pln2 was again shown to affect individual foraging behavior of workers derived from a hybrid backcross of the selected strains. In addition, pln2 was shown to affect the amount of pollen stored in combs of colonies derived from a wide cross of European and Africanized honeybees. This is noteworthy because it demonstrates that we can map QTLs for behavior in interstrain crosses derived from selective breeding and study their effects in unselected, natural populations. The results we present also demonstrate the repeatability of finding QTLs with measurable effects, even after outcrossing selected strains, suggesting that there is a relatively small subset of QTLs with major effects segregating in the population from which we selected our founding breeding populations. The different QTLs, pln1, pln2, and pln3, appear to have different effects, revealing the complex genetic architecture of honeybee foraging behavior.

A polymerase chain reaction (PCR)-based restriction fragment-length polymorphism (RFLP) assay was developed that discriminates among the 4 mitotypes found in North, Central, and South American honey bee racial groups—eastern European (Apis mellifera ligustica Spinola, caucasica Gorbachev, and carnica Pollman), western European (A. m. mellifera Linnaeus), Egyptian (A. m. lamarckii Cockerell Lepeletier), and sub-Saharan African (A. m. scutellata). Before the development of this assay, 13% of southern Californian feral bees collected before the arrival of the Apis mellifera scutellata (Africanized) race were found to contain a non-European mitochondrial genotype that could not be distinguished from that of A. m. scutellata. DNA sequence analysis suggests the unusual mitotype to be that of A. m. lamarckii. An RFLP polymorphism was identified that distinguished this subspecies from all others present in North America. This polymorphism was not found in any of 96 bees collected primarily in Mexico and Central America. Thus, the Egyptian mitochondrial type is either absent or extremely rare in these regions. The PCR assay also distin- guishes A. m. lamarckii from 2 other north African racial types, A. m. intermissa Buttel-Reepen and A. m. sahariensis Baldensperger.

Quantitative trait loci (QTL) mapping procedures were used to identify loci that influence the levels of alarm pheromones found in the stinging apparatus of worker honeybees. An F1 queen was produced from a cross between a queen of European origin and a drone descended from an African subspecies. Haploid drones from the hybrid queen were individually backcrossed to European queens to produce 172 colonies. Samples of stings were taken from backcross workers of these colonies. Alarm pheromone levels were determined by gas chromatography. RAPD markers were scored from the haploid drone fathers of these colonies. The multiple-QTL model (MQM) of MapQTL was used to identify QTLs that influence the levels of four alarm pheromone components. Seven independent, potential QTLs were identified with LOD scores greater than two, and one at LOD 1.88. We identified one QTL for n-decyl acetate, three for n-octanol, four for isopentyl acetate, and one for hexyl acetate. One region of linkage group XI shows a strong influence on body size and the levels of three alarm pheromone components. This locus explained 40% of the variance for the amount of n-decyl acetate (LOD 6.57). In general, the QTLs influencing alarm pheromone levels were independent of previously identified loci that influenced the stinging behavior of these colonies. The only exception was a potential locus influencing levels of n-octanol, which was inversely correlated with stinging behavior.

We propose an Africanized honey bee identification strategy using morphometrics and an improved polymerase chain reaction (PCR)-based mitotyping procedure that distinguishes between feral and commercial bees maternally descendent from 4 racial groups-Eastern European (Apis mellifera ligustica, caucasica, and carnica), Western European (A. m. mellifera), Egyptian (A. m. lamarckii), and other African origins. Mitochondrial genotype is highly correlated with morphology. Ninety-five percent of morphometrically determined Africanized feral colonies collected in Texas, Arizona, California, and Mexico also contained African mitochondria. Sixty-two percent of colonies from commercial or minimally managed apiaries in Mexico and Central America, with Africanized forewing lengths and 17% of colonies with intermediate forewing lengths, had African mitochondria. The strong correlation between non-European morphology and African mitotype, as well as the speed and accuracy of mitotype determination, suggest a 3-step Africanized bee identification procedure. This identification procedure first examines forewing length. Bees with lengths above a given threshold (9.12 mm) have a very high probability of being pure European in origin and are not examined further. Those bees with wing lengths below the threshold are subjected to mitochondrial analysis (mitotyping). Samples having African mitochondria are not examined further. Those bees with small forewing lengths, but European mitotypes, are then identified using detailed morphometric discriminant function analysis. By performing these steps in sequence, the number of bees requiring full morphometric analysis is reduced, saving time and improving the accuracy of Africanized honey bee identification.

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. Funding for the project was provided by a grant to RG from the National Human Genome Research Institute, National Institutes of Health (NHGRI, NIH) U54 HG003273. Contributions from members of the CGE lab were supported by Agriculture and Food Research Initiative Competitive grant no. 2010-65205-20407 from the USDA National Institute of Food Agriculture. AKB was supported by a Clare Luce Booth Fellowship at Georgetown University

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkage groups. We estimate the total genome size to be approximately 3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species.

The amount of pollen stored in honey bee, Apis mellifera, colonies is a selectable trait. Five generations of two-way selection resulted in high and low strains that differed more than six-fold in quantities of stored pollen. Comparisons with hybrid crosses suggested that colony-level, high pollen-hoarding behaviour is inherited as a recessive trait. Colony levels of stored honey, however, showed an over-dominant pattern, with hybrid colonies storing significantly more honey than either of the selected strains. Controlled studies of individual foraging behaviour revealed the same patterns of inheritance at the individual level: high-strain workers specialized on pollen foraging, low-strain workers on nectar, and hybrid workers demonstrated a significantlt greater nectar-collecting bias than workers of the low strain. Genomic mapping studies of colony-level pollen hoarding and individual foraging behaviour have revealed two genomic regions of the honey bee that contain major quantitative trait loci that explain a large portion of the observed variance in pollen hoarding and foraging behaviour of the two strains. The effects of major genes on within- and between-colony variation in individual foraging behaviour are discussed in the context of conducting and interpreting empirical tests of foraging theory.

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.

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

Populations of honey bees and other pollinators have declined worldwide in recent years. A variety of stressors have been implicated as potential causes, including agricultural pesticides. Neonicotinoid insecticides, which are widely used and highly toxic to honey bees, have been found in previous analyses of honey bee pollen and comb material. However, the routes of exposure have remained largely undefined. We used LC/MS-MS to analyze samples of honey bees, pollen stored in the hive and several potential exposure routes associated with plantings of neonicotinoid treated maize. Our results demonstrate that bees are exposed to these compounds and several other agricultural pesticides in several ways throughout the foraging period. During spring, extremely high levels of clothianidin and thiamethoxam were found in planter exhaust material produced during the planting of treated maize seed. We also found neonicotinoids in the soil of each field we sampled, including unplanted fields. Plants visited by foraging bees (dandelions) growing near these fields were found to contain neonicotinoids as well. This indicates deposition of neonicotinoids on the flowers, uptake by the root system, or both. Dead bees collected near hive entrances during the spring sampling period were found to contain clothianidin as well, although whether exposure was oral (consuming pollen) or by contact (soil/planter dust) is unclear. We also detected the insecticide clothianidin in pollen collected by bees and stored in the hive. When maize plants in our field reached anthesis, maize pollen from treated seed was found to contain clothianidin and other pesticides; and honey bees in our study readily collected maize pollen. These findings clarify some of the mechanisms by which honey bees may be exposed to agricultural pesticides throughout the growing season. These results have implications for a wide range of large-scale annual cropping systems that utilize neonicotinoid seed treatments.

A fundamental problem in meta-analysis is how to systematically combine information from multiple statistical tests to rigorously evaluate a single overarching hypothesis. This problem occurs in systems biology when attempting to map genomic attributes to complex phenotypes such as behavior. Behavior and other complex phenotypes are influenced by intrinsic and environmental determinants that act on the transcriptome, but little is known about how these determinants interact at the molecular level. We developed an informatic technique that identifies statistically significant meta-associations between gene expression patterns and transcription factor combinations. Deploying this technique for brain transcriptome profiles from ca. 400 individual bees, we show that diverse determinants of behavior rely on shared combinations of transcription factors. These relationships were revealed only when we considered complex and variable regulatory rules, suggesting that these shared transcription factors are used in distinct ways by different determinants. This regulatory code would have been missed by traditional gene coexpression or cis -regulatory analytic methods. We expect that our meta-analysis tools will be useful for a broad array of problems in systems biology and other fields.

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Background The ectoparasitic mite Varroa destructor has emerged as the primary pest of domestic honey bees (Apis mellifera). Here we present an initial survey of the V. destructor genome carried out to advance our understanding of Varroa biology and to identify new avenues for mite control. This sequence survey provides immediate resources for molecular and population-genetic analyses of Varroa-Apis interactions and defines the challenges ahead for a comprehensive Varroa genome project. Results The genome size was estimated by flow cytometry to be 565 Mbp, larger than most sequenced insects but modest relative to some other Acari. Genomic DNA pooled from ~1,000 mites was sequenced to 4.3× coverage with 454 pyrosequencing. The 2.4 Gbp of sequencing reads were assembled into 184,094 contigs with an N50 of 2,262 bp, totaling 294 Mbp of sequence after filtering. Genic sequences with homology to other eukaryotic genomes were identified on 13,031 of these contigs, totaling 31.3 Mbp. Alignment of protein sequence blocks conserved among V. destructor and four other arthropod genomes indicated a higher level of sequence divergence within this mite lineage relative to the tick Ixodes scapularis. A number of microbes potentially associated with V. destructor were identified in the sequence survey, including ~300 Kbp of sequence deriving from one or more bacterial species of the Actinomycetales. The presence of this bacterium was confirmed in individual mites by PCR assay, but varied significantly by age and sex of mites. Fragments of a novel virus related to the Baculoviridae were also identified in the survey. The rate of single nucleotide polymorphisms (SNPs) in the pooled mites was estimated to be 6.2 × 10-5per bp, a low rate consistent with the historical demography and life history of the species. Conclusions This survey has provided general tools for the research community and novel directions for investigating the biology and control of Varroa mites. Ongoing development of Varroa genomic resources will be a boon for comparative genomics of under-represented arthropods, and will further enhance the honey bee and its associated pathogens as a model system for studying host-pathogen interactions.

The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 1∶1 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.

A prominent theory states that animal phenotypes arise by evolutionary changes in gene regulation, but the extent to which this theory holds true for behavioral evolution is not known. Because “nature and nurture” are now understood to involve hereditary and environmental influences on gene expression, we studied whether environmental influences on a behavioral phenotype, i.e., aggression, could have evolved into inherited differences via changes in gene expression. Here, with microarray analysis of honey bees, we show that aggression-related genes with inherited patterns of brain expression are also environmentally regulated. There were expression differences in the brain for hundreds of genes between the highly aggressive Africanized honey bee compared with European honey bee (EHB) subspecies. Similar results were obtained for EHB in response to exposure to alarm pheromone (which provokes aggression) and when comparing old and young bees (aggressive tendencies increase with age). There was significant overlap of the gene lists generated from these three microarray experiments. Moreover, there was statistical enrichment of several of the same cis regulatory motifs in promoters of genes on all three gene lists. Aggression shows a remarkably robust brain molecular signature regardless of whether it occurs because of inherited, age-related, or environmental (social) factors. It appears that one element in the evolution of different degrees of aggressive behavior in honey bees involved changes in regulation of genes that mediate the response to alarm pheromone.