Behavioral and molecular studies of quantitative differences in hygienic behavior in honeybees.

BACKGROUND: Hygienic behavior (HB) enables honeybees to tolerate parasites, including infection with the parasitic mite Varroa destructor, and it is a well-known example of a quantitative genetic trait. The understanding of the molecular processes underpinning the quantitative differences in this behavior remains limited. RESULTS: We performed gene expression studies in worker bees that displayed quantitative genetic differences in HB. We established a high and low genetic source of HB performance and studied the engagements into HB of single worker bees under the same environmental conditions. We found that the percentage of worker bees that engaged in a hygienic behavioral task tripled in the high versus low HB sources, thus suggesting that genetic differences may mediate differences in stimulated states to perform HB. We found 501 differently expressed genes (DEGs) in the brains of hygienic and non-hygienic performing workers in the high HB source bees, and 342 DEGs in the brains of hygienic performing worker bees, relative to the gene expression in non-hygienic worker bees from the low HB source group. "Cell surface receptor ligand signal transduction" in the high and "negative regulation of cell communication" in the low HB source were overrepresented molecular processes, suggesting that these molecular processes in the brain may play a role in the regulation of quantitative differences in HB. Moreover, only 21 HB-associated DEGs were common between the high and low HB sources. CONCLUSIONS: The better HB colony performance is primarily achieved by a high number of bees engaging in the hygienic tasks that associate with distinct molecular processes in the brain. We propose that different gene products and pathways may mediate the quantitative genetic differences of HB.

On the relevance of technical variation due to building pools in microarray experiments.

BACKGROUND: Pooled samples are frequently used in experiments measuring gene expression. In this method, RNA from different individuals sharing the same experimental conditions and explanatory variables is blended and their concentrations are jointly measured. As a matter of principle, individuals are represented in equal shares in each pool. However, some degree of disproportionality may arise from the limits of technical precision. As a consequence a special kind of technical error occurs, which can be modelled by a respective variance component. Previously published theory - allowing for variable pool sizes - has been applied to four microarray gene expression data sets from different species in order to assess the practical relevance of this type of technical error in terms of significance and size of this variance component. RESULTS: The number of transcripts with a significant variance component due to imperfect blending was found to be 4329 (23 %) in mouse data and 7093 (49 %) in honey bees, but only 6 in rats and none whatsoever in human data. These results correspond to a false discovery rate of 5 % in each data set. The number of transcripts found to be differentially expressed between treatments was always higher when the blending error variance was neglected. Simulations clearly indicated overly-optimistic (anti-conservative) test results in terms of false discovery rates whenever this source of variability was not represented in the model. CONCLUSIONS: Imperfect equality of shares when blending RNA from different individuals into joint pools of variable size is a source of technical variation with relevance for experimental design, practice at the laboratory bench and data analysis. Its potentially adverse effects, incorrect identification of differentially expressed transcripts and overly-optimistic significance tests, can be fully avoided, however, by the sound application of recently established theory and models for data analysis.

Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera).

Honeybees (Apis mellifera), which are important pollinators of plants, display remarkable individual behaviors that collectively contribute to the organization of a complex society. Advances in dissecting the complex processes of honeybee behavior have been limited in the recent past due to a lack of genetic manipulation tools. These tools are difficult to apply in honeybees because the unit of reproduction is the colony, and many interesting phenotypes are developmentally specified at later stages. Here, we report highly efficient integration and expression of piggyBac-derived cassettes in the honeybee. We demonstrate that 27 and 20% of queens stably transmitted two different expression cassettes to their offspring, which is a 6- to 30-fold increase in efficiency compared with those generally reported in other insect species. This high efficiency implies that an average beekeeping facility with a limited number of colonies can apply this tool. We demonstrated that the cassette stably and efficiently expressed marker genes in progeny under either an artificial or an endogenous promoter. This evidence of efficient expression encourages the use of this system to inhibit gene functions through RNAi in specific tissues and developmental stages by using various promoters. We also showed that the transgenic marker could be used to select transgenic offspring to be employed to facilitate the building of transgenic colonies via the haploid males. We present here the first to our knowledge genetic engineering tool that will efficiently allow for the systematic detection and better understanding of processes underlying the biology of honeybees.

Gradual molecular evolution of a sex determination switch through incomplete penetrance of femaleness.

Some genes regulate phenotypes that are either present or absent. They are often important regulators of developmental switches and are involved in morphological evolution. We have little understanding of the molecular mechanisms by which these absence/presence gene functions have evolved, because the phenotype and fitness of molecular intermediate forms are unknown. Here, we studied the sex-determining switch of 14 natural sequence variants of the csd gene among 76 genotypes of the honeybee (Apis mellifera). Heterozygous genotypes (different specificities) of the csd gene determine femaleness, while hemizygous genotypes (single specificity) determine maleness. Homozygous genotypes of the csd gene (same specificity) are lethal. We found that at least five amino acid differences and length variation between Csd specificities in the specifying domain (PSD) were sufficient to regularly induce femaleness. We estimated that, on average, six pairwise amino acid differences evolved under positive selection. We also identified a natural evolutionary intermediate that showed only three amino acid length differences in the PSD relative to its parental allele. This genotype showed an intermediate fitness because it implemented lethality regularly and induced femaleness infrequently (i.e., incomplete penetrance). We suggest incomplete penetrance as a mechanism through which new molecular switches can gradually and adaptively evolve.

Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate.

Division of labor in social insects has made the evolution of collective traits possible that cannot be achieved by individuals alone. Differences in behavioral responses produce variation in engagement in behavioral tasks, which as a consequence, generates a division of labor. We still have little understanding of the genetic components influencing these behaviors, although several candidate genomic regions and genes influencing individual behavior have been identified. Here, we report that mixing of worker honeybees with different genotypes influences the expression of individual worker behaviors and the transcription of genes in the neuronal substrate. These indirect genetic effects arise in a colony because numerous interactions between workers produce interacting phenotypes and genotypes across organisms. We studied hygienic behavior of honeybee workers, which involves the cleaning of diseased brood cells in the colony. We mixed ∼500 newly emerged honeybee workers with genotypes of preferred Low (L) and High (H) hygienic behaviors. The L/H genotypic mixing affected the behavioral engagement of L worker bees in a hygienic task, the cooperation among workers in uncapping single brood cells, and switching between hygienic tasks. We found no evidence that recruiting and task-related stimuli are the primary source of the indirect genetic effects on behavior. We suggested that behavioral responsiveness of L bees was affected by genotypic mixing and found evidence for changes in the brain in terms of 943 differently expressed genes. The functional categories of cell adhesion, cellular component organization, anatomical structure development, protein localization, developmental growth and cell morphogenesis were overrepresented in this set of 943 genes, suggesting that indirect genetic effects can play a role in modulating and modifying the neuronal substrate. Our results suggest that genotypes of social partners affect the behavioral responsiveness and the neuronal substrate of individual workers, indicating a complex genetic architecture underlying the expression of behavior.

Function and evolution of sex determination mechanisms, genes and pathways in insects.

Animals have evolved a bewildering diversity of mechanisms to determine the two sexes. Studies of sex determination genes--their history and function--in non-model insects and Drosophila have allowed us to begin to understand the generation of sex determination diversity. One common theme from these studies is that evolved mechanisms produce activities in either males or females to control a shared gene switch that regulates sexual development. Only a few small-scale changes in existing and duplicated genes are sufficient to generate large differences in sex determination systems. This review summarises recent findings in insects, surveys evidence of how and why sex determination mechanisms can change rapidly and suggests fruitful areas of future research.

Sex determination in honeybees: two separate mechanisms induce and maintain the female pathway.

Organisms have evolved a bewildering diversity of mechanisms to generate the two sexes. The honeybee (Apis mellifera) employs an interesting system in which sex is determined by heterozygosity at a single locus (the Sex Determination Locus) harbouring the complementary sex determiner (csd) gene. Bees heterozygous at Sex Determination Locus are females, whereas bees homozygous or hemizygous are males. Little is known, however, about the regulation that links sex determination to sexual differentiation. To investigate the control of sexual development in honeybees, we analyzed the functions and the regulatory interactions of genes involved in the sex determination pathway. We show that heterozygous csd is only required to induce the female pathway, while the feminizer (fem) gene maintains this decision throughout development. By RNAi induced knockdown we show that the fem gene is essential for entire female development and that the csd gene exclusively processes the heterozygous state. Fem activity is also required to maintain the female determined pathway throughout development, which we show by mosaic structures in fem-repressed intersexuals. We use expression of Fem protein in males to demonstrate that the female maintenance mechanism is controlled by a positive feedback splicing loop in which Fem proteins mediate their own synthesis by directing female fem mRNA splicing. The csd gene is only necessary to induce this positive feedback loop in early embryogenesis by directing splicing of fem mRNAs. Finally, fem also controls the splicing of Am-doublesex transcripts encoding conserved male- and female-specific transcription factors involved in sexual differentiation. Our findings reveal how the sex determination process is realized in honeybees differing from Drosophila melanogaster.

Evidence for the evolutionary nascence of a novel sex determination pathway in honeybees.

Sex determination in honeybees (Apis mellifera) is governed by heterozygosity at a single locus harbouring the complementary sex determiner (csd) gene, in contrast to the well-studied sex chromosome system of Drosophila melanogaster. Bees heterozygous at csd are females, whereas homozygotes and hemizygotes (haploid individuals) are males. Although at least 15 different csd alleles are known among natural bee populations, the mechanisms linking allelic interactions to switching of the sexual development programme are still obscure. Here we report a new component of the sex-determining pathway in honeybees, encoded 12 kilobases upstream of csd. The gene feminizer (fem) is the ancestrally conserved progenitor gene from which csd arose and encodes an SR-type protein, harbouring an Arg/Ser-rich domain. Fem shares the same arrangement of Arg/Ser- and proline-rich-domain with the Drosophila principal sex-determining gene transformer (tra), but lacks conserved motifs except for a 30-amino-acid motif that Fem shares only with Tra of another fly, Ceratitis capitata. Like tra, the fem transcript is alternatively spliced. The male-specific splice variant contains a premature stop codon and yields no functional product, whereas the female-specific splice variant encodes the functional protein. We show that RNA interference (RNAi)-induced knockdowns of the female-specific fem splice variant result in male bees, indicating that the fem product is required for entire female development. Furthermore, RNAi-induced knockdowns of female allelic csd transcripts result in the male-specific fem splice variant, suggesting that the fem gene implements the switch of developmental pathways controlled by heterozygosity at csd. Comparative analysis of fem and csd coding sequences from five bee species indicates a recent origin of csd in the honeybee lineage from the fem progenitor and provides evidence for positive selection at csd accompanied by purifying selection at fem. The fem locus in bees uncovers gene duplication and positive selection as evolutionary mechanisms underlying the origin of a novel sex determination pathway.

Exceptionally high levels of recombination across the honey bee genome.

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.

Patterns of conservation and change in honey bee developmental genes.

The current insect genome sequencing projects provide an opportunity to extend studies of the evolution of developmental genes and pathways in insects. In this paper we examine the conservation and divergence of genes and developmental processes between Drosophila and the honey bee; two holometabolous insects whose lineages separated approximately 300 million years ago, by comparing the presence or absence of 308 Drosophila developmental genes in the honey bee. Through examination of the presence or absence of genes involved in conserved pathways (cell signaling, axis formation, segmentation and homeobox transcription factors), we find that the vast majority of genes are conserved. Some genes involved in these processes are, however, missing in the honey bee. We have also examined the orthology of Drosophila genes involved in processes that differ between the honey bee and Drosophila. Many of these genes are preserved in the honey bee despite the process in which they act in Drosophila being different or absent in the honey bee. Many of the missing genes in both situations appear to have arisen recently in the Drosophila lineage, have single known functions in Drosophila, and act early in developmental pathways, while those that are preserved have pleiotropic functions. An evolutionary interpretation of these data is that either genes with multiple functions in a common ancestor are more likely to be preserved in both insect lineages, or genes that are preserved throughout evolution are more likely to co-opt additional functions.