Emily J. Remnant

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster.

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 change into an Ala301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

Whole Genome DNA Methylation Profile of the Jewel Wasp (Nasonia vitripennis)

The epigenetic mark of DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, has been extensively studied in many mammalian genomes and, although it is commonly found at the promoter regions of genes, it is also involved in a number of different biological functions. In other complex animals, such as social insects, DNA methylation has been determined to be involved in caste differentiation and to occur primarily in gene bodies. The role of methylation in nonsocial insects, however, has not yet been explored thoroughly. Here, we present the whole-genome DNA methylation profile of the nonsocial hymenopteran, the jewel wasp (Nasonia vitripennis). From high-throughput sequencing of bisulfite-converted gDNA extracted from male Nasonia thoraces, we were able to determine which cytosine residues are methylated in the entire genome. We found that an overwhelming majority of methylated sites (99.7%) occur at cytosines followed by a guanine in the 3′ direction (CpG sites). Additionally, we found that a majority of methylation in Nasonia occurs within exonic regions of the genome (more than 62%). Overall, methylation is sparse in Nasonia, occurring only at 0.18% of all sites and at 0.63% of CpGs. Our analysis of the Nasonia methylome revealed that in contrast to the methylation profile typically seen in mammals, methylation is sparse and is constrained primarily to exons. This methylation profile is more similar to that of the social hymenopteran species, the honey bee (Apis mellifera). In presenting the Nasonia methylome, we hope to promote future investigation of the regulatory function of DNA methylation in both social and nonsocial hymenoptera.

The dynamic DNA methylation cycle from egg to sperm in the honey bee Apis mellifera

In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species.

A parent-of-origin effect on honeybee worker ovary size

Apis mellifera capensis is unique among honeybees in that unmated workers can produce pseudo-clonal female offspring via thelytokous parthenogenesis. Workers use this ability to compete among themselves and with their queen to be the mother of new queens. Males could therefore enhance their reproductive success by imprinting genes that enhance fertility in their daughter workers. This possibility sets the scene for intragenomic conflict between queens and drones over worker reproductive traits. Here, we show a strong parent-of-origin effect for ovary size (number of ovarioles) in reciprocal crosses between two honeybee subspecies, A. m. capensis and Apis mellifera scutellata. In this cross, workers with an A. m. capensis father had 30% more ovarioles than genotypically matched workers with an A. m. scutellata father. Other traits we measured (worker weight at emergence and the presence/absence of a spermatheca) are influenced more by rearing conditions than by parent-of-origin effects. Our study is the first to show a strong epigenetic (or, less likely, cytoplasmic maternal) effect for a reproductive trait in the honeybee and suggests that a search for parent-of-origin effects in other social insects may be fruitful.

Parent-of-origin effects on genome-wide DNA methylation in the Cape honey bee (Apis mellifera capensis) may be confounded by allele-specific methylation

BackgroundIntersexual genomic conflict sometimes leads to unequal expression of paternal and maternal alleles in offspring, resulting in parent-of-origin effects. In honey bees reciprocal crosses can show strong parent-of-origin effects, supporting theoretical predictions that genomic imprinting occurs in this species. Mechanisms behind imprinting in honey bees are unclear but differential DNA methylation in eggs and sperm suggests that DNA methylation could be involved. Nonetheless, because DNA methylation is multifunctional, it is difficult to separate imprinting from other roles of methylation. Here we use a novel approach to investigate parent-of-origin DNA methylation in honey bees. In the subspecies Apis mellifera capensis, reproduction of females occurs either sexually by fertilization of eggs with sperm, or via thelytokous parthenogenesis, producing female embryos derived from two maternal genomes.ResultsWe compared genome-wide methylation patterns of sexually-produced, diploid embryos laid by a queen, with parthenogenetically-produced diploid embryos laid by her daughters. Thelytokous embryos inheriting two maternal genomes had fewer hypermethylated genes compared to fertilized embryos, supporting the prediction that fertilized embryos have increased methylation due to inheritance of a paternal genome. However, bisulfite PCR and sequencing of a differentially methylated gene, Stan (GB18207) showed strong allele-specific methylation that was maintained in both fertilized and thelytokous embryos. For this gene, methylation was associated with haplotype, not parent of origin.ConclusionsThe results of our study are consistent with predictions from the kin theory of genomic imprinting. However, our demonstration of allele-specific methylation based on sequence shows that genome-wide differential methylation studies can potentially confound imprinting and allele-specific methylation. It further suggests that methylation patterns are heritable or that specific sequence motifs are targets for methylation in some genes.

A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations

ABSTRACT Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa. This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management. IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales. Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator.

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala(301) site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach.

Reproductive interference between honeybee species in artificial sympatry

Reproductive isolation between closely related species is often incomplete. The Western honeybee, Apis mellifera, and the Eastern hive bee, Apis cerana, have been allopatric for millions of years, but are nonetheless similar in morphology and behaviour. During the last century, the two species were brought into contact anthropogenically, providing potential opportunities for interspecific matings. Hybrids between A. mellifera and A. cerana are inviable, so natural interspecific matings are of concern because they may reduce the viability of A. cerana and A. mellifera populations – two of the worlds most important pollinators. We examined the mating behaviour of A. mellifera and A. cerana queens and drones from Caoba Basin, China and Cairns, Australia. Drone mating flight times overlap in both areas. Analysis of the spermathecal contents of queens with species‐specific genetic markers indicated that in Caoba Basin, 14% of A. mellifera queens mated with at least one A. cerana male, but we detected no A. cerana queens that had mated with A. mellifera males. Similarly, in Cairns, no A. cerana queens carried A. mellifera sperm, but one‐third of A. mellifera queens had mated with at least one A. cerana male. No hybrid embryos were detected in eggs laid by interspecifically mated A. mellifera queens in either location. However, A. mellifera queens artificially inseminated with A. cerana sperm produced inviable hybrid eggs or unfertilized drones. This suggests that reproductive interference will impact the viability of honeybee populations wherever A. cerana and A. mellifera are in contact.

Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

DNA methylation of Kr-h1 is involved in regulating ovary activation in worker honeybees (Apis mellifera)

A fundamental feature of insect colonies is the presence of a sterile worker caste. In the presence of a queen, worker reproduction is suppressed, leading to degradation of the ovaries. However in the absence of the queen, some young workers activate their ovaries and lay eggs that can result in viable male offspring. The genetic and molecular mechanisms that underlie the regulation of worker sterility are only partially understood, but are of great interest since they are fundamental to the evolution of eusociality. Here, we determine whether DNA methylation of the transcription factor, Krüppel homolog 1 (Kr-h1), is associated with ovary activation in workers of honeybee. Workers were reared under queenless conditions and separated into control and CO2-treated cages (CO2 narcosis is known to suppress ovary activation in queenless workers). Patterns of DNA methylation of Kr-h1 in the ovaries of 7-day-old adult workers showed that non-active ovaries had significantly higher levels of methylation at 4 CpG sites compared to workers with active ovaries. CO2 narcosis inhibited ovary activation, but did not alter methylation levels in non-active ovaries. These findings suggest that methylation of Kr-h1 contributes to the regulation of worker sterility in the honeybee. In addition to the overall finding of differential methylation between ovary-activated and non-activated workers, we found evidence of allele-specific methylation at one site.