Genetic diversity and population structure of two subspecies of western honey bees (Apis mellifera L.) in the Republic of South Africa as revealed by microsatellite genotyping.

Apis mellifera scutellata and Apis mellifera capensis, two native subspecies of western honey bees in the Republic of South Africa (RSA), are important to beekeepers in their native region because beekeepers use these bees for honey production and pollination purposes. Additionally, both bees are important invasive pests outside of their native ranges. Recently, whole mitogenome sequencing and single nucleotide polymorphisms were used to study their genetic diversity. To add to our knowledge of the molecular ecology of both bees, we tested the ability of microsatellites to be used as a tool to discriminate between A.m. capensis and A.m. scutellata. We analyzed the genetic variability and overall population structure of both bee subspecies and hybrids of the two by genotyping individuals collected from RSA (N = 813 bees from 75 apiaries) at 19 microsatellite DNA loci. Overall, populations averaged between 9.2 and 11.3 alleles per locus, with unbiased heterozygosity values ranging from 0.81 to 0.86 per population. Bayesian clustering analyses revealed two distinct evolutionary units, though the results did not match those of earlier morphometric and molecular analyses. This suggests that the microsatellites we tested were not sufficient for subspecies identification purposes, especially for Cape and hybrid bees. Nevertheless, the microsatellite data highlight the considerable genetic diversity within both populations and a larger-than-expected hybridization zone between the natural distributions of A.m. capensis and A.m. scutellata.

Assessing the role of dispersed floral resources for managed bees in providing supporting ecosystem services for crop pollination.

Most pollination ecosystem services studies have focussed on wild pollinators and their dependence on natural floral resources adjacent to crop fields. However, managed pollinators depend on a mixture of floral resources that are spatially separated from the crop field. Here, we consider the supporting role these resources play as an ecosystem services provider to quantify the use and availability of floral resources, and to estimate their relative contribution to support pollination services of managed honeybees. Beekeepers supplying pollination services to the Western Cape deciduous fruit industry were interviewed to obtain information on their use of floral resources. For 120 apiary sites, we also analysed floral resources within a two km radius of each site based on geographic data. The relative availability of floral resources at sites was compared to regional availability. The relative contribution of floral resources-types to sustain managed honeybees was estimated. Beekeepers showed a strong preference for eucalypts and canola. Beekeepers selectively placed more hives at sites with eucalypt and canola and less with natural vegetation. However, at the landscape-scale, eucalypt was the least available resource, whereas natural vegetation was most common. Based on analysis of apiary sites, we estimated that 700,818 ha of natural vegetation, 73,910 ha of canola fields, and 10,485 ha of eucalypt are used to support the managed honeybee industry in the Western Cape. Whereas the Cape managed honeybee system uses a bee native to the region, alien plant species appear disproportionately important among the floral resources being exploited. We suggest that an integrated approach, including evidence from interview and landscape data, and fine-scale biological data is needed to study floral resources supporting managed honeybees.

Population genomics and morphometric assignment of western honey bees (Apis mellifera L.) in the Republic of South Africa.

BACKGROUNDS: Apis mellifera scutellata and A.m. capensis (the Cape honey bee) are western honey bee subspecies indigenous to the Republic of South Africa (RSA). Both bees are important for biological and economic reasons. First, A.m. scutellata is the invasive "African honey bee" of the Americas and exhibits a number of traits that beekeepers consider undesirable. They swarm excessively, are prone to absconding (vacating the nest entirely), usurp other honey bee colonies, and exhibit heightened defensiveness. Second, Cape honey bees are socially parasitic bees; the workers can reproduce thelytokously. Both bees are indistinguishable visually. Therefore, we employed Genotyping-by-Sequencing (GBS), wing geometry and standard morphometric approaches to assess the genetic diversity and population structure of these bees to search for diagnostic markers that can be employed to distinguish between the two subspecies. RESULTS: Apis mellifera scutellata possessed the highest mean number of polymorphic SNPs (among 2449 informative SNPs) with minor allele frequencies > 0.05 (Np = 88%). The RSA honey bees generated a high level of expected heterozygosity (Hexp = 0.24). The mean genetic differentiation (FST; 6.5%) among the RSA honey bees revealed that approximately 93% of the genetic variation was accounted for within individuals of these subspecies. Two genetically distinct clusters (K = 2) corresponding to both subspecies were detected by Model-based Bayesian clustering and supported by Principal Coordinates Analysis (PCoA) inferences. Selected highly divergent loci (n = 83) further reinforced a distinctive clustering of two subspecies across geographical origins, accounting for approximately 83% of the total variation in the PCoA plot. The significant correlation of allele frequencies at divergent loci with environmental variables suggested that these populations are adapted to local conditions. Only 17 of 48 wing geometry and standard morphometric parameters were useful for clustering A.m. capensis, A.m. scutellata, and hybrid individuals. CONCLUSIONS: We produced a minimal set of 83 SNP loci and 17 wing geometry and standard morphometric parameters useful for identifying the two RSA honey bee subspecies by genotype and phenotype. We found that genes involved in neurology/behavior and development/growth are the most prominent heritable traits evolved in the functional evolution of honey bee populations in RSA. These findings provide a starting point for understanding the functional basis of morphological differentiations and ecological adaptations of the two honey bee subspecies in RSA.

Turning workers into false queens: the role of exogenous pheromones in regulating reproduction in worker honey bees.

One of the responses that honey bee workers can make in the event of queen loss is to develop into false queens. False queens are workers that exhibit both behavioural and physiological traits similar to those of a true queen. However, the presence of more than one false queen in a colony distorts the established hierarchies. As transformation into a false queen occurs after emergence as an adult, we tested the effect of worker mobile pheromone carriers (PCs) treated with exogenously supplied pheromones on their nestmates. The PCs carried either synthetic mandibular gland pheromones or pheromones extracted from Apis melliferacapensis parasitic workers. Only the PCs attracted retinues of workers, increased pheromone production and activated their ovaries, becoming false queens. Pheromones from A. m.capensis workers were more effective than extracts of commercially available synthetic queen pheromones in eliciting these effects. Using this simple mobile pheromone delivery system, we have shown that carrying amounts of exogenous pheromone can induce pheromone production in the carrier, resulting in the production of false queens within experimental groups. Possible implications of using this technique to modify and regulate worker reproduction in colonies are discussed.

The transcriptomic changes associated with the development of social parasitism in the honeybee Apis mellifera capensis.

Social insects are characterized by the division of labor. Queens usually dominate reproduction, whereas workers fulfill non-reproductive age-dependent tasks to maintain the colony. Although workers are typically sterile, they can activate their ovaries to produce their own offspring. In the extreme, worker reproduction can turn into social parasitism as in Apis mellifera capensis. These intraspecific parasites occupy a host colony, kill the resident queen, and take over the reproductive monopoly. Because they exhibit a queenlike behavior and are also treated like queens by the fellow workers, they are so-called pseudoqueens. Here, we compare the development of parasitic pseudoqueens and social workers at different time points using fat body transcriptome data. Two complementary analysis methods-a principal component analysis and a time course analysis-led to the identification of a core set of genes involved in the transition from a social worker into a highly fecund parasitic pseudoqueen. Comparing our results on pseudoqueens with gene expression data of honeybee queens revealed many similarities. In addition, there was a set of specific transcriptomic changes in the parasitic pseudoqueens that differed from both, queens and social workers, which may be typical for the development of the social parasitism in A. m. capensis.

Firewalls in bee nests-survival value of propolis walls of wild Cape honeybee (Apis mellifera capensis).

The Cape bee is endemic to the winter rainfall region of South Africa where fires are an integral part of the ecology of the fynbos (heathland) vegetation. Of the 37 wild nests in pristine Peninsula Sandstone Fynbos in the Cape Point section of Table Mountain National Park that have been analyzed so far, only 22 could be accessed sufficiently to determine the existence of a propolis wall of which 68% had propolis walls which entirely enclosed their openings. The analysis of the 37 wild nests revealed that 78% occurred under boulders or in clefts within rocks, 11% in the ground, 8% in tree cavities, and 3% within shrubs. The analysis of 17 of these nests following a fire within the park revealed that the propolis walls materially protected the nests and retarded the fire with all the colonies surviving. The bees responded to the smoke by imbibing honey and retreating to the furthest recess of their nest cavity. The bees were required to utilize this honey for about 3 weeks after which fire-loving plants appeared and began flowering. Considerable resources were utilized in the construction of the propolis walls, which ranged in thickness from 1.5 to 40 mm (mean 5 mm). Its physical environment determines the nesting behavior of the Cape bee. The prolific use of propolis serves to insulate the nest from extremes of temperature and humidity, restricts entry, camouflages the nest, and acts as an effective fire barrier protecting nests established mostly under rocks in vegetation subjected to periodic fires.

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

BACKGROUND: Intersexual 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. RESULTS: We 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. CONCLUSIONS: The 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.

The complete mitochondrial genome of the Cape honey bee, Apis mellifera capensis Esch. (Insecta: hymenoptera: apidae).

We characterized the complete mitogenome sequence of the Cape honey bee, Apis mellifera capensis, from South Africa. The circle genome is 16,470 bp in length, with the base composition of 43.2% A, 9.6% C, 5.6% G, and 41.5% T. The assembled mitogenome has 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNA genes, and one control region. All protein-coding genes are initiated by ATT, ATC, ATG or ATA codons and are terminated by the typical stop codon TAA. The heavy strand encodes four protein-coding genes, eight tRNAs, and two rRNAs. The light strand encodes nine protein-coding genes and 14 tRNAs. The complete mitogenome sequence of A.m. capensis is identical to the gene arrangement found in other A. mellifera mitogenomes and it provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis of members of the genus Apis.

The complete mitochondrial genome of the hybrid honey bee, Apis mellifera capensis × Apis mellifera scutellata, from South Africa.

We characterized the complete mitogenome sequence of the South African hybrid honey bee Apis mellifera capensis × Apis mellifera scutellata using genome skimming. The mitochondrial genome was a circular molecule 16,340 bp in length with a gene organization identical to that of the other A. mellifera mitogenomes. The base composition is 43.2% A, 9.7% C, 5.6% G, and 41.5% T, with an A + T content of 84.7%. The mitogenome had 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs genes, and one control region. All PCGs were initiated by ATT, ATG, ATA, and ATC codons and were terminated by a TAA stop codon. The heavy strand encodes four PCGs, eight tRNAs, and two rRNAs. The light strand encodes nine PCGS and 14 tRNAs. A phylogenetic analysis of the PCGs reveals a close relationship between this hybrid honey bee and other Apis spp.

Differences in Varroa destructor infestation rates of two indigenous subspecies of Apis mellifera in the Republic of South Africa.

Varroa destructor Anderson & Trueman (Varroa) is a damaging pest of the Western honey bee, Apis mellifera, in North America, Europe, and Asia. However, Varroa infestations have not produced equivalent colony losses of African subspecies of honey bee throughout Africa and parts of the Americas. We surveyed the Varroa infestation rates (number of Varroa per 100 adult honey bees) in colonies of A. m. scutellata, A. m. capensis, and hybrids of the two subspecies throughout the Republic of South Africa in the fall of 2014. We found that A. m. scutellata colonies had significantly higher Varroa infestations than did A. m. capensis colonies. Furthermore, hybridized colonies of the two subspecies had Varroa infestations intermediate to those of A. m. scutellata and A. m. capensis. This is the first documentation of a clear difference in Varroa infestation rates of A. m. scutellata, A. m. capensis, and hybridized colonies in South Africa. Furthermore, our data confirm that Varroa populations in A. m. scutellata colonies are within the range of populations that are damaging to European honey bees.

Selection on overdominant genes maintains heterozygosity along multiple chromosomes in a clonal lineage of honey bee.

Correlations between fitness and genome-wide heterozygosity (heterozygosity-fitness correlations, HFCs) have been reported across a wide range of taxa. The genetic basis of these correlations is controversial: do they arise from genome-wide inbreeding ("general effects") or the "local effects" of overdominant loci acting in linkage disequilibrium with neutral loci? In an asexual thelytokous lineage of the Cape honey bee (Apis mellifera capensis), the effects of inbreeding have been homogenized across the population, making this an ideal system in which to detect overdominant loci, and to make inferences about the importance of overdominance on HFCs in general. Here we investigate the pattern of zygosity along two chromosomes in 42 workers from the clonal Cape honey bee population. On chromosome III (which contains the sex-locus, a gene that is homozygous-lethal) and chromosome IV we show that the pattern of zygosity is characterized by loss of heterozygosity in short regions followed by the telomeric restoration of heterozygosity. We infer that at least four selectively overdominant genes maintain heterozygosity on chromosome III and three on chromosome IV via local effects acting on neutral markers in linkage disequilibrium. We conclude that heterozygote advantage and local effects may be more common and evolutionarily significant than is generally appreciated.

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.