RESTseq – Efficient Benchtop Population Genomics with RESTriction Fragment SEQuencing

<div><p>We present RESTseq, an improved approach for a cost efficient, highly flexible and repeatable enrichment of DNA fragments from digested genomic DNA using Next Generation Sequencing platforms including small scale Personal Genome sequencers. Easy adjustments make it suitable for a wide range of studies requiring SNP detection or SNP genotyping from fine-scale linkage mapping to population genomics and population genetics also in non-model organisms. We demonstrate the validity of our approach by comparing two honeybee and several stingless bee samples.</p></div

Microsatellite markers for genotyping and detection of drifters.

<p>Microsatellite markers for genotyping and detection of drifters.</p

Number of bees drifting from neighbouring colonies (N = 47).

<p>The distance of “one” means that the bees came from the neighbouring colony, “two” from the colony next to the neighbouring colony and so on. The equation for the relationship is: y = a + b/x where a = 0.716 and b = 13.96.</p

Infection of drifter and native bees.

<p>To assess the effect of pathogens on drifting behavior, viruses and <i>Nosema</i> spp. infections were compared between the drifters and the native bees of the sink colonies (N = 14 colonies).</p

Network map of colonies from Simonswald on accepting drifters according to their <i>indegree centrality</i>.

<p>Here we represent the observed <i>indegree centrality</i> based on the actual number of drifters. Each square represents a colony (“+”high <i>Varroa</i>; “-” low <i>Varroa</i>) whilst the number refers to the colony position at the apiary, with +1 and -1 being the two central hives and +7 and -7 those at the two ends of the row. The arrows represent the flow of drifters and their width is proportional to the number of drifters (from 1 to 4) going from one colony to another. Only Simonswald network is represented since more drifters were found in this site than in Kenzingen, which illustrates better our results.</p

Principal Component Analysis.

<p>Genetic clustering of the <i>Varroa</i> mites based on their genotype at six microsatellite markers. The <i>Varroa</i> mites were sampled in <i>A</i>. <i>cerana</i> (<b>Vc</b>) and <i>A</i>. <i>mellifera</i> (<b>Vm</b>) colonies in the Philippines and Vietnam. The genotypes of the mites sampled in Los Banos (<b>LB</b>) are shown in green (groups 1 and 2). The mites from Lipa city (<b>LC</b>, group 3) and sampled in <i>A</i>. <i>mellifera</i> from Dien Bien (<b>DB</b>, group 5) and Son La (<b>SL</b>, group 7) are represented in orange. The mites from <i>A</i>. <i>cerana</i> colonies located in Vietnam from Dien Bien (<b>DB</b>, group 4), Son La (<b>SL</b>, group 6) and Cat Ba (<b>CB</b>, group 8) are represented in blue. Each dot represents a distinct individual, and each inertia ellipsoid shows the population’s prediction ellipses for each group.</p

Host Specificity in the Honeybee Parasitic Mite, <i>Varroa spp</i>. in <i>Apis mellifera</i> and <i>Apis cerana</i>

<div><p>The ectoparasitic mite <i>Varroa destructor</i> is a major global threat to the Western honeybee <i>Apis mellifera</i>. This mite was originally a parasite of <i>A</i>. <i>cerana</i> in Asia but managed to spill over into colonies of <i>A</i>. <i>mellifera</i> which had been introduced to this continent for honey production. To date, only two almost clonal types of <i>V</i>. <i>destructor</i> from Korea and Japan have been detected in <i>A</i>. <i>mellifera</i> colonies. However, since both <i>A</i>. <i>mellifera</i> and <i>A</i>. <i>cerana</i> colonies are kept in close proximity throughout Asia, not only new spill overs but also spill backs of highly virulent types may be possible, with unpredictable consequences for both honeybee species. We studied the dispersal and hybridisation potential of <i>Varroa</i> from sympatric colonies of the two hosts in Northern Vietnam and the Philippines using mitochondrial and microsatellite DNA markers. We found a very distinct mtDNA haplotype equally invading both <i>A</i>. <i>mellifera</i> and <i>A</i>. <i>cerana</i> in the Philippines. In contrast, we observed a complete reproductive isolation of various Vietnamese <i>Varroa</i> populations in <i>A</i>. <i>mellifera</i> and <i>A</i>. <i>cerana</i> colonies even if kept in the same apiaries. In light of this variance in host specificity, the adaptation of the mite to its hosts seems to have generated much more genetic diversity than previously recognised and the <i>Varroa</i> species complex may include substantial cryptic speciation.</p></div

Parasites and Pathogens of the Honeybee (<i>Apis mellifera</i>) and Their Influence on Inter-Colonial Transmission

<div><p>Pathogens and parasites may facilitate their transmission by manipulating host behavior. Honeybee pathogens and pests need to be transferred from one colony to another if they are to maintain themselves in a host population. Inter-colony transmission occurs typically through honeybee workers not returning to their home colony but entering a foreign colony (“drifting”). Pathogens might enhance drifting to enhance transmission to new colonies. We here report on the effects infection by ten honeybee viruses and <i>Nosema</i> spp., and <i>Varroa</i> mite infestation on honeybee drifting. Genotyping of workers collected from colonies allowed us to identify genuine drifted workers as well as source colonies sending out drifters in addition to sink colonies accepting them. We then used network analysis to determine patterns of drifting. Distance between colonies in the apiary was the major factor explaining 79% of drifting. None of the tested viruses or <i>Nosema</i> spp. were associated with the frequency of drifting. Only colony infestation with <i>Varroa</i> was associated with significantly enhanced drifting. More specifically, colonies with high <i>Varroa</i> infestation had a significantly enhanced acceptance of drifters, although they did not send out more drifting workers. Since <i>Varroa-</i>infested colonies show an enhanced attraction of drifting workers, and not only those infected with <i>Varroa</i> and its associated pathogens, infestation by <i>Varroa</i> may also facilitate the uptake of other pests and parasites.</p></div

Varroa <i>coxI</i> haplotype divergence in the Philippines and Vietnam.

<p>Network representing the amount of substitutions between the different <i>coxI</i> sequences obtained from different <i>Varroa</i> mites sampled in colonies of <i>A</i>. <i>mellifera</i> (in blue with white text) and <i>A</i>. <i>cerana</i> (in orange with black text). The mites were sampled in Vietnam in the surrounding of Dien Bien (<b>DB</b>), Son La (<b>SL</b>) and Cat Ba (<b>CB</b>) and in the Philippines in the city of Los Banos (<b>LB</b>) and Lipa City (<b>LC</b>). The sample size for each haplotype is written in italic between brackets below the sampling location. The grey components represent additional accessions generated by Navajas <i>et al</i>. (2010): AmK1-1 haplotype (<b>K1</b>, accession GQ379056), AcV1-1 (<b>V1</b>, accession GQ379061), AcC1-1 (<b>C1</b>, accession GQ379065) and AmJ1-6 together with AcJ1-4 (<b>J1</b>, accessions GQ379074.1 and GQ379072.1, respectively). Each diamond represents a substitution. The number indicated close to the dotted line represents the number of substitutions not represented on the figure.</p

Pairwise F_ST.

<p>Pairwise comparison of the genetic differentiation using the fixation index (F_ST) between the three main genetic clusters found in this study: the mites from the two honeybees species sampled in Los Banos (<b>Philippines</b>), the mites sampled in <i>A</i>. <i>mellifera</i> colonies in Vietnam and Lipa city (<b>Korea</b>) and the mites sampled in <i>A</i>. <i>cerana</i> colonies in Vietnam (<b>Vietnam</b>).</p><p>***: <i>p</i><0.001,</p><p>**: <i>p</i><0.01.</p><p>Pairwise F_ST.</p