The number of parasite genotypes found in individual hosts.

<p>(<b>a</b>) Workers have a mean of 2.467±0.22 (S.E. n = 45; black bars) genotypes/host. (<b>b</b>) Queens have a mean of 3.65±1.03 (n = 37) genotypes/host. Statistically, the two distributions do not differ from one another, have the same means (glm with quasipoisson: t₈₁ = 1.323, p = 0.19), but different variances (see text). Compared to a zero-truncated Poisson expectation (lines), the observed distribution deviates for both castes (Kolmogorov-Smirnov for workers: D = 0.821, p<0.001; for queens: D = 0.714, p<0.001). In the graphs, the first bars refer to multiplicity = 1 (single infections).</p

The work protocol.

<p>Shown are two examples - <b>Top panel:</b> queen nr. 10.374, with a single infection; <b>Bottom panel:</b> queen nr. 10.179, with mixed-genotype infection. In each case, the infecting population was separated by FACS into clones and grown in microtiter plates (left). The clones (symbols: circles, squares) were then genotyped by five polymorphic microsatellite loci. Entries are the microsatellite alleles (lengths in bp) observed at each locus (<i>C. bombi</i> is diploid). Frequency indicates the number of clones of each type that were found in the infecting population. Note that the allelic pattern of the two “derived” genotypes suggests that nr.3 is a case of allele loss, and nr.4 is a case of recombination as a result of genetic exchange among the two upper genotypes that were considered “primary” due to their higher frequency.</p

Mean genetic relatedness (Queller-Goodnight estimator) of single and mixed-genotype infections from workers and queens, and in different years.

<p>(<b>a</b>) Classes comparing single and mixed-genotype infections between and within hosts. Averages of classes are for ‘single between hosts’: <i>r</i> = −0.063±0.245 (S.D.); ‘mixed-genotype between hosts’: <i>r</i> = −0.081±0.299; ‘mixed-genotype within host’: <i>r</i> = 0.405±0.306; (<b>b</b>) Co-infecting genotypes within hosts that are classified as either primary or derived. Averages of classes are for ‘primary’: <i>r</i> = 0.066±0.348 (S.D.); ‘derived’: <i>r</i> = 0.457±0.263. Error bars represent ±1 S.E. Small figures are sample sizes (numbers of pairs). Different shadings represent different castes and years (see legend). Significant deviations from zero at a level of p<0.05 (t-tests for normalized data) for a given class are marked by an asterisk. Populations are the host individuals defining the genotypic background for the relatedness estimator.</p

Fraction of hosts that showed derived genotypes with mutational modifications (alleles lost or gained), or that were recombinants of primary infections.

<p><i>N</i> is the number of investigated host individuals.</p

Summary statistics of <i>C. bombi</i> infections in worker and queen bees over two years (summer 2008 – spring 2010).

<p><i>N</i> is number of hosts. Note that the study aim was to characterize a typical sample from the field. Sample sizes are thus too limited to generate a statistic for every host species separately.</p>a<p>Comparing prevalence of mixed-genotype infections (queens vs. workers): <i>χ</i>² = 1.357, <i>p</i> = 0.244.</p>b<p>Comparing number of different genotypes (queens vs. workers): <i>t</i>₈₀ = −1.225, <i>p</i> = 0.224.</p>c<p>Comparing number of primary infections (queens vs. workers): <i>t</i>₈₀ = 3.156, <i>p</i> = 0.002.</p

Total number of samples collected in the study years.

a<p>Queens in 2008 are daughter queens of that season.</p>b<p>Prevalence of infections. Castes differ for 2008 (Likelihood ratio = 8.772, <i>P</i> = 0.033), and for 2009 (LR = 101.49, <i>P</i><0.001).</p

Probing Mixed-Genotype Infections I: Extraction and Cloning of Infections from Hosts of the Trypanosomatid <em>Crithidia bombi</em>

<div><p>We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single <em>Crithidia bombi</em> cells from bumblebee faeces using flow cytometry. We report a precision of >99% in obtaining single trypanosomatid cells for further culture and analysis (“cloning”). In the study, we have investigated the use of different liquid media to cultivate <em>C. bombi</em> and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve <em>C. bombi</em> cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.</p> </div

Quality control for the sorting method.

<p>A standard mixture of five clones was sorted with our method and genotyped. A total of 651 wells could be genotyped.</p>1<p>Each plate is a replicate for the same mixture.</p>2<p>Clones: for genotypes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone-0049046-t002" target="_blank">Tab. 2</a>.</p>3<p>Errors occur when two clones are found in a single well (double); wells without cells are not errors of the sorting process in this sense but remain empty, and thus are unambiguous.</p>4<p>Coefficient of variation.</p

Scatter plot of defined cell populations targeted for sorting.

<p>Axes represent relative differences in size of cells (FSC-A), and relative differences in complexity or granularity of cells (SSC-A). The envelopes (solid lines) define a “gate” (P1) from within which cells will be collected by the procedure. The examples in the panels show (a) the gate for the pure clone no. 08.076, which is a cryo-preserved clone from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone.0049046-Wu1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone.0049046-Wu2" target="_blank">[37]</a>. Gates (b)–(d) refer to <i>C. bombi</i> cells from raw faeces. For (a) and (b), the defined collection gates P1 are identical;(c) and (d) have adjusted P1 gates, as different clones can vary in shape and granularity. Only cells within a defined gate will be selected and sorted into the well plates. Gates can be defined and adjusted based on how variable the targeted population is. All additional cell populations contained in the sample (noise spreading along the SSC-A axis) which could be anything from microbial or fungal origin to cell debris, will be excluded. Note that this noise is much less substantial in the pure clone (a), where the only noise could be dead cells, compared to the faeces samples, which contain cells of various origins.</p

Preparation of optimized culture medium for <i>C. bombi</i>.

(1)<p>autoclave and store at 4°C.</p>(2)<p>aliquot to 5.0 ml and store at −20°C.</p>(3)<p>aliquot to 5.0 ml and store at −20°C.</p>(4)<p>aliquot to 50 µl.</p