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

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

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

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

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

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

Synopsis of tests with various media used for growing <i>C. bombi</i> in culture (see Information S1) for details on the composition of media), after [38].

1<p>FBS: Fetal Bovine Serum (Biological Industries, K Beth Haemek, Israel). FBS could not be replaced by horse serum, or calf serum, as cells agglutinated and perished in these alternatives.</p>2<p>Composition of media can be found in the following references.</p><p>- Grace (Gibco 11605) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone.0049046-Grace1" target="_blank">[68]</a>, developed for insect cell cultures.</p><p>- SDM-79 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone.0049046-Brun1" target="_blank">[69]</a>, used for cultivation of pro-cyclic forms of <i>Trypanosoma brucei</i>.</p><p><i>-</i> SM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone.0049046-Cunningham1" target="_blank">[70]</a>, used for cultivation of trypanosomatids.</p

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