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

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

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

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

Summary of clones derived from 31 infected queens.

<p>One clone per queen was analysed here to show the diversity of genotypes. Entries are alleles (fragment length in bp) at five loci. Note that <i>C. bombi</i> is diploid. Sites are Neunforn (N) and Aesch (A).</p

Tested classical protocols for the isolation of <i>C. bombi</i> from the host (after [38]).

<p>Tested classical protocols for the isolation of <i>C. bombi</i> from the host (after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049046#pone.0049046-Bouquet1" target="_blank">[38]</a>).</p