A Draft Genome of the Honey Bee Trypanosomatid Parasite <i>Crithidia mellificae</i>

<div><p>Since 2006, honey bee colonies in North America and Europe have experienced increased annual mortality. These losses correlate with increased pathogen incidence and abundance, though no single etiologic agent has been identified. <i>Crithidia mellificae</i> is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. <i>C. mellificae</i> is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (<i>e.g</i>., the bumble bee pathogen <i>Crithidia bombi</i>), as well as species that infect both invertebrate and vertebrate hosts including human pathogens (<i>e.g.,Trypanosoma cruzi</i>, <i>T. brucei</i>, and <i>Leishmania spp.</i>). To better characterize <i>C. mellificae</i>, we sequenced the genome and transcriptome of strain SF, which was isolated and cultured in 2010. The 32 megabase draft genome, presented herein, shares a high degree of conservation with the related species <i>Leishmania major.</i> We estimate that <i>C. mellificae</i> encodes over 8,300 genes, the majority of which are orthologs of genes encoded by <i>L. major</i> and other <i>Leishmania</i> or <i>Trypanosoma</i> species. Genes unique to <i>C. mellificae,</i> including those of possible bacterial origin, were annotated based on function and include genes putatively involved in carbohydrate metabolism. This draft genome will facilitate additional investigations of the impact of <i>C. mellificae</i> infection on honey bee health and provide insight into the evolution of this unique family.</p></div

<i>Crithidia mellificae</i> genome assembly statistics.

<p><i>Crithidia mellificae</i> genome assembly statistics.</p

The gene catalogues of <i>Leishmania major</i> and <i>Crithidia mellificae</i> are compared after ortholog analysis by INPARANOID [60].

<p>*Truncated genes at contig ends were included in this analysis for a total of 9,971 ORFs. Approximately 17% of these ORFs are incomplete ends of the same presumed gene, resulting in ∼8,300 actual genes (see Results).</p

<i>Crithidia mellificae</i>, a trypanosomatid parasite of honey bees.

<p>(A) Majority consensus tree of select members of the Trypanosomatidae derived from Bayesian analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Yang2" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Lewis1" target="_blank">[56]</a> (<i>i.e.,</i> MrBayes v3.1.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Ronquist1" target="_blank">[57]</a>) of a <i>glyceraldehyde 3-phosphate dehydrogenase</i> (<i>GAPDH</i>) nucleotide alignment (799 nt). <i>T. cruzi</i> was selected as the outgroup based on results from previous phylogenetic analyses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Maori1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Baer1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Simpson3" target="_blank">[50]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095057#pone.0095057-Drummond1" target="_blank">[52]</a>. Numbers on branches are Bayesian posterior probabilities (0–1); scale bar corresponds to the proportion of nucleotide change. The genus and species names are consistent with the GenBank accession numbers in the figure; we note that <i>Crithidia deanei</i> was renamed <i>Angomonas deanei</i>. (B) Composite of light and fluorescent microscope images of <i>C. mellificae</i> illustrate the flagellum, kinetoplast (smaller, brighter DAPI stained organelle; yellow arrow) and nucleus (white arrow) of the crithidial stage and (C) additional life-stages in culture.</p

Predicted genes unique to <i>Crithidia mellificae.</i>

<p>*tblastx of each predicted gene was performed using all Trypanosomatidae sequences in the nr database, a tblastx threshold of an E-value ≤10⁻⁶ was selected and alignments that scored within this threshold and had an associated accession number and annotation are reported above. Significant alignments are reported as follows, “<i>C. mellificae</i> only” indicates that no other annotated sequence aligned with this gene, “Trypanosomatidae chromosome” indicates additional unannotated chromosomal sequences from trypanosomes, often very large data files, have a region within them that aligns with the annotated sequence from <i>C. mellificae</i>.</p

Predicted secondary structure is associated with recombination frequency.

<p>Marker pairs are binned based on the Unafold calculated RNA folding energy of the marker pair and the flanking pairs (52-nt fragments). Biological replicates are shown in black and grey. Statistical associations are determined by Student's t-test after multiple testing correction.</p

Mapping statistics.

<p>Mapping statistics.</p

Features of the GC-rich construct.

<p>Features of the GC-rich construct.</p

AU- and GC-tract frequency in Picornavirus species.

<p>Type strains of picornavirus species were analyzed for the presence of AU or GC 4-mers, with the absolute number of AU- or GC-tracts displayed on the positive and negative y-axes.</p

Bee microbiome paired-end, part A

The paired-end deep-sequence data from this paper was generated as a single lane of Illumina data, but was split up into 6 parts (A-F) for Dryad submission. This is part A. The file will unzip into two files containing the paired-end reads for the first 5.5M sequenced clusters. Phred+64 quality score encoding. Library prep details can be found in the Methods of the linked manuscript