A draft genome of the honey bee trypanosomatid parasite Crithidia mellificae

Abstract

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. Crithidia mellificae is a unicellular eukaryotic honey bee parasite that has been associated with colony losses in the USA and Belgium. C. mellificae is a member of the family Trypanosomatidae, which primarily includes other insect-infecting species (e.g., the bumble bee pathogen Crithidia bombi), as well as species that infect both invertebrate and vertebrate hosts including human pathogens (e.g.,Trypanosoma cruzi, T. brucei, and Leishmania spp.). To better characterize C. mellificae, 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 Leishmania major. We estimate that C. mellificae encodes over 8,300 genes, the majority of which are orthologs of genes encoded by L. major and other Leishmania or Trypanosoma species. Genes unique to C. mellificae, 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 C. mellificae infection on honey bee health and provide insight into the evolution of this unique family.

Figure 1. Crithidia mellificae, a trypanosomatid parasite of honey bees.

(A) Majority consensus tree of select members of the Trypanosomatidae derived from Bayesian analysis , (i.e., MrBayes v3.1.2 [57]) of a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) nucleotide alignment (799 nt). T. cruzi was selected as the outgroup based on results from previous phylogenetic analyses , , –. 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 Crithidia deanei was renamed Angomonas deanei. (B) Composite of light and fluorescent microscope images of C. mellificae 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.

Figure 2. Assembly and annotation of C. mellificae contig 175.

(A) Read coverage of the gDNA library used for assembly. A gene duplication of the GAPDH gene is highlighted on the right. (B) RNA-seq coverage aligned to the contig. (C) Genes predicted by the Maker pipeline in C. mellificae with assigned putative functions. The homologous and syntenic region of L. major is shown below, with nucleotide identity of the C. mellificae genes to L. major color-coded by nt identify (green ≥70%, yellow ≥60%, red <60%). A putative bacterial xenolog of a sulfate transporter, sulfate permease JQ247792, is noted (sixth from the right).

Figure 3. The gene catalogues of Leishmania major and Crithidia mellificae are compared after ortholog analysis by INPARANOID .

*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).

Figure 4. Urea and polyamine synthesis in trypanosomatids.

The putative origin of genes involved in urea and polyamine synthesis is indicated by color; eukaryotic (blue) or bacterial (red) origin. For C. mellificae, genes conserved from L. major are displayed on the left column and unique genes on the right; the compounds are numbered as follows: 1. aspartic acid, 2. citrulline, 3. argininosuccinate, 4. arginine, 5. ornithine, 6. putrescine.