Cecal_1_100uL_SDSTCAUrea_2d_24hr_OrbiVelos_Run3_032310_02

Second salt pulse for ceca 1, run

Cecal_1_100uL_SDSTCAUrea_2d_24hr_OrbiVelos_Run3_032310_01

First salt pulse for Ceca 1, run

<i>Onchocerca flexuosa</i> sequences related to <i>de novo</i> purine and pyrimidine biosynthesis.

<p>KEGG pathway modules are outlined for inosine monophosphate (A) and uridine monophosphate (B) biosynthesis (module entries M00048 and M00051, respectively). Boxes representing enzymes present in nematodes (e.g., <i>C. elegans</i>), <i>Wolbachia</i>, or both are colored blue, red and purple, respectively. Enzymes sharing sequence identity with <i>O. flexuosa</i> peptide translations are highlighted in yellow. Enzymes identified from <i>B. malayi</i> are marked with an asterisk.</p

Localization of the putative LolC peptide by immunohistology and <i>in situ</i> hybridization in <i>Onchocerca flexuosa</i>.

<p>The putative LolC peptide and transcript were localized in adult worm tissues via immunohistology (A-G) and <i>in situ</i> hybridization (H-K), respectively. Negative control antibodies against keyhole limpit hemocyanin (carrier protein) showed no specific staining in <i>O. flexuosa</i> (A). Antibodies against a peptide from MS protein 1591, a putative homolog of <i>Wolbachia</i> LolC, stained the fibrillar portions of the somatic muscles (arrow) in a cross section of an adult male worm (B). The somatic muscles (solid arrows) and excretory cell (dashed arrow) are stained in cross sections of young adult females (C, D). Magnification of the excretory cell shows intense staining of the cell membrane (dashed arrow) and more diffuse staining in adjacent muscles (solid arrow) (E). The uterine muscles of an older female (arrow) are clearly stained (F), as are the intrauterine stretched microfilaria (F, G). The LolC sense RNA probe produced no signal in <i>in situ</i> hybridizations (H), while the antisense RNA probe labeled the lateral chords (arrows) and developing sperm within the male testes (I, J). The antisense RNA probe also labeled the lateral chords (arrow), intestine, and uteri of a young adult female (K). Abbreviations: m, muscle; lc, lateral chords; i, intestine; ut, uterus; mf, microfilariae; hy, hypodermis; t, testes; vd, vas deferens. Scale bars = 25 µm.</p

Western blots detecting the putative LolC peptide in <i>Onchocerca flexuosa</i> adult worm lysate.

<p>Affinity-purified rabbit antibodies raised against a peptide from MS protein 1591, a putative LolC homolog, bound a 38 kDa band in Western blots of <i>O. flexuosa</i> adult worm lysate (lane 1). Purified IgG from pre-immune serum (lane 2) and antibodies against the keyhole limpit hemocyanin carrier (lane 3) did not bind to proteins in this size range.</p

Selected KEGG pathway modules in <i>B. malayi</i> and <i>O. flexuosa</i>. Table lists the number of module enzymes represented in each dataset.

<p>Selected KEGG pathway modules in <i>B. malayi</i> and <i>O. flexuosa</i>. Table lists the number of module enzymes represented in each dataset.</p

<i>Wolbachia</i>-like peptides identified by mass spectroscopy (MS).

<p><i>Wolbachia</i> genes are shown in red with MS peptides highlighted in blue and peptide sequences shown in black text. One peptide with two charge states was mapped to a LolC protein found in several insect <i>Wolbachia</i> strains, including the <i>Wolbachia</i> endosymbiont of <i>Drosophila simulans</i> (wRi) (A), and two unique peptides were mapped to a HlyD family protein from the <i>Wolbachia</i> endosymbiont of <i>Culex</i> quinquefasciatus (wCq) (B). A region with sequence identity to the LolC gene was identified in the <i>O. flexuosa</i> genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045777#pone.0045777-McNulty1" target="_blank">[14]</a>, but this locus would not be capable of producing the exact peptide identified in our MS experiment (consensus shown in blue text). Polyclonal antibodies were raised against the underlined portion of the LolC peptide.</p

Results of BLAST searches comparing <i>O. flexuosa</i> transcripts to various databases.

<p>Results of BLAST searches comparing <i>O. flexuosa</i> transcripts to various databases.</p

Sequence conservation between <i>Onchocerca flexuosa</i> and <i>O. volvulus</i>.

<p><i>O. flexuosa</i> transcripts containing <i>Wolbachia</i>-like sequences were compared with the latest version of the <i>O. volvulus</i> genome assembly. Eighteen <i>O. flexuosa</i> isogroups (24 isotigs) and eight singletons with <i>Wolbachia</i>-like sequences shared at least 80% sequence identity with sequences from <i>O. volvulus</i> over 80% of the length of the <i>O. flexuosa</i> transcript. For example, <i>O. flexuosa</i> isotig05747 shares 90% sequence identity with a 1344 bp portion of <i>O. volvulus</i> scaffold245.1; both of these sequences contain a similar <i>Wolbachia</i>-like insert (A). Another nine <i>O. flexuosa</i> isogroups (9 isotigs) and one singleton matched <i>O. volvulus</i> sequences, except the latter did not contain <i>Wolbachia</i>-like inserts. For example, the sequences before and after the <i>Wolbachia</i>-like insert in <i>O. flexuosa</i> isotig09655 share 84% and 89% sequence identity with corresponding regions of <i>O. volvulus</i> scaffold14.1, respectively; however, the <i>Wolbachia</i>-like sequence present in <i>O. flexuosa</i> is not present in <i>O. volvulus</i> (B). Regions sharing sequence identity with <i>Wolbachia</i> are indicated in red while regions similar to nematode genes are indicated in blue. Arrows indicate directionality of high-scoring segment pairs. The grey dashed line represents a gap in the sequence alignment between the two species.</p

Results of sequencing, assembly, and translation of the <i>O. flexuosa</i> transcriptome.

<p>Results of sequencing, assembly, and translation of the <i>O. flexuosa</i> transcriptome.</p