<i>S. stercoralis</i> ILP promoters are active in the nervous system and other tissues.

<p>Transgenic <i>S. stercoralis</i> post-free-living larvae expressing enhanced green fluorescent protein (EGFP) under the control of three insulin-like peptide (ILP) promoters were assessed for tissue-specific expression. (A–D) Transgenic larvae carrying the <i>Ss-ilp-1</i> promoter::<i>egfp</i> reporter construct; (A,C) DIC images and (B,D) fluorescent images. The <i>Ss-ilp-1</i> promoter is active in the hypodermis/body wall and a single pair of head neurons (D, arrow). (E–H) Transgenic larvae carrying the <i>Ss-ilp-6</i> promoter::<i>egfp</i> reporter construct; (E,G) DIC images and (F,H) fluorescent images. The <i>Ss-ilp-6</i> promoter is active in the hypodermis/body wall and several head neurons. (I–L) Transgenic larvae carrying the <i>Ss-ilp-7</i> promoter::<i>egfp</i> reporter construct; (I,K) DIC images and (J,L) fluorescent images. The <i>Ss-ilp-7</i> promoter is active in the intestine and a single pair of head neurons, with a single process that extends dorsally almost to the anterior portion of the intestine (L, arrow), most consistent with the SIAV neurons in <i>C. elegans</i>.</p

Location of EGFP expression in transgenic <i>S. stercoralis</i> post-free-living larvae under the control of ILP promoters.

<p>Location of EGFP expression in transgenic <i>S. stercoralis</i> post-free-living larvae under the control of ILP promoters.</p

Transcript abundance of <i>S. stercoralis</i> homologs of <i>C. elegans</i> chemosensory 7TM GPCRs.

<p>Transcript abundance of <i>S. stercoralis</i> homologs of <i>C. elegans</i> chemosensory 7TM GPCRs.</p

Identification of <i>S. stercoralis</i> heterotrimeric G protein orthologs.

<p>Identification of <i>S. stercoralis</i> heterotrimeric G protein orthologs.</p

Regulation of <i>C. elegans</i> dauer development by cellular signaling pathways.

<p>Dauer entry in <i>C. elegans</i> is regulated by four signaling pathways: a cyclic guanosine monophosphate (cGMP) signaling pathway, an insulin/IGF-1-like signaling (IIS) pathway, a dauer transforming growth factor β (TGFβ) signaling pathway, and a DAF-12 nuclear hormone receptor (NHR) regulated by dafachronic acid (DA) steroid ligands. Unfavorable conditions stimulate dauer entry (left panel) by down-regulating cGMP production, increasing expression of antagonistic insulin-like peptides that down-regulate IIS, decreasing expression of the dauer TGFβ ligand, and inhibiting production of DAs. When dauer larvae encounter favorable conditions, these pathways are hypothesized to act in reverse. Dotted lines signify down-regulated signaling through the pathway, while black lines signify up-regulated signaling through the pathway. Colored proteins are active; grayed out proteins are inactive. Grayed out ligands are absent. Adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-Fielenbach1" target="_blank">[12]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-McGrath1" target="_blank">[32]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-Wollam1" target="_blank">[43]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-Cornils1" target="_blank">[91]</a>.</p

<i>S. stercoralis</i> L3i are activated by 8-bromo-cGMP.

<p>The membrane-permeable cGMP analog, 8-bromo-cGMP, induced resumption of feeding, a hallmark of activation, in <i>S. stercoralis</i> L3i. Feeding was assessed by ingestion of a FITC dye after incubation at 37°C and 5% CO₂ in air for 24 hours for all conditions. (A) At 200 µM, 8-bromo-cGMP dissolved in M9 buffer results in potent resumption of feeding in L3i, with 85.1% (±2.2, SD) of larvae feeding after 24 hours. In comparison, host-like cues consisting of DMEM, 10% canine serum (S), and 12.5 mM reduced glutathione (G), resulted in 43.9% (±2.6, SD) of L3i feeding, while the M9 buffer negative control resulted in 0.6% (±0.3, SD) of L3i feeding, after 24 hours. (B) Kinetics of activation were determined for both 200 µM 8-bromo-cGMP and host-like cues, consisting of DMEM, 10% canine serum, and 3.75 mM reduced glutathione, after incubation for 4, 6, 12, 18, or 24 hours. All conditions were incubated for a total of 24 hours at 37°C and 5% CO₂ in air. Error bars represent ±1 standard deviation (SD).</p

Protein sequence diversity and temporal regulation of <i>S. stercoralis</i> insulin-like peptides.

<p>(A) A predicted protein sequence alignment of seven <i>S. stercoralis</i> insulin-like peptides (ILPs), <i>Ss</i>-ILP-1 through -7, was constructed using human insulin (<i>Hs</i>-INSULIN) and <i>Lymnaea stagnalis</i> molluscan insulin-related peptide I (<i>Ls</i>-MIP-1) as the references. Cysteine residues, which are predicted to form disulfide bonds, are in red letters. Predicted signal sequences are highlighted in yellow, predicted furin recognition motifs are highlighted in red, hydrophobic residues important for helix formation are highlighted in green, and a conserved glycine is highlighted in blue. Predicted C peptides are highlighted in gray with dibasic predicted cleavage sites underlined. The B peptide is N-terminal of the C peptide, while the A peptide is C-terminal of the C peptide. (B-H) Transcript abundances were determined for the coding region of seven <i>S. stercoralis</i> ILP-encoding genes (<i>Ss-ilp-1</i> through <i>-7</i>) in seven developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), <i>in vivo</i> activated third-stage larvae (L3+), parasitic females (P Female), post-parasitic first-stage larvae (PP L1), and post-parasitic third-stage larvae (PP L3). Transcript abundances were calculated as fragments per kilobase of coding exon per million mapped reads (FPKM) and log transformed. Error bars represent 95% confidence intervals. The y-axes were scaled from 0 to 3.5 to aid comparison between genes.</p

RNAseq Analysis of the Parasitic Nematode <i>Strongyloides stercoralis</i> Reveals Divergent Regulation of Canonical Dauer Pathways

<div><p>The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode <i>Strongyloides stercoralis</i> differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode <i>Caenorhabditis elegans</i> has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in <i>S. stercoralis</i>, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for <i>S. stercoralis</i> homologs of <i>C. elegans</i> dauer genes identified by BLAST searches of the <i>S. stercoralis</i> genome as well as <i>de novo</i> assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to <i>C. elegans</i>, <i>S. stercoralis</i> has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven <i>S. stercoralis</i> genes encoding homologs of the single <i>C. elegans</i> dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in <i>S. stercoralis</i> and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.</p></div

John Nichols to Sir James Edward Smith

Notes that although [Emanuel Mendez] da Costa's [(1717-1791), naturalist] manuscripts are chiefly concerned with mineralogy there are several on botany from [George] Edwards [(1694-1773)], [Georg Dionysius] Ehret [(1708-1770)], Knolles, [John] Martyn [(1699-1768)], [Philip] Miller, [Richard] Pulteney, and some others, and are available for Smith's use [for "A selection of the correspondence of Linnaeus" (1821)]

<i>S. stercoralis</i> RNAseq mean library sizes and number of reads aligning to the genome.

<p>A total of 21 libraries were derived from polyadenylated RNA and sequenced from seven developmental stages, each in biological triplicate. Paired-end 100 base-pair (bp) reads were generated from the following developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), <i>in vivo</i> activated third-stage larvae (L3+), parasitic females (P Female), predominantly (>95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults and enriched for females (PP L3). The mean number of reads generated per replicate refers to the mean number of 100 bp reads sequenced (black bars) per biological replicate from each developmental stage. The mean number of mapped reads per replicate refers to the mean number of 100 bp reads aligned to <i>S. stercoralis</i> genomic contigs using TopHat (white bars) per biological replicate from each developmental stage. Error bars represent +1 standard deviation.</p