Schematic illustration and surface electron microscopy (SEM)-analyses of tegument solubilisation by TS-solution treatment.

<p><b>A)</b> Untreated control males (upper left) and females (upper right) showing intact tegument (TE) with spines (SP), pits (PI), and sensory endings (SE). <b>B)</b> The tegument was completely removed due to detergent treatment exposing the outer circular muscles (CM) and the basis of the (male-specific) tubercles (TU). Membranocalyx (MC), plasma membrane (PM), longitudinal muscles (LM), basal membrane (BM), musculature (MU), parenchyma (PA); modified according to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd.0002336-Braschi3" target="_blank">[107]</a>; dashed arrow = continued in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-g002" target="_blank">Figure 2</a>.</p

Protein patterns of <i>S. mansoni</i> organs/tissues and adults.

<p>1.2 µg total protein from male (Mars symbol), female (Venus symbol), testes (Te), ovaries (Ov), and male tegument (T) were separated by 13% SDS-PAGE and visualised by silver staining. Marker (M) = PageRuler Plus Prestained Protein Ladder (Fermentas).</p

Quantitative and qualitative microfluid analysis of total RNA.

<p>RNA-analyses exemplarily shown for RNA isolated from adult males (<b>A</b>), testes (<b>B</b>), and ovaries (<b>C</b>) obtained by the organ isolation procedure were used. The figure shows a “gel-like image” consisting of the RNA-ladder and the appropriate total RNA sample (left) and the corresponding electropherogram (right); fluorescent units (FU), retention time (s).</p

Summary of gene-specific expression patterns.

<p>* = current study, <b>+</b> = detected, <b>−</b> = not detected, <b>nd</b> = not determined.</p

Target genes and RT-PCR primers.

<p>Target genes and RT-PCR primers.</p

Northern-blot analyses of SmDia transcripts.

<p>Northern-blot analyses with RNA (left side) of adult schistosomes (mixed-sex). As probe for the filter, a radioactively labeled part of SmDia (fragments S3, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006998#pone-0006998-g004" target="_blank">Fig. 4A</a>) was used (right side). Sizes (kb) are indicated. M = RNA molecular weight marker.</p

Gonad protein-specific immunoblots.

<p>15 µg of total protein per lane isolated from adult worms (Mars and Venus symbol), testes (Te), ovaries (Ov), and tegumental proteins of both genders (T) were analysed by immunoblotting employing immune sera directed against SmSPRM1hc (Permease 1 heavy chain), SmHSP70 (Heat shock protein 70), SmAQP (Aquaporin), and SmFKBP12 (FK506-binding protein); for references see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-t001" target="_blank">Table 1</a>.</p

Schematic illustration and bright-field microscopy (BF) of gonad tissues following tegument solubilisation and protease treatment.

<p><b>A)</b> Crude preparation of intact testes (TE) together with a part of an incompletely digested male worm body (MB) and different types of cells (CE) (left) and an mature ovary (Om) surrounded mainly by S4-vitelline cells (VC) from the vitellarium (right); immature ovary (Oi) and ootype (OT) with vitelloduct (VD) and oviduct (OD) isolated from a unisexual female; the ootype was contrasted by brief staining with Ponceau S; asterisk: “hymen”-like morphological structure typical for ootypes of unisexual females <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd.0002336-Beckmann1" target="_blank">[18]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd.0002336-Beckmann2" target="_blank">[108] </a><b>B)</b> Mechanical transfer by pipetting led to the enrichment of pure testes (TE), mature ovaries (Om) after collecting and concentrating. TL (testes lobe), Op (ovary - posterior part containing mature primary oocytes in the case of mature ovaries), Oa (ovary - anterior part containing immature, stem cell-like oogonia); vitellarium (VI) with vitelline lobes (VL); dashed arrow = continued from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-g001" target="_blank">Figure 1</a>.</p

SmShb interacts with ligand-activated SmVKR1 and SmVKR2 receptors.

<p>(A) cRNA encoding V5-tagged SmVKR1 WT or SmVKR1 Y₉₇₉F were injected with or without cRNA encoding HA-tagged SmShb in <i>Xenopus</i> oocytes. Following their expression, full-length receptors were activated by L-Arg to induce their auto-phosphorylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163283#pone.0163283.ref011" target="_blank">11</a>]. Proteins were then analysed by immunoprecipitation (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results demonstrated that only phosphorylated SmVKR1 WT bound to and phosphorylated SmShb as revealed by PY20 antibodies. (B) cRNA encoding V5-tagged SmVKR2 WT or SmVKR2 F₉₄₉Y were injected with or without cRNA encoding HA-tagged SmShb in <i>Xenopus</i> oocytes. Following their expression, full-length receptors were activated by Ca⁺⁺ to induce their auto-phosphorylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163283#pone.0163283.ref011" target="_blank">11</a>]. Proteins were then analysed by immunoprecipiration (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) analysis using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results indicated that phosphorylated SmVKR2 WT did not bind SmShb whereas the mutated V5-SmVKR2 F₉₄₉Y bound to and phosphorylated SmShb.</p

Phylogenetic analyses showing the unique status of plathyhelminth α-integrins.

<p>Phylogram of the analysis of the full-length sequences of the <i>S. mansoni</i> α-integrin receptors Smα-Int1, Smα-Int2, Smα-Int3, and other α-integrin receptors using CLUSTAL X (<a href="http://www.clustal.org" target="_blank">www.clustal.org</a>) and TreeViewX. The phylogenetic relationship was deduced using the Bootstrap Neighbour-Joining (N–J) method and the bootstrap values were generated based on 1000 bootstrap trails with a random number generator seed of 100. Sequences were obtained from the National Centre for Biotechnology Information using the WWW Entrez Browser (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>), Swiss-Prot (<a href="http://www.uniprot.org" target="_blank">www.uniprot.org</a>), GeneDB (<a href="http://www.genedb.org" target="_blank">www.genedb.org</a>), and the <i>Schmidtea mediterranea</i> Genome Database (<a href="http://smedgd.neuro.utah.edu/" target="_blank">http://smedgd.neuro.utah.edu/</a>). The corresponding protein numbers are: Sha a1 (α-integrin 1 receptor, <i>S. haematobium</i>; Sha_102401), Sm a1 (α-integrin 1 receptor, <i>S. mansoni</i>; FR749887), Sjp a1 (α-integrin 1 receptor, <i>S. japonicum</i>; Sjp_0037690), Cs a5 (α-integrin 5 receptor, <i>Clonorchis sinesis</i>; GAA56616.1), Em a1 (α-integrin 1 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000215000 ), Sm a4 (α-integrin 4 receptor, <i>S. mansoni</i>; Smp_1735401, Smp_181010), Sha a4 (α-integrin 4 receptor, <i>S. haematobium</i>; Sha_104436, Sha_106831), Sjp a4 (α-integrin 4 receptor, <i>S. japonicum</i>; Sjp_0046780, Sjp_0046790), Cs a4 (α-integrin 4 receptor, <i>Clonorchis sinesis</i>; GAA28731), Em a4 (α-integrin 4 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000573500), Sha a2 (α-integrin 2 receptor, <i>S. haematobium</i>; Sha_106921), Sm a2 (α-integrin 2 receptor, <i>S. mansoni</i>; FR749888), Sjp a2 (α-integrin 2 receptor, <i>S. japonicum</i>; Sjp_0069490), Cs a-ps (α-integrin-ps receptor, <i>Clonorchis sinesis</i>; GAA54095, GAA49531, GAA49530), Em a2 (α-integrin 2 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000192500 ), Smed a3 (α-integrin 3 receptor, <i>Schmidtea mediterranea</i>; lcl|mk4.000046.14.01), Smed a1 (α-integrin 1 receptor, <i>Schmidtea mediterranea</i>; lcl|mk4.001411.00.01), Smed a2 (α-integrin 2 receptor, <i>Schmidtea mediterranea</i>; lcl|mk4.003797. 00.01), Sha a3 (α-integrin 3 receptor, <i>S. haematobium</i>; Sha_102914), Sm a3 (α-integrin 3 receptor, <i>S. mansoni</i>; FR749889, Smp_156610, Smp_156620), Sjp a3 (α-integrin 3 receptor, <i>S. japonicum</i>; Sjp_0063430, Sjp_0063420), Cs a7 (α-integrin 7 receptor, <i>Clonorchis sinesis</i>; GAA52225.1), Em a3 (α-integrin 3 receptor, <i>Echinococcus multilocularis</i>; EmuJ_000782500), Sp aP (α-integrin P receptor, <i>Strongylocentrotus purpuratus</i>, AF177914), Dm aPS2 (α-integrin PS2 receptor, <i>Drosophila melanogaster</i>, Q24247), Mm a2b (α-integrin 2b receptor, <i>Mus musculus</i>; EDL34136.1), Hs a2b (α-integrin 2b receptor, <i>Homo sapiens</i>; EAW51595.1), Xl a2b (α-integrin 2b receptor, <i>Xenopus laevis</i>; NP_001088223.1), Mm a5 (α-integrin 5 receptor, <i>Mus musculus</i>; CAA55638.1), Rn a5 (α-integrin 5 receptor, <i>Rattus norvegicus</i>; NP_001101588.1), Hs a5 (α-integrin 5 receptor, <i>Homo sapiens</i>; NP_002196.2), Xl a5 (α-integrin 5 receptor, <i>Xenopus laevis</i>; NP_001081072.1), Hs aV (α-integrin V receptor, <i>Homo sapiens</i>; P06756), Hs a8 (α-integrin 8 receptor, <i>Homo sapiens</i>; P53708), Ce a-pat2 (α-integrin pat-2, <i>Ceanorhabditis elegans</i>; P34446), Gc a (α-integrin receptor, <i>Geodia cydonium</i>; X97283), Hs a1 (α-integrin 1 receptor, <i>Homo sapiens</i>; P56199), Hs a2 (α-integrin 2 receptor, <i>Homo sapiens</i>; P17301), Hs a10 (α-integrin 10 receptor, <i>Homo sapiens</i>; O75578), Hs a11 (α-integrin 11 receptor, <i>Homo sapiens</i>; Q9UKX5), Hs aD (α-integrin D receptor, <i>Homo sapiens</i>; Q13349), Hs aX (α-integrin X receptor, <i>Homo sapiens</i>; P20702), Hs aM (α-integrin M receptor, <i>Homo sapiens</i>; P11215), Hs aL (α-integrin L receptor, <i>Homo sapiens</i>; P20701), Hs aE (α-integrin E receptor, <i>Homo sapiens</i>; P38579), Hs a4 (α-integrin 4 receptor, <i>Homo sapiens</i>; P13612), Hs a9 (α-integrin 9 receptor, <i>Homo sapiens</i>; Q13797), Mm a7 (α-integrin 7 receptor, <i>Mus musculus</i>; AAA16600.1), Rn a7 (α-integrin 7 receptor, <i>Rattus norvegicus</i>; NP_110469.1), Hs a7 (α-integrin 7 receptor, <i>Homo sapiens</i>; EAW96822.1), Hs a6 (α-integrin 6 receptor, <i>Homo sapiens</i>; P23229), Hs a3 (α-integrin 3 receptor, <i>Homo sapiens</i>; P26006), Dm aPSI (α-integrin PSI receptor, <i>Drosophila melanogaster</i>, Q24247), and Ce a-ina1 (α-integrin ina1, <i>Ceanorhabditis elegans</i>; Q03600).</p