DataSheet_1_Molecular characterization of Smtdc-1 and Smddc-1 discloses roles as male-competence factors for the sexual maturation of Schistosoma mansoni females.zip

IntroductionSchistosomes are the only mammalian flatworms that have evolved separate sexes. A key question of schistosome research is the male-dependent sexual maturation of the female since a constant pairing contact with a male is required for the onset of gonad development in the female. Although this phenomenon is long known, only recently a first peptide-based pheromone of males was identified that contributes to the control of female sexual development. Beyond this, our understanding of the molecular principles inducing the substantial developmental changes in a paired female is still rudimentary.ObjectivesPrevious transcriptomic studies have consistently pointed to neuronal genes being differentially expressed and upregulated in paired males. These genes included Smp_135230 and Smp_171580, both annotated as aromatic-L-amino-acid decarboxylases (DOPA decarboxylases). Here, we characterized both genes and investigated their roles in male–female interaction of S. mansoni.Methodologies/findingsSequence analyses indicated that Smp_135230 represents an L-tyrosine decarboxylase (Smtdc-1), whereas Smp_171580 represents a DOPA decarboxylase (Smddc-1). By qRT-PCR, we confirmed the male-specific and pairing-dependent expression of both genes with a significant bias toward paired males. RNA-interference experiments showed a strong influence of each gene on gonad differentiation in paired females, which was enhanced by double knockdown. Accordingly, egg production was significantly reduced. By confocal laser scanning microscopy, a failure of oocyte maturation was found in paired knockdown females. Whole-mount in situ hybridization patterns exhibited the tissue-specific occurrence of both genes in particular cells at the ventral surface of the male, the gynecophoral canal, which represents the physical interface of both genders. These cells probably belong to the predicted neuronal cluster 2 of S. mansoni.ConclusionOur results suggest that Smtdc-1 and Smddc-2 are male-competence factors that are expressed in neuronal cells at the contact zone between the genders as a response of pairing to subsequently control processes of female sexual maturation.</p

Current and projected (2011-2040) climatic suitability for five sand fly species in Bavaria (southern Germany).

<p>Currently, the climatic suitability for sand fly species is rather low or moderate in Bavaria. The projections for future climate change refer to the A1B emission scenario of greenhouse gases, expecting an economical growth in a globalized world with a balanced use of fossil and non-fossil energy resources. During the next decades it can be expected that the suitability will increase. This is true for all analyzed sand fly species. A rather high climatic suitability can be expected for the valley of the river Main, especially for Lower Franconia in the most north-western part of Bavaria, along the river Danube and for the alpine foothills.</p

Map of southern Germany with sampling regions of the sand fly survey in the years 2009 and 2010.

<p>The insert shows a map of Germany with the Federal States of Bavaria and Baden-Württemberg highlighted. CDC light traps for sand fly trapping were put up in seven sampling areas in Bavaria (indicated by the numbers 1-7 and shaded in gray) which were allocated to the following administrative districts: 1, Aschaffenburg/Main-Spessart; 2, Würzburg/Kitzingen; 3, Erlangen-Höchstadt/Forchheim; 4, Regensburg/Kelheim; 5, Aichach-Friedberg/Landsberg am Lech/Weilheim-Schongau; 6, Passau; 7, Lindau am Bodensee/Oberallgäu. Sites of reported cases of autochthonous leishmaniasis in Bavaria and Baden-Württemberg and some of the reported sites of sand flies in Baden-Württemberg are marked [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081088#B12" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081088#B14" target="_blank">14</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081088#B16" target="_blank">16</a>].</p

Inhibition of AChE enzymatic activity by harmonine.

(A, B) Expression of SmAChE1 (Smp_154600) and SmAChE2 (Smp_125350) in male and female schistosomes after 72 h treatment with different concentrations of harmonine as determined by qPCR, expressed as relative expression vs. the geometric mean of three reference genes. The expression in control worms was set to 100%. A summary of two experiments with SEM is shown. (C) Enzymatic activity over time in protein lysates of paired males after adding different concentrations of harmonine (0 – 100 μM). One representative out of three similar experiments is shown. (D) Enzymatic activity of AChE from a model organism (electric eel) after addition of different concentrations of harmonine (0 – 100 μM). One representative out of two similar experiments is shown. (E) Relative AChE activity of electric eel at 30 min after adding different concentrations of harmonine, with the activity at 0 μM set as 100%. Mean values of two experiments were used. IC50 was calculated by non-linear least squares curve fitting using the ic50.tk tool. Significant differences as determined by the unpaired t-test are indicated with * pp<0.001.</p

Reduction in the number of proliferating neoblasts upon harmonine treatment.

S. mansoni couples were treated and processed for EdU labeling of proliferating cells as described in Fig 7. (A) Representative images of a control and a harmonine-treated female showing proliferating cells (neoblasts) in the parenchyma and/or vitellarium. Scale bar: 100 μm. BF, bright field. (B, C) Expression of the stem cell marker nanos-2 (Smp_051920) in harmonine-treated (B) or hydroxyurea-treated (C) females related to the expression in untreated control females (set to 100%) as determined by qPCR (summary of two experiments). An unpaired t-test was performed to reveal significant differences (* pp<0.001).</p

Effect of harmonine on gonadal tissue structure.

For CLSM-analysis of gonadal tissues, S. mansoni couples were cultured for 72 h with 10 μM harmonine (E-G) or solvent as a control (B-D), and stained with carmine red. (A) Bright-field microscopic images indicating the localization of ovary and testes in worms. (B, E) Confocal images showing part of the intact vitellarium of a control female (still paired with a male) (B) compared to the porous appearance of the vitellarium after harmonine treatment (E). (C, F) Well-defined immature (iO) and mature (mO) parts of a control ovary (C), compared to the disintegrated structure of an ovary in a harmonine-treated female (F). (D, G) Seminal vesicle (sv) filled with spermatozoa, and testes lobes filled with spermatogonia of a control male (D), compared with the gonad of a harmonine-treated male with reduced number of spermatozoa and partially disintegrated lobes (arrows) (G). Scale bar: 50 μm.</p

Tissue damage induced by harmonine.

S. mansoni couples were cultured for 72 h with 5 μM harmonine with a change of medium and compound every 24 h. (A, B) Bubble formation (arrows) on the tegument surface in a representative female (A) and male (B) worm. (C) Gut dilatation (arrow) in a female worm, visualized by bright-field microscopy. (D, E) CLSM images of carmine red-stained worms showing the gut lumen of a control male (D) and the dilated lumen (arrow) of a harmonine-treated male (E). Scale bars are 100 μm (A-C) or 50 μm (D, E).</p

Effect of harmonine on motility, viability and pairing.

S. mansoni couples were treated with different concentrations of harmonine for 72 h and microscopically screened every 24 h. As a negative control, the solvent DMSO was added in an amount as present in the highest harmonine concentration. (A) Representative images of a control couple and worms after treatment with 10 μM harmonine for 72 h. Scale bar: 100 μm. H. axyridis [33] and the structure of the alkaloid harmonine are shown in the middle. (B) Percentage of paired worms after harmonine treatment. (C) Dose-response curve for pairing stability (expressed as % pairing) after 72 h of treatment. (D) Worm motility reflected by motility scores 3 = normal motility, 2 = reduced motility, 1 = almost no movements, 0 = dead. (E) Dose-response curve for the reduction of motility by harmonine after 72 h of treatment. EC50 values were calculated by non-linear least squares curve fitting using the ic50.tk tool. A-C show a summary of two experiments with 10 worm couples per experiment and condition; error bars: SEM. Triangles indicate a value of zero. Significant differences to control worms at the respective time point are indicated with ** pp<0.001 (students t-test).</p

Reduction of gonadal stem cell proliferation upon harmonine treatment.

S. mansoni couples were treated with 20 μM harmonine in vitro for 6 days. As control, an equivalent volume of the solvent DMSO was used. EdU was added for the last 24 h of culture, worm couples were separated and images were processed using the IMARIS software as described in the text and Fig 6. (A, B) Representative images (A) and summary of the analyses of four ovaries (B) from either control or harmonine-treated females. A significant reduction of stem cells was found after treatment, expressed as percentage of EdU-positive per Hoechst-positive cells. (C, D) Representative images (C) and summary of four testes (C) from control and harmonine-treated males showing a significant reduction of the percentage of stem cells following treatment. Scale bar: 30 μm. (E, F) Expression of the gonadal stem cell marker nanos-1 (Smp_055740) in females (E) and males (F) after treatment with 20 μM harmonine compared to control worms as determined by qPCR. The expression in control worms was set to 100%. Summary of two experiments. An unpaired t-test was performed to reveal significant differences (** pp<0.001).</p

Effect of harmonine on egg production.

S. mansoni couples were cultured with different concentrations of harmonine for a period of 72 h. Medium and compound were renewed every 24 h. (A, C) Effect of harmonine on reproduction was assessed by counting the number of total eggs being laid by 10 couples per 24 h period (A) and their percentage of deformed eggs (C). (B) Dose-response curve for the reduction of egg numbers being laid in the last 24 h of the 72 h culture. Summary of two experiments with 10 couples of S. mansoni per experiment and condition; error bars: SEM. (D) Representative images of eggs from control couples and after 72 h treatment with 5 μM harmonine. Deformed eggs were e.g. smaller and often lacked an oocyte, a spine, or a normal amount of vitelline cells. Scale bar is 50 μm. Significant differences to the control at the respective time point are indicated with * ppp<0.001 (students t-test).</p