The hitchhiker's guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi.

Wolbachia is a maternally inherited and ubiquitous endosymbiont of insects. It can hijack host reproduction by manipulations such as cytoplasmic incompatibility (CI) to enhance vertical transmission. Horizontal transmission of Wolbachia can also result in the colonization of new mitochondrial lineages. In this study, we present a 15-year-long survey of Wolbachia in the cherry fruit fly Rhagoletis cerasi across Europe and the spatiotemporal distribution of two prevalent strains, wCer1 and wCer2, and associated mitochondrial haplotypes in Germany. Across most of Europe, populations consisted of either 100% singly (wCer1) infected individuals with haplotype HT1, or 100% doubly (wCer1&2) infected individuals with haplotype HT2, differentiated only by a single nucleotide polymorphism. In central Germany, singly infected populations were surrounded by transitional populations, consisting of both singly and doubly infected individuals, sandwiched between populations fixed for wCer1&2. Populations with fixed infection status showed perfect association of infection and mitochondria, suggesting a recent CI-driven selective sweep of wCer2 linked with HT2. Spatial analysis revealed a range expansion for wCer2 and a large transition zone in which wCer2 splashes appeared to coalesce into doubly infected populations. Unexpectedly, the transition zone contained a large proportion (22%) of wCer1&2 individuals with HT1, suggesting frequent intraspecific horizontal transmission. However, this horizontal transmission did not break the strict association between infection types and haplotypes in populations outside the transition zone, suggesting that this horizontally acquired Wolbachia infection may be transient. Our study provides new insights into the rarely studied Wolbachia invasion dynamics in field populations.

Diversity and activity of sugar transporters in nematode-induced root syncytia.

The plant-parasitic nematode Heterodera schachtii stimulates plant root cells to form syncytial feeding structures which synthesize all nutrients required for successful nematode development. Cellular re-arrangements and modified metabolism of the syncytia are accompanied by massive intra- and intercellular solute allocations. In this study the expression of all genes annotated as sugar transporters in the Arabidopsis Membrane Protein Library was investigated by Affymetrix gene chip analysis in young and fully developed syncytia compared with non-infected Arabidopsis thaliana roots. The expression of three highly up-regulated (STP12, MEX1, and GTP2) and three highly down-regulated genes (SFP1, STP7, and STP4) was analysed by quantitative RT-PCR (qRT-PCR). The most up-regulated gene (STP12) was chosen for further in-depth studies using in situ RT-PCR and a nematode development assay with a T-DNA insertion line revealing a significant reduction of male nematode development. The specific role of STP12 expression in syncytia of male juveniles compared with those of female juveniles was further shown by qRT-PCR. In order to provide evidence for sugar transporter activity across the plasma membrane of syncytia, fluorescence-labelled glucose was used and membrane potential recordings following the application of several sugars were performed. Analyses of soluble sugar pools revealed a highly specific composition in syncytia. The presented work demonstrates that sugar transporters are specifically expressed and active in syncytia, indicating a profound role in inter- and intracelluar transport processes.

Starch serves as carbohydrate storage in nematode-induced syncytia.

The plant parasitic nematode Heterodera schachtii induces specific syncytial feeding sites in the roots of Arabidopsis thaliana from where it withdraws all required nutrients. Therefore, syncytia have to be well supplied with assimilates and generate strong sinks in the host plant's transport system. Import mechanisms and consequent accumulation of sucrose in syncytia were described recently. In this work, we studied the starch metabolism of syncytia. Using high-performance liquid chromatography and microscopic analyses, we demonstrated that syncytia store carbohydrates by starch accumulation. Further, we monitored the expression of genes involved in the starch metabolic pathway by gene chip analysis and quantitative reverse transcription-PCR. Finally, we provide functional proof of the importance of starch synthesis for nematode development using T-DNA insertion lines. We conclude that syncytia accumulate starch as a carbohydrate buffer to compensate for changing solute uptake by the nematode and as long-term storage during juvenile development.

Value of counting white blood cells (WBC) in semen samples to predict the presence of bacteria.

OBJECTIVES: To investigate the correlation between the presence of white blood cells (WBC) without the use of specific stain to differentiate leukocytes and the presence of bacteria in semen samples of infertile men. METHODS: A total of 143 semen samples of men who attended an andrologic clinic for the evaluation of fertility were investigated using routine semen analysis (according to WHO laboratory guidelines) and bacterial culture. RESULTS: WBC were found in 43.4% (62/143). There were no WBC in 56.6% (81/143) of the samples (group I) while WBC were found in 43.4% (62/143) of the samples (group II). Pathogenic bacteria were detected in 48.2% (39/81) in group I and in 54.9% (34/62) in group II, all in all Bacteriospermia was present in 51.1% (73/143). The most common bacteria were Ureaplasma urealyticum, Enterococcus faecalis and Escherichia coli (23.8%, 16.8%, and 7.0% of samples, respectively). The sensitivity/specificity for detecting bacteria was 0.47/0.60 at a cut-off level of 0.25 Mio/mL WBC and 0.16/0.84 at a cut-off level of WBC 1 Mio/mL, representing likelihood ratios of 1.16 and 1.04, respectively. The greatest ratio between sensitivity and specificity (0.37/0.72) was found at a cut-off level of 0.5 Mio/mL WBC, with a likelihood ratio of 1.29. CONCLUSIONS: Counting WBC instead of a specific stain for the detection of leukocytes has only a poor sensitivity/specificity for the detection of bacteria.

DD3PCA3 RNA analysis in urine--a new perspective for detecting prostate cancer.

OBJECTIVES: Serum tPSA lacks specificity. The DD3(PCA3) gene is highly specific for prostate cancer and is detectable in prostate cancer cells shed into urine after rectal palpation. A newly developed nucleic acid sequence based amplification assay (uPM3) for detecting DD3(PCA3) RNA in urine samples was evaluated prospectively in patients referred for prostate cancer detection. METHODS: The uPM3 assay simultaneously detects the relative expression of DD3(PCA3) RNA and PSAmRNA as a marker for prostate cells in urine. Urine samples were collected after attentive digital rectal palpation prior to transrectal guided prostate biopsy. Samples were provided as a single void specimen (20-30 ml), stabilized in phosphate buffer and centrifuged. Lysis was performed on cell pellets DD3(PCA3) RNA and PSAmRNA were extracted and amplification was performed using isothermic nucleic acid based amplification (NASBA). The two targets were detected in real-time using specific beacons as probes in a thermostated spectrofluorimeter. Parameters of the amplification curve were defined after a logistic curve fitting routine and a classification tree model was constructed to predict the outcome of patients (i.e. cancer and non-cancer). RESULTS: 201 patients were included in this prospective study. 158/201 analyzed urine samples contained enough prostate cells sufficient for DD3(PCA3) analysis (79% adequacy rate). Prostate cancer was found in 62 (39%) of the evaluable patients. Overall sensitivity, specificity, positive predictive value and negative predictive value for the uPM3 assay at a cut-off 0.5 probability were 82%, 76%, 67% and 87% respectively as compared to 98%, 5%, 40% and 83% respectively for tPSA (at a cutoff of 2.5 ng/ml). In the tPSA categories <4, 4-10 and >10 ng/ml sensitivity was 73%, 84% and 84% and specificity was 61%, 80% and 70%, respectively. The AUC (area under the curve) was 0.87 (CI 0.81-0.92). CONCLUSION: The uPM3 assay showed excellent clinical performances and a specificity far superior to tPSA.