TEP1 occurrence in the testes during spermatogonial development.

<p>The <i>DSX</i> transgenic line [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002255#pbio.1002255.ref012" target="_blank">12</a>] that expresses <i>GFP</i> (green) under the <i>β-tubulin</i> promoter in the meiotic stages starting from spermatocytes but not in the mitotic germline stem cells (GSC) and spermatogonia. Nuclei were colored with DAPI (blue). (A) Male reproductive organs. (B) Organization of spermatogonial compartments in the testis (dotted lines). (C–E) TEP1 (red) recruitment to the spermatogonia (C–C”‘) and to the spermatozoa’s head (D–D”‘) and tail (E–E”). (F) Occurrence of testes with TEP1-positive cells during the first week after adult emergence. Testes were dissected for immunofluorescence analyses at the indicated time points. (G) Effect of mating on the percentage of testes with TEP1-positive cells. Virgin males (7-d-old) were collected in copula, and 2 d later their testes were dissected for immunofluorescence analyses. Significant differences (<i>p</i> < 0.05, χ² test) are shown by an asterisk. Vertical bars show standard deviation, <i>n</i> ≥ 30 testes. Data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002255#pbio.1002255.s001" target="_blank">S1 Data</a>.</p

Effect of radiation on TEP1-mediated removal of damaged sperm.

<p>A <i>DSX</i> transgenic line expressing <i>GFP</i> (green) under the <i>β-tubulin</i> promoter was used. Nuclei were colored with DAPI (blue). (A,B) 3-d-old virgin males that emerged from irradiated pupae were mated with 3-d-old virgin females, and the number of (A) laid eggs and the (B) larval hatching rates per female were gauged. Means ± standard error of the mean (SEM) are plotted for <i>n</i> ≥ 25. (C) The proportion of testes with TEP1-positive spermatogonia in irradiated males (40 Gy), <i>n</i> ≥ 30. (D,E) Radiation (40 Gy) reduces the size of the spermatogonial compartment (white dotted line) in 1- and 3-d-old males. (F,G) Colocalization of TEP1 (red) and TUNEL (white) signals in (F–F”) spermatogonia and (G–G”) the GSC in irradiated 1-d-old males. (H) Occurrence of TEP1 in spermatogonia of irradiated (40 Gy) males (<i>DSX)</i> injected with <i>dsTEP1</i>, <i>dsLRIM1</i>, <i>dsHPX2</i>, and <i>dsLacZ</i> (control). Males depleted for <i>TEP1</i> (<i>7b</i> line) served as positive controls. The proportion of testes with TEP1 signal was gauged 2 d later. Mean ± standard error (SE) is shown; N, number of testes. (i) Accumulation of TUNEL-positive spermatogonia after irradiation in the testes of control and TEP1-depleted males (progeny of reciprocal crosses between <i>7b</i> and <i>DSX</i>) was examined 1 and 3 d after emergence. Each dot represents one testis. Significant differences (<i>p</i> < 0.05, χ² test) are shown by an asterisk and by characters above the corresponding values. Data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002255#pbio.1002255.s001" target="_blank">S1 Data</a>.</p

Effect of radiation and TEP1 depletion on male fertility.

<p>Pupae were irradiated (40 Gy), and the resulting 3-d-old males were mated with 3-d-old females. The mean ± SEM of laid eggs and the proportions of hatched larvae are plotted. N, number of oviposited females. (A) After irradiation, TEP1 depletion (<i>7b</i> line) decreases hatching rates as compared to controls (<i>T4</i> line). (B) The proportion of testes with apoptotic cells was examined by TUNEL staining in irradiated <i>TEP1-</i>homozygous (<i>S1</i>/<i>S1</i>, <i>S2</i>/<i>S2</i>, or <i>R1</i>/<i>R1</i>) 1-d-old males. Each dot represents one testis. (C) Irradiated <i>TEP1</i>-homozygous (<i>S1</i>/<i>S1</i>, <i>S2</i>/<i>S2</i>, or <i>R1</i>/<i>R1</i>) 3-d-old males were mated with <i>TEP1*S1/S1</i> females. (D) <i>TEP1</i> expression was silenced in the males of F₁ reciprocal crosses between <i>7b</i> and each of the <i>TEP1</i>-homozygogus lines. Irradiated F₁ 3-d-old males were mated with <i>TEP1*S1/S1</i>-homozygogus females. The results of two-way analysis of variance (ANOVA) tests are shown in tables below the corresponding graphs. Post hoc Tukey’s test: * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001. Data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002255#pbio.1002255.s001" target="_blank">S1 Data</a>.</p

Allele-specific function of TEP1 in male fertility.

<p>Males (3- and 9-d-old) were mated with 3-d-old females. The mean ± SEM of laid eggs and the proportions of hatched larvae are plotted. N, number of oviposited females. (A) Fertility rates of TEP1-depleted (<i>7b</i> line) and TEP1-control (<i>T4</i> line) males. (B) The fertility of <i>TEP1</i>-homozygous (<i>S1</i>/<i>S1</i>, <i>S2</i>/<i>S2</i>, or <i>R1</i>/<i>R1</i>) males mated with <i>TEP1*S1/S1</i> females. (C) 1-d-old <i>TEP1</i>*<i>S2/R1</i> males were injected with <i>dsTEP1*S2</i>- (<i>dsS</i>) or with <i>dsTEP1*R1</i>-(<i>dsR</i>), and 12 d later, relative expression levels of <i>TEP1</i> were gauged by allele-specific quantitative reverse transcription PCR (qRT-PCR). Injection of <i>dsLacZ</i> and <i>dsTEP1</i> served as a negative and a positive control, respectively. Expression of a gene encoding ribosomal protein L19 (<i>RpL19</i>) was used for normalization. Results of three independent experiments are plotted. (D) 1-d-old <i>TEP1*S2</i>/<i>R1</i> males were injected with <i>dsTEP1*S2</i>- (<i>dsS</i>) or with <i>dsTEP1*R1</i>-(<i>dsR</i>) and mated 8 d later with <i>TEP1*S1</i>/<i>S1</i> females. Injection of <i>dsLacZ</i> and <i>dsTEP1</i> served as a negative and a positive control, respectively. Results of two-way ANOVA tests are shown in tables below the corresponding graphs. Statistically significant differences in (A,B): * <i>p</i> < 0.05; *** <i>p</i> < 0.001, post hoc Tukey’s test, and in (D): <i>p</i> < 0.05 (Fisher’s LSD test), are indicated by characters above the corresponding values. Data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002255#pbio.1002255.s001" target="_blank">S1 Data</a>.</p

Distribution of cases identified within and outside case-clusters from 1990 to 2014.

<p>A case-cluster is defined as two or more infected persons epidemiologically linked by place [within 150m (200m till 2002)] with respect to their home address and time (within 14 days). Numbers above bars represent percentage of cases outside clusters [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005667#pntd.0005667.ref030" target="_blank">30</a>].</p

Infection rate and viral genome copies per infected <i>Ae</i>. <i>malayensis</i> 14 days after injection of saliva collected from DENV- or CHIKV-infected <i>Ae</i>. <i>malayensis</i>.

<p>nd; not detected.</p

Infection and dissemination abilities for DENV and CHIKV in <i>Ae</i>. <i>aegypti</i>, <i>Ae</i>. <i>albopictus</i> and <i>Ae</i>. <i>malayensis</i>.

<p>Mosquitoes were orally infected with the virus and analyzed after 14 days. (A) Infection and dissemination rates for DENV. (B) DENV genome copies per infected tissue in abdomen and thorax. (C) Infection and dissemination rates for CHIKV. (D) CHIKV genome copies per infected tissue in abdomen and thorax. (A and C) Each point represents an independent repeat of 25 mosquitoes. Bars show mean ± s.e.m. T-test significant differences between mosquito species are shown. (B and D). Table below indicates the results from a two-ANOVA testing the impact of species and tissues on viral genome copies per infected tissues.</p

Virus titer in salivary glands is increased for mosquitoes infected with the chimeric virus containing the high epidemiological fitness 3’UTR sequence.

<p>Mosquitoes were orally infected with 10⁶ pfu / ml of the chimeric viruses, IC6452 or IC315022, containing the high or low EF 3’UTR sequence, respectively. At 14 days p.i., salivary glands were dissected and virus titer was quantified using plaque assay. (A) Infection rate in salivary glands. Bars represent percentages ± s.e. (B) plaque forming unit (pfu) per infected salivary glands. Only infected samples were represented, used to calculate the average and to perform t-test. Each point represents a pair of salivary glands. Bars show mean ± s.e.m. N, number of samples analyzed.</p

Ratios of sfRNA:gRNA in human cells and mosquitoes correlate well.

<p>HuH-7 cells were infected with the isolates. (A) gRNA copies relative to GAPDH expression and (B) sfRNA:gRNA ratio were quantified using RT-qPCR. Bars with a different letter were significantly different following Tukey’s test. (C) Correlation between sfRNA:gRNA ratios in HuH-7 cells and whole mosquitoes was plotted. The dotted line represents an exponential regression, for which the R² and equation is provided within the graph. The data in HuH-7 cells have been previously used for comparing averages of PR-1 and PR-2B strains [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006535#ppat.1006535.ref030" target="_blank">30</a>] but never shown for each individual strains.</p

Infection rate of saliva is increased for mosquitoes infected with the viruses containing the high epidemiological fitness 3’UTR sequence.

<p>Mosquitoes were orally infected with 10⁶ pfu / ml of viruses, and let to expectorate in blood. DENV gRNA was quantified in mosquitoes and saliva. (A) DENV gRNA copies per infected mosquitoes, (B) DENV gRNA copies per infected saliva and (C) saliva infection rate 10 days p.i. with the high epidemiological fitness (EF) PR6452 or low EF PR315022. (D) DENV gRNA copies per infected mosquitoes, (E) DENV gRNA copies per infected saliva and (F) saliva infection rate 14 days p.i. with IC6452, containing the high EF 3’UTR, or IC315022, containing the low EF 3’UTR. N, number of infected mosquitoes that contained blood in their abdomen. (A, B, D, E) Bars show geometric mean ± 95% C.I. Only infected samples are represented. (C, F) Bars represent percentages ± s.e. Statistical differences between percentages were calculated using Z-test.</p