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

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

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

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

<div><p><i>Anopheles gambiae</i>, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene <i>TEP1</i> using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several <i>TEP1</i> mutant <i>A. gambiae</i> lines. These mutations inhibited protein production and rendered <i>TEP1</i> mutants hypersusceptible to <i>Plasmodium berghei</i>. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.</p> </div

TALEN mutagenesis of the <i>TEP1</i> gene.

<p>A: Fragment from the <i>TEP1</i> gene showing the target site of the TALEN pair. Nucleotides bound by each TALEN are underlined, TALEN repeats are color-coded to show repeat/nucleotide specificity. The <i>Nco</i>I restriction site centrally located at the TALEN cleavage site is highlighted. Inset: scheme of the entire TEP1 protein showing the location of TALEN-induced mutations (SP: signal peptide; CUB: CUB domain interrupted by the TED: thioester domain; the star indicates the position of the thioester site). B: PCR assay to identify <i>TEP1</i> mutant mosquitoes. A PCR product spanning the TALEN target site is generated from individual mosquitoes (small larva or a leg from a living adult) and incubated with <i>Nco</i>I. Full cleavage of the PCR product (w) denotes a wild-type individual. Partial cleavage (h) denotes a heterozygous <i>TEP1</i> mutant. Absence of cleavage (H) corresponds to a homozygous <i>TEP1</i> mutant. C: TALEN-induced mutations in the TEP1 gene. Left and right TALEN target nucleotide sequences are shown in green and blue respectively, with the 15bp spacer sequence between the TALENs in black. The <i>Nco</i>I restriction site is highlighted in orange. Deletions are designated by a red dash or by Δ+number of missing bases. Insertions are shown in lowercase red letters. Uppercase red letters correspond to natural polymorphisms between multiple <i>TEP1</i> alleles.</p

<i>TEP1</i> mutant mosquitoes are hypersusceptible to <i>P. berghei</i>.

<p>Mosquito females from five different homozygous mutant mosquito lines and from control parental lines were offered a blood meal on a <i>P. berghei</i>-infected mouse. Seven days after infection, the midgut was dissected and the number of oocysts developing in each midgut was evaluated. The statistical significance of differences in mean parasite numbers was measured with a Mann-Whitney test (mutant versus control) and with a Kruskall-Wallis test followed by Dunn’s post-test (to compare all groups in [d]). (a) M^∆T mosquitoes are compared to the two parental, non-mutant TALEN lines. (b) 4 different mutant lines are compared to the parental mosquito line that initially served to produce TALEN transgenic lines. This control line was verified to show the same level of susceptibility to <i>P. berghei</i> as the two TALEN daughter lines (not shown). In this experiment, three mutant lines showed significantly elevated parasite numbers compared to the control while the M^∆ct2 line did not, presumably due to a different physiological condition of this mosquito culture. In two independent experiments (c, d), we used mosquitoes of different genotypes marked with distinct fluorescence markers and cultured the larvae together in the same water to eliminate potential confounding factors due to rearing conditions. On the day of dissection, genotypes were separated on the basis of fluorescence. The same M^∆ct2 line shows significantly elevated parasite numbers. (d) Heterozygous M^∆ct2 mosquitoes are compared to control and homozygous M^∆ct2 mosquitoes: the susceptibility phenotype of the heterozygote is intermediate. (e) Control mosquitoes of the parental line, mosquitoes of the parental line injected with <i>TEP1</i> double-stranded RNA, and homozygous <i>TEP1</i> mutant mosquitoes of the M^∆T line are compared. TEP1 mutant and dsRNA-injected mosquitoes show comparable susceptibility to <i>P. berghei</i>.</p

TEP1 mutant proteins.

<p>A: Fragment of the TEP1 protein encompassing the TALEN-induced mutations is shown for the wild-type (WT) and for those mutants that we maintain as homozygous mosquito lines (M^∆T, M^∆MV, M^∆3+6, M^∆ct1 and M^∆ct2). Gaps in the protein sequence denote amino acid deletions. Inserted exogenous amino acids are shown in red. Nonsense amino acids followed by a stop codon (*), resulting from frame-shift mutations, are shown in blue. B: Immunoblots to evaluate the presence of mutant TEP1 protein in whole mosquito extract (top panels) or hemolymph. Hemolymph prophenoloxidase (PPO) serves as a loading control. In the control samples, both TEP1 full-length (full) and C-terminal fragment (cleaved) are visible. Cross-reacting background bands, some running in close proximity to TEP1 fragments, are marked on the left with ‘>’ signs.</p

Correlation between sporozoite numbers and luciferase activity in whole mosquitoes, and dissected midgut and salivary gland sporozoites of <i>uis3::GFP-LUC</i> and <i>glyc::GFP-LUC</i>.

<p>Mosquitoes were infected with either <i>uis3::GFP-LUC</i> (A and C) or <i>glyc::GFP-LUC</i> (B and D) and sporozoites extracted from salivary glands (sgs), midguts (mgs) or whole mosquitoes were collected 18–19 dpi. The sporozoites and whole mosquitoes were lysed and dilution series of cell extracts were generated and used to perform luciferase assays. Luciferase activity was measured and plotted as the value after subtraction of the baseline against the sporozoite number (RLU). Goodness of the linear curve fit is given as r². Graphs representing two independent experiments are shown. 10⁴ sgs correspond to 1.1 mosquito equivalent in (C) and 1.2 in (D). Outliers have been omitted from the curve fit (i.e. highest sgs concentrations for isolated sgs for (C) and whole mosquitoes (D)).</p

GFP detection in blood and mosquito stages by fluorescence analysis.

<p>(A) For blood stage images, blood of infected mice was diluted and stained with the blue nuclear dye Hoechst 33258. Ookinetes were cultured, pelleted and stained with Hoechst 33258 before imaging. To image oocysts and salivary gland sporozoites, mosquitoes were infected with the transgenic parasites. Midguts containing oocysts were dissected directly into PBS/Hoechst 33258 11–21 dpi; infected salivary glands were dissected into PBS/Hoechst 33258 16–21 dpi. Oocysts and sporozoites were stained with the nuclear dye for 15–30 min. The GFP was visualized using the GFP fluorescence channel, the nuclei using the UV channel. The scale bars represent 5 µm for all the blood stages, ookinetes and sporozoites and 10 µm for oocysts.</p