Additional file 1: Table S1. of Isolation of fungi from dead arthropods and identification of a new mosquito natural pathogen

Number of occurrences of each isolated fungus in the collected cadavers. (DOCX 22 kb

Transcription analysis of <i>CPIJ005623</i> in the <i>Culex pipiens</i> complex.

<p><b>A–B</b>. <i>CPIJ005623</i> transcription in adult female (A) and male (B) <i>Culex quinquefasciatus</i>, Pel (<i>Wolbachia</i>-infected) line relative to the Wolbachia-uninfected PelU, over time (days post pupal eclosion). Similar decreasing expression dynamics was seen in both sexes. <b>C–D</b>. Tissue analysis of <i>CPIJ005623</i> transcription at 4 d in ovaries (C) and 1 d in testes (D) in <i>C. quinquefasciatus Wolbachia</i>-infected lines Pel and Cxq (<i>30</i>), their <i>Wolbachia</i>-free counterparts PelU and CxqT, and <i>Wolbachia</i>-infected Italy line <i>C. molestus</i>. Upregulation of <i>CPIJ005623</i> expression is seen in all <i>Wolbachia</i>-infected lines. Average of the mean values of four biological repeats (+/− standard error-SE) are presented. Two-way ANOVA statistical analysis was used to determine effect of <i>Wolbachia</i> infection status (wis) and time on <i>CPIJ005623</i> expression. Wilcoxon rank-sum test was used to determine differences between <i>Wolbachia</i> infection status in <i>CPIJ005623</i> expression (C–D): *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Knockdown analysis of <i>CPIJ005623</i> in <i>C. quinquefasciatus</i> Pel females.

<p><b>A</b>. Diagram representing experimental design of knockdown (KD) experiments, d-day post pupal eclosion, BF-Blood feed, T-time point. <b>B</b>. KD assessment of i<i>CPIJ005623</i> in Pel ovaries. Reduction of <i>CPIJ005623</i> mRNA levels similar to uninfected ovaries was observed in infected ovaries when CI developmental progression was seen. <b>C</b>. Increased early embryo (stageI) developmental arrest is detected after i<i>CPIJ005623</i> in compatible Pel male cross.</p

Knockdown analysis of <i>CPIJ005623</i> in <i>C. molestus</i> Italy females.

<p><b>A</b>. Diagram representing experimental design of knockdown (KD) experiments, d-day post pupal eclosion, T-time point. <b>B</b>. KD assessment of i<i>CPIJ005623</i> in Italy ovaries. Reduction of <i>CPIJ005623</i> mRNA levels similar to uninfected ovaries was observed in infected ovaries when CI developmental progression was seen. <b>C</b>. Picture examples of developmental progression in <i>Culex</i> embryos undergoing CI, Stage I: early arrest, Stage II and III: late arrest. <b>D</b>. Increased percentage of unhatched embryos mimic a reduced rescue function. Also increased early embryo (stageI) developmental arrest is detected after i<i>CPIJ005623</i> in compatible Italy male cross.</p

Knockdown analysis of <i>CPIJ005623</i> in <i>C. pipiens</i> males.

<p><b>A</b>. Diagram representing experimental design of knockdown (KD) experiments, d-day post pupal eclosion, BF- Blood feed, T-time point. <b>B</b>. KD assessment of i<i>CPIJ005623</i> in Pel testis. Reduction of <i>CPIJ005623</i> mRNA levels was observed in testis when CI developmental progression was seen. <b>C–F</b> Embryo developmental progression after i<i>CPIJ005623</i> males in an incompatible Italy female cross. Timepoints under dsRNA early effect (C-T1; D-T2) show an increased proportion of Stage II&III embryos compared to control KDs. No effect was seen at later time points (E-T3; F-T4). <b>G</b>. KD assessment of i<i>CPIJ005623</i> in Italy testes. <b>H</b>. Double KD analysis of <i>CPIJ005623</i> in Pel females crossed to Italy males. Increased developmental progression is detected in female, male and double <i>iCPIJ005623</i>.</p

<i>CPIJ005623</i> KD effects in <i>C. molestus</i> Italy and <i>C. quinquefasciatus</i> Pel females in compatible crosses.

<p><i>CPIJ005623</i> KD effects in <i>C. molestus</i> Italy and <i>C. quinquefasciatus</i> Pel females in compatible crosses.</p

Immune gene expression and challenges with <i>Brugia pahangi</i> in <i>Ae. aegypti</i> somatically infected with <i>w</i>MelPop, and effects of immune knockdown on <i>Wolbachia</i> density.

<p>A) The expression of four immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, <i>PGRPS1</i>; cecropin D, <i>CECD</i>; CLIP-domain serine protease, <i>CLIPB37</i>; and a C-type galactose-specific lectin. Adult females were injected with <i>w</i>MelPop or the buffer alone, approximately seven days post-eclosion. RNA was extracted from these adults eight days after injection. Expression was normalized to non-injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing eight adult females (total of 24 mosquitoes per condition). B) The mean numbers of L3 stage (infective) larvae per mosquito are shown following <i>B. pahangi</i> challenge in <i>Ae. aegypti</i> Ref^m strain previously injected with <i>w</i>MelPop or buffer; * <i>P</i><0.05. Numbers above bars show the prevalence of filarial infection as a proportion of mosquitoes that contained at least one L3 <i>Brugia</i> larva over the total number of mosquitoes dissected in each category. C) We measured the levels <i>Wolbachia ftsZ</i> gene expression as a proxy for <i>Wolbachia</i> density and normalized the qRT-PCR data to the mosquito <i>Actin5C</i> gene. Two sets of three females per time point injected with either dsLacZ or dsRel2 were assayed. <i>ftsZ</i> gene expression was found to be higher in dsRel2-injected mosquitoes than in dsLacZ-injected mosquitoes at both six and ten days post injection. The mean level of <i>Rel2</i> transcript in dsRel2-injected mosquitoes was confirmed to be approximately 40% of that in dsLacZ injected mosquitoes at both time points. These data suggest that the immune effectors controlled by the Imd pathway (<i>Rel2</i>-controlled) can influence <i>Wolbachia</i> densities.</p

Model of possible effects of <i>w</i>MelPop on malaria vectorial capacity.

<p>Vectorial capacity is a measure that describes the transmission potential of a mosquito population and is independent of <i>Plasmodium</i> prevalence. It can be thought of as proportional to the number of infectious bites that occur per day after a single infectious human arrives in a previously malaria-free area. If we assume recruitment to the adult mosquito stage is constant then vectorial capacity can be written (<i>A b</i> (1−<i>μ</i>)^τ)/<i>μ</i> where <i>b</i> is the ability of the mosquito to transmit <i>Plasmodium</i>, <i>μ</i> is adult daily survival, <i>τ</i> is the length of the intrinsic incubation period of the <i>Plasmodium</i> and all other parameters are combined in <i>A </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001143#ppat.1001143-Smith1" target="_blank">[42]</a>. The figure plots vectorial capacity as transmission (<i>b</i>) and daily survival (<i>μ</i>) are each reduced because of the presence of <i>Wolbachia</i> by a multiplicative factor (1−<i>x</i>) where <i>x</i> varies in the range 0 to 1 (parameters: <i>b</i> = 1; <i>μ</i> = 0.1; <i>τ</i> = 1; <i>A</i> = 1). A more advanced analysis tailored to a specific system might want to include age-specific adult mortality, the effect of <i>Wolbachia</i> on mosquito population dynamics and seasonality.</p

Oligonucleotide primers used in quantitative PCR experiments and dsRNA synthesis.

<p>Previously published oligonucleotides are indicated by the reference number following the gene name.</p

Immune gene expression in <i>An. gambiae</i> somatically infected with <i>w</i>MelPop.

<p>The expression of six immune genes were analyzed by qRT-PCR: leucine-rich repeat immune protein, <i>LRIM1</i>; thioester-containing protein, <i>TEP1</i>; cecropin, CEC1; defensin, <i>DEF1</i>; C-type lectin, <i>CTL4</i>; and clip-domain serine protease, <i>CLIPB3</i>. Adult <i>An. gambiae</i> females were injected with <i>E. coli</i>, <i>w</i>MelPop or the buffer alone, 2–3 days post-eclosion, and RNA was extracted from these adults eight days after injection. Expression was normalized to non-injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing eight adult females (total of 24 mosquitoes per condition).</p