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Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility.

Pinto SB, Stainton K, Harris S, Kambris Z, Sutton ER, Bonsall MB, Parkhill J, Sinkins SP - PLoS Pathog. (2013)

Bottom Line: Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line.Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses.The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible.

View Article: PubMed Central - PubMed

Affiliation: Peter Medawar Building for Pathogen Research and Nuffield Department of Medicine (NDM), University of Oxford, Oxford, United Kingdom ; Department of Zoology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.

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Transcription analysis of CPIJ005623 in the Culex pipiens complex.A–B. CPIJ005623 transcription in adult female (A) and male (B) Culex quinquefasciatus, Pel (Wolbachia-infected) line relative to the Wolbachia-uninfected PelU, over time (days post pupal eclosion). Similar decreasing expression dynamics was seen in both sexes. C–D. Tissue analysis of CPIJ005623 transcription at 4 d in ovaries (C) and 1 d in testes (D) in C. quinquefasciatus Wolbachia-infected lines Pel and Cxq (30), their Wolbachia-free counterparts PelU and CxqT, and Wolbachia-infected Italy line C. molestus. Upregulation of CPIJ005623 expression is seen in all Wolbachia-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 Wolbachia infection status (wis) and time on CPIJ005623 expression. Wilcoxon rank-sum test was used to determine differences between Wolbachia infection status in CPIJ005623 expression (C–D): *p<0.05, **p<0.01, ***p<0.001.
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ppat-1003647-g001: Transcription analysis of CPIJ005623 in the Culex pipiens complex.A–B. CPIJ005623 transcription in adult female (A) and male (B) Culex quinquefasciatus, Pel (Wolbachia-infected) line relative to the Wolbachia-uninfected PelU, over time (days post pupal eclosion). Similar decreasing expression dynamics was seen in both sexes. C–D. Tissue analysis of CPIJ005623 transcription at 4 d in ovaries (C) and 1 d in testes (D) in C. quinquefasciatus Wolbachia-infected lines Pel and Cxq (30), their Wolbachia-free counterparts PelU and CxqT, and Wolbachia-infected Italy line C. molestus. Upregulation of CPIJ005623 expression is seen in all Wolbachia-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 Wolbachia infection status (wis) and time on CPIJ005623 expression. Wilcoxon rank-sum test was used to determine differences between Wolbachia infection status in CPIJ005623 expression (C–D): *p<0.05, **p<0.01, ***p<0.001.

Mentions: Relative transcription of CPIJ005623 was measured in members of the C. pipiens complex using quantitative reverse transcription PCR (qRT-PCR). We first looked at the transcription of the gene in whole adults over time to understand its expression dynamics in individual mosquitoes that carry Wolbachia and their antibiotic-treated Wolbachia-free genetic counterparts. In Wolbachia-infected C. quinquefasciatus Pel adult females, CPIJ005623 transcription showed a peak of over two-fold upregulation compared to the Wolbachia-cured line PelU at 4 days post pupal eclosion (dpe), which then decreased to similar levels as the PelU line by 8 dpe (Figure 1A). Differences in CPIJ005623 transcript levels were found, with an interaction between Wolbachia infection status (wis) and developmental time (Two-way Anova: wis:time- F = 7.66, Df = 1, p<0.01), where the difference between wis contributes most strongly to the interaction (Two-way Anova: wis- F = 13.53, Df = 1, p<0.001). In males (Figure 1B), there was an interaction between Wolbachia infection status (wis) and developmental time (Two-way Anova: wis:time- F = 5.79, Df = 1, p = 0.025) which was independent from any difference seen at 6 dpe between the Wolbachia-infected and uninfected males (Welch's t-test: t = 0.78, Df = 4.833, p = 0.4716).


Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility.

Pinto SB, Stainton K, Harris S, Kambris Z, Sutton ER, Bonsall MB, Parkhill J, Sinkins SP - PLoS Pathog. (2013)

Transcription analysis of CPIJ005623 in the Culex pipiens complex.A–B. CPIJ005623 transcription in adult female (A) and male (B) Culex quinquefasciatus, Pel (Wolbachia-infected) line relative to the Wolbachia-uninfected PelU, over time (days post pupal eclosion). Similar decreasing expression dynamics was seen in both sexes. C–D. Tissue analysis of CPIJ005623 transcription at 4 d in ovaries (C) and 1 d in testes (D) in C. quinquefasciatus Wolbachia-infected lines Pel and Cxq (30), their Wolbachia-free counterparts PelU and CxqT, and Wolbachia-infected Italy line C. molestus. Upregulation of CPIJ005623 expression is seen in all Wolbachia-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 Wolbachia infection status (wis) and time on CPIJ005623 expression. Wilcoxon rank-sum test was used to determine differences between Wolbachia infection status in CPIJ005623 expression (C–D): *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814344&req=5

ppat-1003647-g001: Transcription analysis of CPIJ005623 in the Culex pipiens complex.A–B. CPIJ005623 transcription in adult female (A) and male (B) Culex quinquefasciatus, Pel (Wolbachia-infected) line relative to the Wolbachia-uninfected PelU, over time (days post pupal eclosion). Similar decreasing expression dynamics was seen in both sexes. C–D. Tissue analysis of CPIJ005623 transcription at 4 d in ovaries (C) and 1 d in testes (D) in C. quinquefasciatus Wolbachia-infected lines Pel and Cxq (30), their Wolbachia-free counterparts PelU and CxqT, and Wolbachia-infected Italy line C. molestus. Upregulation of CPIJ005623 expression is seen in all Wolbachia-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 Wolbachia infection status (wis) and time on CPIJ005623 expression. Wilcoxon rank-sum test was used to determine differences between Wolbachia infection status in CPIJ005623 expression (C–D): *p<0.05, **p<0.01, ***p<0.001.
Mentions: Relative transcription of CPIJ005623 was measured in members of the C. pipiens complex using quantitative reverse transcription PCR (qRT-PCR). We first looked at the transcription of the gene in whole adults over time to understand its expression dynamics in individual mosquitoes that carry Wolbachia and their antibiotic-treated Wolbachia-free genetic counterparts. In Wolbachia-infected C. quinquefasciatus Pel adult females, CPIJ005623 transcription showed a peak of over two-fold upregulation compared to the Wolbachia-cured line PelU at 4 days post pupal eclosion (dpe), which then decreased to similar levels as the PelU line by 8 dpe (Figure 1A). Differences in CPIJ005623 transcript levels were found, with an interaction between Wolbachia infection status (wis) and developmental time (Two-way Anova: wis:time- F = 7.66, Df = 1, p<0.01), where the difference between wis contributes most strongly to the interaction (Two-way Anova: wis- F = 13.53, Df = 1, p<0.001). In males (Figure 1B), there was an interaction between Wolbachia infection status (wis) and developmental time (Two-way Anova: wis:time- F = 5.79, Df = 1, p = 0.025) which was independent from any difference seen at 6 dpe between the Wolbachia-infected and uninfected males (Welch's t-test: t = 0.78, Df = 4.833, p = 0.4716).

Bottom Line: Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line.Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses.The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible.

View Article: PubMed Central - PubMed

Affiliation: Peter Medawar Building for Pathogen Research and Nuffield Department of Medicine (NDM), University of Oxford, Oxford, United Kingdom ; Department of Zoology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.

Show MeSH
Related in: MedlinePlus