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Anopheles gambiae miRNAs as actors of defence reaction against Plasmodium invasion.

Winter F, Edaye S, Hüttenhofer A, Brunel C - Nucleic Acids Res. (2007)

Bottom Line: Twelve of them are expressed ubiquitously across the body, independently of gender, while the other six exhibited an expression pattern restricted to the digestive system.Strikingly, the expression patterns of four miRNAs, including the three unique to mosquito, are affected by the presence of Plasmodium.Altogether, these data support an involvement of miRNAs as new layers in the regulation of Anopheles defence reaction.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université Louis Pasteur, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue Descarte, 67084 Strasbourg, France.

ABSTRACT
The path Plasmodium takes across the Anopheles midgut constitutes the major bottleneck during the malaria transmission cycle. In the present study, using a combination of shot-gun cloning and bioinformatic analysis, we have identified 18 miRNAs from Anopheles gambiae including three miRNAs unique to mosquito. Twelve of them are expressed ubiquitously across the body, independently of gender, while the other six exhibited an expression pattern restricted to the digestive system. Strikingly, the expression patterns of four miRNAs, including the three unique to mosquito, are affected by the presence of Plasmodium. We also show that knocking down Dicer1 and Ago1 mRNAs led to an increased sensitivity to Plasmodium infection. Altogether, these data support an involvement of miRNAs as new layers in the regulation of Anopheles defence reaction.

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Survival of P. berghei in drosha, Dicer1 and Argonaute1 depleted mosquitoes. Females were infected 4 days after injection, and midguts were dissected 10 days later and fixed. (A) Representative composite image between the green fluorescent channel and a differential interference contrast (DIC) image of dissected midguts showing parasite development in the dsRNA-injected mosquitoes. (B–D) Survival of P. berghei parasites in control ds-lacZ and in ds-drosha-, ds-Dicer1- or ds-Ago1-injected mosquitoes. Data were collected from three independent experiments. The mean of parasite number observed in each experiment was similar so that the data were pooled. Statistically significant differences between samples were evaluated by Mann–Whitney test and P-values are indicated on the graph. Black bars represent mean parasite numbers per group. Here, n represents the number of guts, m the average number of parasites per gut and me the median number of parasites per gut.
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Figure 4: Survival of P. berghei in drosha, Dicer1 and Argonaute1 depleted mosquitoes. Females were infected 4 days after injection, and midguts were dissected 10 days later and fixed. (A) Representative composite image between the green fluorescent channel and a differential interference contrast (DIC) image of dissected midguts showing parasite development in the dsRNA-injected mosquitoes. (B–D) Survival of P. berghei parasites in control ds-lacZ and in ds-drosha-, ds-Dicer1- or ds-Ago1-injected mosquitoes. Data were collected from three independent experiments. The mean of parasite number observed in each experiment was similar so that the data were pooled. Statistically significant differences between samples were evaluated by Mann–Whitney test and P-values are indicated on the graph. Black bars represent mean parasite numbers per group. Here, n represents the number of guts, m the average number of parasites per gut and me the median number of parasites per gut.

Mentions: No evidence has been published yet establishing that mosquito genes of the silencing pathway would affect parasite development in the midgut. We conducted a functional screen using the recently established gene silencing technique for adult A. gambiae (30). To achieve gene silencing, drosha, Argonaute1, Dicer1 or lacZ double-stranded RNAs were injected in the body cavity of newly emerged female mosquitoes of the G3 strain that are susceptible to the model rodent parasite P. berghei. Injection of lacZ dsRNA was used as a control to ensure that the observed effects did not simply reflect the dsRNA treatment. The reduction in drosha, Argonaute1 or Dicer1 transcript levels was determined 4 days later by real-time PCR. After injection of drosha dsRNA, the transcript level was unchanged. Transcript levels were reduced to 63% (±9%) after injection of Dicer1 dsRNA and to 50% (±12%) after injection of Argonaute1 dsRNA compared to transcript levels in the lacZ dsRNA control. At the same time point, 4 days after injection, the mosquitoes were allowed to feed on a mouse infected by a carrying-GFP parasite (26). We counted the number of live GFP-expressing oocysts on the dissected midguts 10 days post-infection. Correlating with the inefficiency of the silencing of drosha mRNA, no effect was observed on the parasite count after injection of drosha dsRNA. Depletion of Dicer1 and Argonaute1 led to a substantial increase in oocysts numbers: a significant 2-fold increase in the parasite number was observed (Figure 4).Figure 4.


Anopheles gambiae miRNAs as actors of defence reaction against Plasmodium invasion.

Winter F, Edaye S, Hüttenhofer A, Brunel C - Nucleic Acids Res. (2007)

Survival of P. berghei in drosha, Dicer1 and Argonaute1 depleted mosquitoes. Females were infected 4 days after injection, and midguts were dissected 10 days later and fixed. (A) Representative composite image between the green fluorescent channel and a differential interference contrast (DIC) image of dissected midguts showing parasite development in the dsRNA-injected mosquitoes. (B–D) Survival of P. berghei parasites in control ds-lacZ and in ds-drosha-, ds-Dicer1- or ds-Ago1-injected mosquitoes. Data were collected from three independent experiments. The mean of parasite number observed in each experiment was similar so that the data were pooled. Statistically significant differences between samples were evaluated by Mann–Whitney test and P-values are indicated on the graph. Black bars represent mean parasite numbers per group. Here, n represents the number of guts, m the average number of parasites per gut and me the median number of parasites per gut.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Survival of P. berghei in drosha, Dicer1 and Argonaute1 depleted mosquitoes. Females were infected 4 days after injection, and midguts were dissected 10 days later and fixed. (A) Representative composite image between the green fluorescent channel and a differential interference contrast (DIC) image of dissected midguts showing parasite development in the dsRNA-injected mosquitoes. (B–D) Survival of P. berghei parasites in control ds-lacZ and in ds-drosha-, ds-Dicer1- or ds-Ago1-injected mosquitoes. Data were collected from three independent experiments. The mean of parasite number observed in each experiment was similar so that the data were pooled. Statistically significant differences between samples were evaluated by Mann–Whitney test and P-values are indicated on the graph. Black bars represent mean parasite numbers per group. Here, n represents the number of guts, m the average number of parasites per gut and me the median number of parasites per gut.
Mentions: No evidence has been published yet establishing that mosquito genes of the silencing pathway would affect parasite development in the midgut. We conducted a functional screen using the recently established gene silencing technique for adult A. gambiae (30). To achieve gene silencing, drosha, Argonaute1, Dicer1 or lacZ double-stranded RNAs were injected in the body cavity of newly emerged female mosquitoes of the G3 strain that are susceptible to the model rodent parasite P. berghei. Injection of lacZ dsRNA was used as a control to ensure that the observed effects did not simply reflect the dsRNA treatment. The reduction in drosha, Argonaute1 or Dicer1 transcript levels was determined 4 days later by real-time PCR. After injection of drosha dsRNA, the transcript level was unchanged. Transcript levels were reduced to 63% (±9%) after injection of Dicer1 dsRNA and to 50% (±12%) after injection of Argonaute1 dsRNA compared to transcript levels in the lacZ dsRNA control. At the same time point, 4 days after injection, the mosquitoes were allowed to feed on a mouse infected by a carrying-GFP parasite (26). We counted the number of live GFP-expressing oocysts on the dissected midguts 10 days post-infection. Correlating with the inefficiency of the silencing of drosha mRNA, no effect was observed on the parasite count after injection of drosha dsRNA. Depletion of Dicer1 and Argonaute1 led to a substantial increase in oocysts numbers: a significant 2-fold increase in the parasite number was observed (Figure 4).Figure 4.

Bottom Line: Twelve of them are expressed ubiquitously across the body, independently of gender, while the other six exhibited an expression pattern restricted to the digestive system.Strikingly, the expression patterns of four miRNAs, including the three unique to mosquito, are affected by the presence of Plasmodium.Altogether, these data support an involvement of miRNAs as new layers in the regulation of Anopheles defence reaction.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, Université Louis Pasteur, CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue Descarte, 67084 Strasbourg, France.

ABSTRACT
The path Plasmodium takes across the Anopheles midgut constitutes the major bottleneck during the malaria transmission cycle. In the present study, using a combination of shot-gun cloning and bioinformatic analysis, we have identified 18 miRNAs from Anopheles gambiae including three miRNAs unique to mosquito. Twelve of them are expressed ubiquitously across the body, independently of gender, while the other six exhibited an expression pattern restricted to the digestive system. Strikingly, the expression patterns of four miRNAs, including the three unique to mosquito, are affected by the presence of Plasmodium. We also show that knocking down Dicer1 and Ago1 mRNAs led to an increased sensitivity to Plasmodium infection. Altogether, these data support an involvement of miRNAs as new layers in the regulation of Anopheles defence reaction.

Show MeSH
Related in: MedlinePlus