Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

<div><p>Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, <i>Anopheles stephensi</i>. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.</p></div

Blockade of <i>P</i>. <i>berghei</i> transmission in transgenic mosquitoes.

<p>Blockade of <i>P</i>. <i>berghei</i> transmission in transgenic mosquitoes.</p

Oocyst numbers of <i>P</i>. <i>berghei</i> in wild-type and AAPP-mBax mosquitoes.

<p>Midguts were dissected 10–12 days after blood feeding and oocyst numbers were counted by microscopy. Homozygous and heterozygous transgenic mosquitoes were tested. (A) Results of line 1. Six independent experiments (Exp 1–6) were shown. (B) Results of line 3. Four independent experiments (Exp 1–4) were shown. The status of transgenic mosquitoes (homozygous or heterozygous) is represented below the numbers of the experiment. These experiments were performed using separate generations of mosquitoes. (*****: <i>P</i> < 0.000001, ***: <i>P</i> < 0.0001, **: <i>P</i> < 0.001, *: <i>P</i> < 0.01, calculated by the Mann-Whitney <i>U</i> test). The line shows the median.</p

The gene structure of the <i>piggyBac</i> transformation vector, pBac[pAAPP-mBax; 3xP3-EGFP], TG mosquito lines, and insertion sites.

<p>(A) The gene construct derived from the <i>piggyBac</i>-based vector contains <i>piggyBac</i> Left-arm (L) and Right-arm (R) with an inverted terminal repeat (ITR). The <i>T7-mBax</i> gene is expressed under the control of the <i>An</i>. <i>stephensi aapp</i> promoter (pAAPP) and <i>An</i>. <i>gambiae trypsin</i> terminator (Tryter). The transformation marker, <i>EGFP</i> is expressed under the control of the <i>3xP3</i> promoter. A double line represents the probe region for a Southern blot analysis. The restriction enzyme (<i>Msp</i> I) site is represented below the scheme. The red arrow represents the primer sites for a RT-PCR analysis in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005872#ppat.1005872.g002" target="_blank">Fig 2</a>. (B) A Southern blot analysis of AAPP-mBax lines. Genomic DNA from AAPP-mBax mosquito lines (lines 1 and 3) was digested with <i>Msp</i> I, and hybridized with a fragment corresponding to the <i>piggyBac</i> R region. (C) Insertion sites of the transgene in AAPP-mBax lines 1 and 3. The blue bars show the local DNA region within each genomic scaffold. Black boxes represent the annotated protein-coding region in the VectorBase (<a href="https://www.vectorbase.org/" target="_blank">https://www.vectorbase.org/</a>). Double-headed arrows show the <i>piggyBac</i> construct. L: <i>piggyBac</i> Left-arm, R: <i>piggyBac</i> Right-arm.</p

Analysis of transgene expression in salivary glands of female AAPP-mBax lines.

<p>(A) Expression of the <i>T7-mBax</i> mRNA in the AAPP-mBax mosquito. The salivary glands (SG), midgut (Mg), and carcass (Ca) of 1-day-old adult females were used in analyses. The whole wild-type (WT) female sample (WB) used for analyses was extracted from 1-3-day-old adults. The <i>ribosomal protein S7</i> (<i>rpS7</i>) gene was used as a control for ubiquitous expression. (B) The expression profile of <i>T7-mBax</i> mRNA in AAPP-mBax line 1 female mosquitoes. One-day-old female pupae and female adults (within 4 h, 1 day, 2 days, 7 days, 9 days, 10 days after eclosion) were used in analyses. Nine-day- and 10-day-old adult females were fed blood 7 days after eclosion (BF). Whole female bodies were used in analyses. The <i>ribosomal protein S7</i> (<i>rpS7</i>) gene was used as a control for ubiquitous expression. (C) Detection of the T7-mBax protein in the salivary gland of AAPP-mBax mosquitoes (line 1) by immunoblotting with anti-T7 antibodies. An anti-alpha-tubulin antibody was used as the loading control. The age of mosquitoes (days post-eclosion) is indicated above. (D) Reductions in the amount of proteins in the salivary glands of female AAPP-mBax mosquitoes (line 1). Silver staining of salivary gland proteins separated by SDS-PAGE. Samples of salivary glands from wild-type (WT) and AAPP-mBax mosquitoes were loaded. The age of mosquitoes (days) is indicated above. (E) The gel loading of the same samples in (D) was analyzed by immunoblotting with the serum of a mouse repeatedly bitten by <i>An</i>. <i>stephensi</i> (anti-saliva).</p

Analysis of the blood feeding behavior of AAPP-mBax line (line 1) mosquitoes.

<p>(A) Measurements of probing times by wild-type and AAPP-mBax mosquitoes. Each dot corresponds to one female mosquito. The probing time is defined as the time taken from the initial insertion of the mouthpart into the skin until the initial observation of the ingestion of blood in the abdomen. Probing times were significantly longer in AAPP-mBax mosquitoes than in wild-type mosquitoes. Data from two independent experiments using separate generations of mosquitoes were pooled. The number and ratio of blood-fed mosquitoes within 420 seconds are indicated below (n = 50, ***: <i>P</i> < 0.0001, calculated by the Mann-Whitney <i>U</i> test). (B) Comparison of the amount of ingested blood between wild-type and AAPP-mBax mosquitoes. The amount of ingested blood was evaluated using hemoglobin contents in the abdomens of mosquitoes allowed access to the same mice for 30 min. The hemoglobin content was represented as OD₅₇₅ units. The abdomens of unfed wild-type mosquitoes were used as the negative control. No significant difference was observed between AAPP-mBax and wild-type mosquitoes. n.s., not significant (<i>P</i> = 0.3007, calculated by the Mann-Whitney <i>U</i> test).</p

Comparison of the <i>P</i>. <i>berghei</i> load in the midgut of mosquitoes.

<p>The expression of the <i>P</i>. <i>berghei Pb47</i> gene in wild-type (WT) and AAPP-mBax (line 1) mosquitoes after blood feeding was analyzed by quantitative RT-PCR. Relative expression levels are shown, with the average value of wild-type mosquitoes being 1. The expression levels of <i>Pb47</i> were normalized using that of the <i>An</i>. <i>stephensi GAPDH</i> gene. Two independent experiments were performed. No significant differences were observed between AAPP-mBax and wild-type mosquitoes. n.s., not significant (<i>P</i> = 0.9377 in Exp 1 and <i>P</i> = 0.9672 in Exp 2).</p

Exflagellation-inducing activity in salivary glands of AAPP-mBax and wild-type mosquitoes.

<p>The homogenate of the salivary glands (equivalent to 10 pairs of salivary glands) with PBS (pH 7.2) was mixed with <i>P</i>. <i>berghei</i>-infected blood. The number of exflagellation bodies per 4 fields (approximately 6,000 RBC) was counted. An experiment using PBS (pH 7.2) was the negative control (n = 4 experiments, ***: <i>P</i> < 0.0001, calculated by the Student’s <i>t</i>-test).</p

Inhibition of infection by <i>P</i>. <i>berghei</i> sporozoites in salivary glands of transgenic mosquitoes.

<p>Inhibition of infection by <i>P</i>. <i>berghei</i> sporozoites in salivary glands of transgenic mosquitoes.</p