Site-Specific Integration and Expression of an Anti-Malarial Gene in Transgenic Anopheles gambiae Significantly Reduces Plasmodium Infections

Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the Streptomyces phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against Plasmodium yoelii nigeriensis, reducing average parasite intensity by 85%. Similar protection was observed against Plasmodium falciparum in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade

Evaluation et controle au laboratoire du pouvoir entomopathogene de Bacillus thuringiensis israelensis et de Bacillus sphaericus sur larves de Culicidae (Dipt. Nematoceres)

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Effect of antimicrobial peptides against <i>P. berghei</i> infections in <i>Anopheles stephensi</i>.

<p>Mosquitoes were provided with a gametocytaemic blood meal mixed with 50 µM of peptide, performed in triplicate. Prevalence (the proportion of infected mosquitoes with total numbers in parentheses) and intensity (mean number of oocysts with the range in parentheses) of infections with paired controls are shown. N/S indicates non-significance. Significant differences are indicated by probability values with (▴) representing oocyst numbers significantly higher than control and (▾) representing oocyst numbers lower than control.</p

Effect of Melittin on <i>Plasmodium</i> development in mosquitoes.

<p>Mosquitoes were fed blood containing gametocytes of rodent malaria (fed to <i>An. stephensi</i>) or human malaria (fed to <i>An. gambiae</i>) supplemented with the AMP Melittin. Fully engorged females were maintained in standardized conditions for 7–8 days prior to dissection for oocyst burdens. Each experiment was performed in triplicate with control feeds containing no AMP. Individual value plots for each dissected midgut are shown. Black diamonds represent the median oocyst burden for each group. Approximately 40–50 individuals were dissected for <i>P. berghei</i> infections and 30 individuals for <i>P. falciparum</i> infections (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003790#ppat-1003790-t005" target="_blank">Tables 5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003790#ppat-1003790-t006" target="_blank">6</a> for full details). A. 50 µM of Melittin added to blood containing <i>P. berghei</i> gametocytes. B. 50 µM of Melittin added to blood containing <i>P. falciparum</i> gametocytes.</p

Effect of antimicrobial peptides on field parasites and mosquitoes.

<p>Peptides (final concentration of 50 µM) were mixed with human blood containing <i>P. falciparum</i> parasites from gametocyte carriers in the village of Nyaganabougou. This was membrane-fed to the progeny of <i>An. gambiae</i> mosquitoes collected from neighbouring areas. For each replicate, oocyst prevalence (number of oocyst positive mosquitoes, total number in parentheses) and oocyst intensity (mean number of oocysts present per gut, range in parentheses) were recorded. An equivalent volume of water without peptide was used for the control. N/D indicates not determined. N/S indicates non-significance.</p

Antimicrobial peptide used in the current study: origin and size.

<p>A description of all antimicrobial peptides tested in the current study. AMPs were sourced from a variety of organisms displaying antibacterial, antifungal and/or anti-parasitic properties. Custom peptides were also included.</p

Effects of AMPs on <i>Anopheles gambiae</i> Sua 4.0 cell viability after 48 hrs.

<p>Effect of AMPs on viability of <i>Anopheles gambiae</i> Sua 4.0 cells. After 48 h of culture, 100 cells were counted in triplicate wells for each peptide for erythrosin B exclusion. Melittin caused 100% lysis of cells, and therefore viability was not assessed. Differences between viability in peptide and control wells were assessed using one-way ANOVA. N/D indicates not determined.</p

Effects of AMPs on <i>Anopheles gambiae</i> Sua 4.0 cell growth.

<p>Effect of AMPs on growth of <i>Anopheles gambiae</i> Sua 4.0 cells. Cells were seeded at a density of 30×10⁴/ml with the addition of 25 µM AMP. Cells in different duplicate wells were counted after 3, 24 and 48 h to assess cell growth. Experiments were repeated three times. Differences between cell numbers in peptide and control wells were assessed using one-way ANOVA.</p

Effect of AMPs on <i>P. berghei</i> ookinetes after 30 minutes.

<p>Peptides were incubated with ookinetes for 30 min to assess their speed of action and efficacy. This table summarizes peptides with significant effects on ookinete viability in these conditions (n = 150 ookinetes per treatment in each replicate). Where 100% mortality was not achieved at 50 µM, peptides were doubled in concentration (100 µM) or added in combination with another peptide to total 50 µM (25 µM of each peptide). All results were significant for Wilcoxon Rank sign test at p<0.009.</p

Survival plot for mosquitoes fed with TP10 dimer.

<p>Mosquitoes were fed blood containing 50 µM of TP10 dimer, or control on days 0 and 7 (arrows). Fully engorged females from each treatment group were separated into cages of 25 individuals to facilitate counting. Mosquito deaths, from a starting total of 100, were recorded daily. This figure shows the survival plot for one replicate with 50 µM of TP10 dimer, using the Kaplan-Meier method. The black line indicates mosquitoes fed with TP10 dimer, the red line, control mosquitoes.</p