Anopheline mosquitoes vary extensively in their innate ability to support development of the malaria parasite. The antiparasitic response is extremely efficient in several strains of genetically selected mosquitoes where parasite development in the mosquito is blocked early after infection (Fig 1). A key question is therefore to understand why some mosquitoes are resistant to malaria parasites, while others, within the same species, support parasite development and transmit the disease.
When we started this work, the long-lasting hunt for genetic factors that control resistance was limited to the mapping of intervals contributing to resistance, and more recently, to the identification of candidate genes within these intervals. Nevertheless, even when these genes were polymorphic and when polymorphisms correlated with mosquito resistance to malaria parasites, it was still unclear whether polymorphism in the identified gene was responsible for resistance or simply linked to a resistance polymorphism nearby. To address this issue, we designed a new approach termed reciprocal allele-specific RNA interference (rasRNAi), which enabled us to assess the contribution of different alleles of the same gene to a given trait (Fig 2A).
Using genome-wide mapping of mosquito crosses infected with the rodent parasite Plasmodium berghei, we recently demonstrated that resistant and susceptible mosquitoes differed in a major region on the third chromosome, named Pbres1 for P. berghei resistance locus 1 (19Mb). Of note, one of the genes located in Pbres1 encodes the complement like protein TEP1 (Fig 3), which we had previously characterised as a key antiparasitic gene. Using rasRNAi, we showed that polymorphisms in the TEP1 gene that is located in Pbres1, modulate the efficiency of parasite killing, thus identifying the first quantitative trait gene (QTG) for resistance to malaria parasites (Fig 2B).
Figure 2. TEP1*R1 is more efficient than TEP1*S3 in parasite killing. (A) rasRNAi. Each box represents a gene. With the use of short dsRNA probes specifically directed against *R1 (dsR) or *S3 (dsS), each TEP1 allele is silenced separately in F1 mosquitoes (open box), allowing us to compare the function of each allele in the same genetic background. (B) Parasite counts in the F1 progeny of L3-5 × G3 treated with allele-specific dsRNA probes and with dsLacZ and dsTEP1 as negative and positive controls, respectively. Mosquitoes expressing only TEP1*S3 (dsR) carry more parasites than those expressing TEP1*R1 (dsS) exclusively, demonstrating that polymorphisms at the TEP1 locus contribute to determine mosquito resistance to malaria parasites. **P < 0.001; ns, not significant (modified from Blandin et al., 2009).
Figure 3. Crystal structure and domain arrangement of TEP1*R1 compared to complement factor C3. The different domains are colored. MG, macroglobulin domain; LNK, linker; TED, thioester domain; ANK, anchor; the position of the thioester bond (TE) is indicated (modified from Baxter et al., 2007).
Still, an important observation from our work was that polymorphism in TEP1 does not account for the entire observed variability in parasite killing, indicating that loci other than TEP1 are necessary to make mosquitoes completely resistant to P. berghei in mosquitoes. We aim at further dissecting the complex genetic networks that sustain mosquito resistance to P. berghei and P. falciparum in laboratory models of infection and in the field.
With the new possibilities offered by high-throughput technologies for fine-mapping of quantitative trait loci (QTLs), and with the rasRNAi assay to precisely identify the causative genes in mapped intervals now in hand, we are performing a fine-resolution screen to identify the genetic components controlling parasite transmission in A. gambiae, and to compare the genetic networks governing resistance to the rodent and human malaria parasites P. berghei and P. falciparum.
The resistance genes and networks identified in controlled laboratory conditions will be tested in the context of natural P. falciparum infections in collaboration with Dr. I. Morlais (IRD/OCEAC, Yaoundé, Cameroon). Our goal is to identify genes that contribute to resistance to P. falciparum in natural populations, and to investigate how we may exploit this natural mosquito resistance to parasites to reduce malaria transmission.
Drosophila transgenesis has been instrumental for the understanding of many aspects of the fruit fly biology, allowing over-expression of gene of interests —in a tissue-specific and timed manner thanks to the Gal4/UAS system and its refinements—; transposon-mediated mutagenesis for the discovery of key player genes in a given biological process; expression of proteins of interest fused to reporters such as the Green Fluorescent Protein (GFP) to follow their cellular fate; fine genetic manipulations such as gene knockout; targeted gene knockdown by transgenic RNAi…
Similarly, transgenesis can be expected to help unravel key processes in Anopheles biology including that of vector/Plasmodium interactions. Thus, we are particularly keen on exploiting Anopheles transgenesis to address our biological queries. In the future, transgenic mosquitoes may even help fight malaria, as transgenesis could also be used to render mosquitoes resistant to Plasmodium parasites or in strategies designed to decrease mosquito vector populations, such as the Sterile Insect Technique (SIT).
We are interested in exploring the potential of all these aspects of mosquito transgenesis. Therefore, we worked hard to establish A. gambiae transgenesis in our laboratory and are now able to produce transgenic mosquitoes in a routine fashion. Key factors in this success were the optimization of each step of transposon-mediated transgenesis; the establishment of robust attP docking lines for phage PhiC31-mediated transgenesis and the construction of efficient helper plasmids; the use of automated larval sorting to distinguish between mosquito larvae carrying 0, 1 or 2 copies of a given transgene in order to rapidly generate stable homozygous lines and monitor subsequent transgene stability; and the use of multiple transgenesis selection markers to gain time by generating several distinct transgenic lines simultaneously.
With these tools at hand, we are currently (i) testing the effect of various transgenes on the outcome of a Plasmodium infection; (ii) establishing targeted mutagenesis of the Anopheles gambiae genome using TALEN synthetic endonucleases; (iii) setting up automated protocols to generate large populations of mosquitoes carrying the desired transgenes, or single-sex mosquito populations carrying no transgene at all. Pure male populations obtained in this manner may be used in SIT or in population replacement intervention schemes, aimed at increasing the frequency of disease resistant mosquitoes in the general mosquito population.
In the future, we will further explore how transgenesis can help obtain mutant (but non-transgenic) mosquitoes that are unable to transmit disease. For example, bringing small modifications to mosquito proteins that Plasmodium needs to colonize its vector could render the vector Plasmodium-resistant. The transgenes used to obtain these genetic modifications can be subsequently eliminated by genetic crosses and selection, and the obtained non-transgenic, disease-resistant mutant lines may be evaluated for population-replacement intervention strategies.
Transgenic mosquito larvae are identified with the help of fluorescent proteins (cyan, green, yellow or red) expressed in the nervous system
Expression of various marker genes in transgenic mosquito larvae allow the automated selection of the desired genotypes
The Group and its members currently receive funding from : INSERM, CNRS, INVESTISSEMENT d’AVENIR, ERC, ANR, INFRAVEC, FONDATION pour la RECHERCHE MEDICALE.