Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

I am George Dimopoulos. I'm an assistant professor at the Johns Hopkins School of Public Health Department of Molecular Microbiology and Immunology at the Malaria Research Institute. My research group is working on the mosquito and Gambia that transmit malaria, and we are studying the molecular biology of the mosquito with a particular interest in the mosquitoes innate immune system that is involved in killing malaria parasites.

We want to understand how this immune system functions and which genes and molecules are important for killing the parasite. We believe that by understanding the biology of this system, we would be able to use the knowledge to develop Malaria control strategies in the future. Malaria is one of the world's most serious Diseases.

It has been with mankind forever, really, and it's a shame that we still have it. Malaria infect 400 million people every year and kill 2 million people, mostly in the Sub-Saharan African region, but we also find it in other developing Countries in South America and Asia. So malaria is caused by a protozoan Parasite of the genus odium, and there are four odium species that can cause human malaria.

The most important of those is plasmodium falciparum. Plasmodium falciparum is transmitted mostly by mosquitoes belonging to the anos genus, and the most important vector mosquitoes for malaria transmission in Sub-Saharan Africa belong to the Ous Gambia Species Complex, and they are mainly distributed In the Sub-Saharan region of Africa. The dengue virus is a flay Virus, and it's mostly transmitted by the mosquito Iida gti.

It's geographic distribution is quite similar to malaria. We find it in the Americas in Southeast Asia and in Africa. Dengue virus causes hemorrhagic fever.

There are about 2.5 billion people at risk from dengue virus, and the virus causes hemorrhagic fever in millions of people.Yearly. Dengue hemorrhagic fever has emerged one of the most serious vector-borne diseases In the past 10 years. So the malaria parasite has A complex lifecycle in both the mosquito vector that transmitted between human host and in the human host.

And my research group is studying the biology of malaria transmission in the mosquito. Now, when a mosquito will ingest an infected blood meal, it'll also ingest the plasmodium falciparum parasites. The gametocytes of the parasite will fertilize in the midgut and produce a MT tile or kin it at the round 18 to 35 hours after ingestion of the blood.

These Orkin needs will invade the mosquitoes gut epithelium here. Most Orkin needs are killed by the mosquitoes immune system At this stage, and this is where we are focusing our studies, and this is also a confocal microscopy image of the or kinine, the or kinine that manage to get through the mid gut will form an cyst on the basal side here, and within this cyst, thousands of sporozoite stage parasites will develop and these por wide stages will then migrate at the round 15 days after the mosquito has taken the blood meal, they will migrate to the sali glands and from the sali glands, the sporozoites can be transmitted to a new host. The mosquito's immune system is also involved in killing Sporozoites at this stage when they are released In the mosquitoes hemo seal.

The mosquitoes Genome was recently sequenced in 2002, and this has allowed us to develop novel molecular biology and genomics tools to study gene expression of all the mosquitoes, approximately 14, 000 genes at different conditions of infection on so-called microarrays. This microarray here, which we developed a couple of years ago, contain the mosquitoes entire transcriptome, all the mosquito genes that were known at that time, as well as the plasmodium parasite genes. So we can assay the activity of genes in both the mosquito and the parasite simultaneously during infection.

Once we identify genes that are activated, when the mosquito is infected with plasmodium parasites, we need to test whether these genes are involved in killing the parasite, and we do that through a method called RNA interference gene silencing. We basically inject RNA for the genes that we want to target, and this double stranded RNA will degrade the mRNAs of the genes in the mosquito that we target, and thereby these genes will not produce protein. Then we will infect the mosquito with a parasite.

Now, if we have silenced or and inactivated a certain gene in the mosquito, and upon this silencing, the parasite can establish a higher infection than normal in the non-treated mosquitoes. That would suggest that this gene is in some way used in killing malaria parasites. And this is really the major method that we are using to test whether genes are involved in killing parasites In the mosquito.

Most plasmodium parasites are Killed in the mosquito, and we have strong evidence that the mosquitoes in innate immune system is mainly responsible for this killing. Our laboratory and our collaborators laboratories have identified a variety of genes that are involved in this. Odium killing, genetically selected mosquito strains also exist that do not transmit malaria because they kill all the parasites in the midgut through innate immune defense mechanisms.

It seems like the mosquito has a variety of different components and mechanisms to target the parasite, and the greatest challenge today is to understand how these functions and how we can manipulate those to Develop resistant mosquitoes. The GTI mosquito is using Different mechanisms to control the dengue virus infection than what an awful Gambia is using controlled odium infection. The aegypti mosquito, as all the eukaryotes have an RNA dependent defense mechanism that is called RNA interference.

It is basically a defense mechanism that is capable of targeting the RNA of the virus degraded, and in this way it protects itself against virus infection. We are trying to understand this defense mechanism, how we can manipulate it in order to generate mosquitoes that Are resistant to the virus. In the past 10 years, The molecular biology has experienced some really dramatic advances, mainly methodological advances that has speeded up our research tremendously.

One of the major advance is the sequencing technologies that have allowed for high throughput sequencing of entire genomes. Thanks to these technologies, we now have the ous Gambia and the A, this gyp mosquito genomes completely sequenced. This allows us to identify genes that are involved in controlling infections much easier than previously.

Other very important advances in the functional genomics field is the high throughput gene expression analysis using the microarray. Microarrays are small glass slides on which thousands of DNA fragments have been immobilized, and we can use these so-called DNA chips to monitor gene expression for very large number of genes. Another very important advance in the field is the RNA interference gene silencing methodology, which allows us to target genes and inactivate them in the mosquito, and thereby use a so-called reverse genetic approach to study gene function and how our genes of interest are Important in killing the parasite.

The mosquitoes that we and our collaborators Have conducted most of these studies on are coming from mosquito colonists that have been maintained at laboratory conditions for as long as 30 years.Now. These mosquitoes have obviously differentiated quite a bit from the mosquitoes that we find in nature. The mosquitoes in nature are under a continuous selective pressure from the different microbes and other parameters that vary a lot in the field compared to the quite stable conditions we have in the laboratory.

So one could argue that our studies would probably be much more informative if they were performed directly on field mosquitoes. Unfortunately, though, that is not as easily done. As said, we don't have the infrastructure in the field and it, it is also very complicated to collect mosquitoes from the field and develop colonies out of those mosquitoes.

Therefore, the best biological models we have today are the lab models, but we will of course have to go into the field at some point and verify and confirm the major findings that come out from our discoveries With our lab mosquitoes. We know that the mosquitoes defense Systems are involved in killing the human pathogens that these insects transmit. Now, if we can understand how this killing occurs, which genes are involved and which mechanisms are involved, we could potentially manipulate them and use that manipulation to develop genetically modified mosquitoes that are unable to transmit the pathogen.

Once we have developed such genetically modified mosquitoes, we can start to think about how we can actually introduce them into the field and make them a dominant species against the susceptible mosquitoes. One strategy that has been widely discussed to achieve that is basically to spread resistant genes in natural populations, genes that would render the mosquitoes resistant to the pathogens, and that has to be achieved through so-called genetic drive mechanisms. And much research is focused today on the development of Such genetic drive mechanisms.

There are, of course, many ethical considerations One has to make for the release of any type of genetically modified organism in nature. It's important to remember that genetic modification occurs naturally all the time in the field. The three mutations there is little, there is little reason to believe that we would end up with a mosquito that will be better in transmitting a pathogen or would have some other non desirable characteristic.

The worst thing that could possibly happen is that the pathogen would develop resistance to this transgenic mosquito at some point, and we would, of course have to do a lot of field testing and a lot of laboratory testing before we would ever think about releasing a genetically modified mosquito in the field. We would also have to educate the people on these controlled strategies and make sure that everyone is feeling comfortable and is on Board with this type of strategy. There are still many obstacles for us To achieve a disease control strategy based on blocking the pathogen in the mosquito vector.

Some of these obstacles relate to technical issues, experimental issues that we need to solve. One obstacle is, for instance, the development of genetic drive mechanisms, mechanism that efficiently would allow these resistant genes to spread in populations. The scientific field of the biology of disease vectors has experienced a traumatic progress in the past 10 to 15 years, mostly because of a recognition that the understanding of the biology of insect pathogen interaction could actually help us to develop control strategies.

And this recognition led to an increased funding towards research in this field. It is extremely important that this funding is continued and the commitment is continued so that we can maintain the momentum that we have achieved and complete our mission by understanding the biology of vector pathogen interaction and how we can use this biology To develop strategies to control diseases.