There are two primary methods used to prevent the transmission of malaria using genetic modification: reducing the amount of vectors and reducing their ability to pass on the malaria parasite. One research team sought to decrease the numbers of wild anopheline mosquitoes, and therefore the rate of malaria transmission, by using CRISPR to disrupt a gene in anopheles gambiae mosquitoes that females need to survive. Since males aren’t affected, they continue to pass on the gene, which keeps it from immediately dying out in wild population. Additionally, males don’t bite and therefore can’t transmit malaria. At the time this paper was released, mechanisms to kill female mosquitoes had been developed, but not effectively for anopheline mosquitoes, which transmit malaria. Though there are methods under development, they face a whole host of obstacles that prevent their implementation, leading scientists to seek an alternative. 

Males are sometimes released by vector control campaigns but that means breeding a bunch of mosquitoes and sorting out the males from the females. For A. gambiae, sorting them is difficult because males and females cannot be differentiated based on pupal size, and there are obstacles in many other mechanisms of identification and separation. As a result, researchers have investigated the use of genetic engineering to separate males from females. For example, genetic modification can be used to indicate sex through the presence of fluorescent proteins and mosquitoes can be sorted from there. Even more efficient is modifying a gene that females need to survive, which would kill all the females and save time and money from physically sorting them, especially in this instance where females are not desired.

A gene was recently discovered that is essential to the development of female anopheles mosquitoes that could be removed to kill female mosquitoes before they reproduce. The research team used a CRISPR-based gene tool they called Ifegenia to target the gene, which is called femaleless (fle), causing the offspring of the modified mosquitoes to have mutations in fle, killing them in the early larval stage. They created a gene that codes for a gRNA that locates a spot in the beginning of the fle gene and a gRNA that targets the first RNA recognition motif, which is essential to the function of the protein containing this motif. To ensure the effectiveness of the transgene, the research team bred hetero- and homozygous GM Ifegenia males with and both hetero- and homozygous Cas9-positive females and found no female pupae, supporting the ability of the fle gene to inhibit female development.

The research team then chose to confirm that disrupting the fle gene killed females, because it is possible that instead, it made females appear as males, which is called androgenization. To eliminate that possibility, they bred GM males with Cas9 females and no female GM mosquitoes were identified, yet the proportion of GM males identified did not differ from Mendelian predictions. This indicates that females were killed and not androgenized, because if they were androgenized, the reported proportion of GM males would be double compared to other phenotypes. Additionally, the researchers conducted PCR (polymerase chain reaction) tests with DNA from randomly selected GM males and identified a Y chromosome in every sample.

To find out which stage of the life cycle GM females are killed at, researchers monitored the proportion of GM females in larvae that were one day old and found no variation from Mendelian expectations, indicating that GM females survived embryogenesis. They then compared the mortality rates from hatching to pupation between all offspring and concluded that most of the GM females died during the larval stage.

The researchers then sought to ensure that GM males had no impairments to fitness, so they compared two different strains of GM males, gFLEG/Cas9 and gFLEJ/Cas9, with wt (wild type) males, and found that one of the GM strains had a lower lifespan, but overall both GM strains had similar reproductive viability to wild. They bred equal numbers of wt and GM males with wt females and found that 45.7% of male offspring carried the fle transgene, which is close to the 50% expected if they were equally competitive.

To predict how effective the introduction of GM males would be on population suppression, the researchers used data from several generations of breeding to simulate the impact of a weekly release of 500 Ifegenia eggs per WT adult, both of which included males and females, over the course of 52 weeks. They used conservative estimates of the ability of transgenic males to decrease population size, such as a mating competitiveness of 75% in comparison to wt males and reduced target site cutting rates. Even with conservative estimates, it was simulated that the population decreased by more than 90% in several circumstances and persisted for more than two years. They also considered how population size would be affected by the development of resistance to the transgene and found that increasing the number of target sites substantially reduces the threat of resistance.

A similar method of mosquito population suppression through genetic modification is the implementation of fsRIDL, a transgene that prevents affected females from flying. It has been researched thoroughly for Aedes mosquitoes and will likely have similar effects as the fle transgene, so the success of fsRIDL in field applications can be used to predict the efficiency of Ifegenia.

The researchers note that the study of Ifegenia could increase the knowledge surrounding the fle gene and similar alleles, which could be used to manage populations of other insects for agricultural and epidemiological purposes. The development of Ifegenia and the fle gene could be incredibly useful in preventing transmission of malaria by decreasing the number of vectors spreading it from host to host, and also save time and resources that would otherwise go towards separating male and female mosquitoes. Additionally, a single female mosquito can have at least 200 GM male offspring, which is ideal for producing large numbers of Ifegenia males to be released and affect wild population sizes.

Once again, I found myself annoyed that I wasn’t able to access tables that contained precise information about certain details regarding the experiments discussed in this paper. They mentioned a few tables that I wanted to look into, but when I tried to download them, the data wouldn’t load. I’m not sure if this is the fault of my computer, but either way I was able to write a somewhat comprehensive summary without it.

I’ve yet to choose my final scientific article, but I think it will be either relatively short or simple. I’ve really enjoyed learning about mosquitoes, but I have such a difficult time understanding and summarizing these articles that it takes me several hours. I’ve been able to balance my honors project with my workload, but as the semester comes to a close and I have to prepare for finals, I’m not sure how much time and energy I can dedicate to my mosquito paper. That being said, I look forward to learning more, so stay tuned to learn with me!

https://www.science.org/doi/10.1126/sciadv.ade8903