Malaria is a disease responsible for killing a shocking percentage of the human race, and continues to take thousands of lives every day. As drug and insecticide resistance grows, scientists have been searching for methods to prevent transmission of malaria in novel ways, such as utilizing symbiotic bacteria, implementing gene drives, preventing females from being born, creating a toxic fungus. and editing host mosquitoes’ immune response to malaria. These tools have huge implications for curbing the spread of malaria, and if given sufficient support and implemented correctly, have the potential of saving countless lives.

Malaria is caused by a parasitic protozoan called Plasmodium that is transmitted by anopheline mosquitoes. Of the five main species that infect humans, P. falciparum is the most virulent, meaning it is the most deadly, and is extremely common in impoverished countries. Malaria fatalities have fallen in recent years, which owes some credit to the efforts of the UN, which decided that malaria was a critical issue in 2021 and began a global initiative to reduce global fatalities to malaria. As the project went on, more money was raised and global  mortality dropped from an estimated 895,709 in 2000 to 608,000 in 2022, 95% of whom lived in the WHO African Region (Roser and Ritchie; “Fact Sheet About Malaria”). Malaria has plagued humanity for much of human history; its ancient presence in Africa likely resulted in the popularity of sickle-cell anemia and thalassemia in endemic areas, which provide resistance to malaria. Malaria’s terrorization of Africa is further exemplified by the prevalence of the Duffy antigen, a genetic trait that provides resistance to Plasmodium vivax, which had been endemic in Africa when the gene was estimated to have evolved 42,000 years ago. As a result, P. vivax is not common in Africa, especially in comparison to P. falciparum, which the Duffy antigen does not protect against.

Malaria is not considered a contagious disease because the life cycle of Plasmodium relies on both its host organism and its vector, anopheline mosquitoes. When a mosquito takes a blood meal from an infected individual, it ingests gametocytes, which are the reproductive cells of Plasmodium. Inside the midgut lumen of the mosquito, the gametocytes differentiate and fertilize to form a zygote, which contains the genetic information of a female and male gametocyte. The zygote then transforms into an ookinete, which burrows into the wall of the mosquito’s midgut and forms an oocyst on the other side. Inside the oocyst, sporozoites multiply until the oocyst ruptures and the sporozoites flood to the salivary glands so they can be transmitted to a new host. Once inside the new host, sporozoites travel to the organism’s liver and multiply, differentiating and multiplying until some eventually turn into gametocytes, which are ingested by mosquitoes when they take a blood meal and the cycle continues. 

Even after malaria seems to have left a patient, the disease can return in two ways: recrudescence and relapse. Recrudescence occurs when faint levels of parasites remain in the host, which can happen when a balance is formed by the immune system where the parasites aren’t completely eliminated, but condition doesn’t worsen, and that balance can be kept for long periods of time. The problem arises when the host’s immune system is disrupted by another ailment and parasite populations are no longer controlled, allowing them to reestablish and cause the illness to return. Relapse can occur even after all parasites seem to be absent in human hosts, as P. vivax has been observed to form hypnozoites, dormant cells that differentiate from sporozoites, which can colonize in the liver, and can survive even as other P. vivax cells have been wiped out by antimalarial treatment. Patients can also be asymptomatic, which is particularly common in endemic regions because immunity is acquired, meaning they carry Plasmodium gametocytes without experiencing symptoms. This is actually dangerous because if they move to an area with little or no cases of malaria, they can unknowingly cause a resurgence of cases if a mosquito takes a blood meal from them and transmits it to other individuals, or if they donate blood or organs, as undetected parasite populations can grow and cause illness in the new host.

There are a variety of methods in use and being researched to reduce the rates of cases and deaths from malaria, including the application of insecticides, bed nets, treatments, vaccines, and various other tools. One of the first known treatments for malaria is quinine, a compound found in the bark of the cinchona tree native to South America used in the 1600s. Since then, chemotherapy and five classes of antimalarial drugs have been developed that inhibit Plasmodium in different ways, but over time, strains of Plasmodium have evolved resistance to all of them. Strains of P. falciparum resistant to several antimalarial drugs often have defects in DNA mismatch repair, which means they experience a higher rate of mutations. This is usually harmful for fitness, but under the strong selection pressure of new treatments, the likelihood of mutating resistant genes increases, which can render new drugs ineffective. 

One mechanism under current investigation is the MEP pathway in Plasmodium, which produces IPP, isopentenyl pyrophosphate, a molecule the parasite needs to survive. A chemical called fosmidomycin inhibits this pathway, which prevents the production of IPP and therefore inhibits the development of P. falciparum, which provides a potential new mechanism for fighting malaria. However, concerns have arisen because of one related species that also relies on the production of IPP in the MEP pathway but has resistance to fosmidomycin. This species, cryptosporidium, gets its IPP from its host, so if Plasmodium mutates and evolves to use the same system, it also will be resistant to the chemical. Application of fosmidomycin has potential to kill parasites and limit the spread, but other tools such as genetic modification should be looked into to avoid a selection pressure that encourages evolution of resistance.

Preventing the spread of malaria by anopheline mosquitoes can be done in two ways: completely eradicating all Anopheles mosquitoes, which is next to impossible and would have environmental repercussions, or making them ineffective vectors. Though decreasing vector populations has drawbacks, it would decrease cases in the short term while scientists develop long-term solutions or even be used in tandem with other mechanisms to enhance their effectiveness. Insecticides have been used for many years to reduce vector populations in high-impact areas because mosquitoes can’t bite and spread malaria if they’re dead. Unfortunately, after years of use, mosquitoes have evolved resistance to insecticides, as the individuals most susceptible to the insecticides are killed, leaving more resistant mosquitoes to survive and produce offspring, which will also carry resistance. As a result, citizens of areas strongly affected by malaria are seeking an alternative to insecticides that can reduce vector populations.

One possibility is the Metarhizium fungus, which is a fungal pathogen that has strains that are toxic to adult mosquitoes yet safe for the environment. One study introduced the spores of the fungus to houses in a community in Tanzania and found that the number of infectious bites experienced by residents decreased, and to further improve its effectiveness, researchers chose to edit a strain of the fungus, Metarhizium pingshaense, to be more virulent. Their goal is to kill more mosquitoes while requiring a lower load of spores, so residents can more easily implement the spores into their community whilst further decreasing their susceptibility to contracting malaria. This GM strain is considered safe for the environment and its modification followed guidelines set by the EPA, but a contained field trial must be conducted to observe how results may differ from lab results.

The study was conducted in Burkina Faso, a country where malaria is a major issue, placing among nine other high-impact countries that failed to meet the goals set by the WHO Global Technical Strategy for malaria. The research team accessed local mosquito breeding sites and gathered Anopheles coluzzii mosquitoes that had resistance to insecticides. They then mixed the the genetically engineered M. pingshaense strain, which they called Mp-Hybrid, and wt fungi with sesame oil and applied the mixture to black cotton sheets, which optimized the period of spore production. Those sheets were then spread along surfaces that the mosquitoes rested on and 100 females were released into the enclosure at dusk with a calf they could feed on and captured the following morning. Of the 2,402 mosquitoes collected and assessed over several trials, 93.2% had fed from the calf, and their tendencies to rest on certain surfaces did not differ between wt and GM fungus treatment or a control with no spores, suggesting that mosquitoes weren’t deterred by the application of either fungus. 

To compare the effectiveness of the GM fungus to the natural strain, Mp-Hybrid was edited to express GFP (green fluorescent protein), while the wt strain was edited to express RFP (red fluorescent protein). The implementation of genes for these different proteins allow researchers to easily differentiate between the natural and GM strain during their tests. Infection was measured by examining mosquito corpses for microscopic fungal colonies and recording the quantities that fluoresced red and green respectively. The transgenic fungus proved to be more infectious than the natural strain, as 79.5% of the mosquitoes caught in the enclosure with the Mp-Hybrid sheets had been infected by the fungus, opposed to the 72.3% infected by the wt strain. About a quarter of the mosquitoes survived, and a sample of the survivors were rinsed and the water was cultured on petri dishes, but no fluorescent colonies formed. This suggests that the mosquitoes that survived did not prevail against the infection, but simply never came into contact with the spores over the night, as the mosquitoes that were infected were covered in about 130 conidia, a type of fungal spore. The absence of fungal growth on the rinse culture in contrast to the high sporogenic load on infected mosquitoes implies that the fungi were extremely virulent, which is ideal when it comes to the elimination of disease vectors.

When mosquitoes were exposed to the GM and wt fungi along with the negative control treatment for two weeks and mortality rates were monitored, it was found that the GM fungus killed significantly more mosquitoes than the control or wt fungus starting on day two and continuing for the remainder of the trial. As displayed in the graph below, the gap between the GM and wt fungi was widest halfway through the experiment, with mortality rates differing by more than 25% from days 6-8, and the gap narrowed by the end of the two weeks, though still significantly different. Even though the final survival rates were not wildly different between the GM and wt fungi, the disparity noted halfway through the experiment can have extreme effects on future generations, as Anopheles mosquitoes mature into adults in just 2-3 days, so an earlier death means less opportunities to reproduce and increase the population of the subsequence generation (“Life Cycle of Anopheles Species Mosquitoes”). Additionally, experimental groups killed drastically more mosquitoes by the end of the experiment than the control group with no fungi.

To assess the impact of the GM fungus over populations over multiple generations, 1000 virgin males and 500 virgin females in the GM, wt, and control conditions were monitored for reproductive success. The experiment lasted about 45 days and was replicated for a total of three sets of data. All experimental groups were given access to a calf three times a week and measured daily over the course of two generations for levels of larvae, pupae, and emerging and surviving adults. In the control group, the first generation of offspring resulted in 921 new adult mosquitoes, followed by 1396 in the next generation. The group exposed to the wt fungus increased by 436 and 455 new adults respectively, whereas the population with the Mp-Hybrid fungus had a growth of 399 and 13 new adults respectively. The decrease in offspring in the second generation in comparison to the first suggests a collapsed population, as the numbers of progeny was not sustainable for the population in the long term.

One interesting observation noted in this study is that in two of the three replicates, larvae in the wt group appeared one day before the control group, and larvae appeared 1-2 days before the control group in all three repeats for the Mp-Hybrid group. This was likely due to the tendency for dying pregnant mosquitoes to lay their eggs early, presumably so they don’t die before they get the chance to lay their eggs. As a result, the amount of eggs laid by mothers and their hatch rates was significantly lower than normal rates, as the median proportion of females exposed to the Mp-Hybrid fungus that laid eggs was 24.7%, compared to the 77.8% observed in the control group. Additionally, 53.3% of control group mosquitoes’ eggs made it to adulthood, but only 12.9% of the eggs from the group exposed to Mp-Hybrid survived to adulthood. This mechanism to improve fitness, where females accelerate their pregnancy, poses a concern for evolutionary adaptation, but the amount of successful offspring hatched is drastically less than typical rates, offering less opportunities for evolution. 

Researchers examined how long applied fungi would pose a threat to mosquitoes for and found that the GM fungus was more persistent than the wt fungus, as it maintained a mosquito mortality rate of more than 80% even six weeks after application, whereas the wt group failed to stay at 80% after four weeks. In fact, mortality rates caused by the Mp-Hybrid strain doubled that of the wt strain during weeks 5-7.

To ensure its safety to the environment, a similar GM Metarhizium strain was tested in a separate study with honeybees to see if the spores would be damaging to other insects, and none of them died to the exposure, even when they were sprayed with a high concentration of 100 million spores per milliliter. However, further research with different species of insect is warranted before it is implemented to decrease transmission of malaria.

The experiments conducted by the research team suggest that the genetically modified strain of Metarhizium fungus is significantly more productive, virulent, and persistent than the wt strain, yet remains safe to its environment. Through gene editing, a new potential solution to devastating rates of malaria transmission has been developed, and could fight in the battle against the disease. This is desperately needed, especially considering the rising rates of insecticide resistance observed in malaria vectors. Perhaps mosquitoes with enhanced resistance to malaria could be given features that make them less susceptible to death by the fungal spores, which would eliminate wild mosquitoes but not transgenic ones, decreasing the transmission of malaria while maintaining mosquito populations, which are a part of the food chain that predators rely on. There are several ways to make a mosquito more resistant to Plasmodium, and one method that has been studied is infection by symbiotic bacteria that inhibit Plasmodium development in hosts.

In 2013, it had been known that mosquitoes’ microbiome affects their susceptibility to disease, as studies had shown that the presence of certain bacteria affected the development of some diseases. One example is the Wolbachia bacterium, which is vertically transmitted from female mosquitoes to their offspring, which was the focus of several studies due to its ability to increase resistance to many diseases in the mosquitoes it infected. For the two decades prior, scientists had been trying to see how Wolbachia infection could affect Plasmodium resistance in anophelines, but Anopheles mosquitoes aren’t naturally infected by the bacterium, and researchers were not able to induce and maintain Wolbachia infection. However, one research team eventually identified a Wolbachia strain called wAlbB that came from Aedes mosquitoes and could persist in A. stephensi mosquitoes.

Researchers injected A. stephensi embryos with wAlbB amplified the DNA from the females that developed from the embryos. They created a line of mosquitoes that descended from one infected female and measured infection frequency for 34 generations, and found that every descendant was infected with the wAlbB strain. This showcases the stability of the infection caused by the mechanism used, supporting its potential for implementation in wild populations. Transmission efficiency was tested by randomly selecting 60 total offspring in generations 9, 10, and 11 and performing diagnostic PCR, all of which were shown to carry wAlbB, along with using FISH (fluorescent in situ hybridization), to visualize the site of infection inside cells. FISH utilizes fluorescent probes that mark specific DNA sequences, such as ones found in wAlbB. Observing the location of the fluorescent probes showed researchers how many offspring were infected, which was 100%, along with allowing them to compare bacterial colonization sites with other mosquito species, which was consistent with A. albopictus and A. aegypti mosquitoes. After ensuring that infection would persist over several generations, researchers introduced varying ratios (5, 10, and 20%) of infected females to cages of 100 uninfected (LIS) mosquitoes of equal proportions of males and females, along with 100 infected males at every generation. By the eighth generation, the entire population was infected regardless of the ratio of infected females and stayed that way for subsequent generations. The ability for infection to overtake and persist uninfected populations show the potential of wAlbB as a means for decreasing malaria transmission, but this mechanism requires a large amount of infected males to be regularly released, so mechanisms should be investigated to isolate mass amounts of male mosquitoes.

To test if wAlbB infection suppresses the development of Plasmodium, researchers fed a highly concentrated gametocyte culture of the parasite to infected mosquitoes, LIS mosquitoes, and infected mosquitoes that had been treated with tetracycline, an antibiotic that kills wAlbB. They recorded Plasmodium development at various stages and found that the quantity of oocysts that formed in the infected mosquitoes was significantly smaller than in both of the controls. The quantity of sporozoites in the salivary glands of infected mosquitoes was 3.4 times smaller than the LIS mosquitoes, which is crucial because sporozoites in the salivary glands are what are transmitted to human hosts. To identify the way wAlbB inhibits Plasmodium development, researchers monitored levels of hydrogen peroxide in infected and LIS mosquitoes. Hydrogen peroxide is a ROS (reactive oxygen species), which has been shown to impair Plasmodium development in anophelines. Previous studies have shown that Wolbachia infection increases the production of ROS in Aedes, so detecting enhanced levels of ROS would indicate the mechanism of wAlbB for decreasing Plasmodium success. Researchers found that ROS levels were heightened throughout the fatbody, midgut, and the entire body in infected mosquitoes in comparison to LIS mosquitoes, supporting the concept that it is responsible for suppressing Plasmodium development.

However, use as an antimalarial tool only works if the presence of infected mosquitoes can persist in wild populations, so impacted fitness could jeopardize success. Researchers monitored fitness by recording what percent of laid eggs hatched and found that descendants of the original infected female had impaired hatching rates. While LIS mosquitoes experienced a hatch rate of 91.0%, only 52.4% of eggs from two infected parents hatched. However, when infected males mated with LIS females, only 1.2% of eggs hatched, which is ideal for inhibiting malaria transmission. Since infection is only vertically transmitted by the mother, a low hatch rate is ideal for LIS offspring, as it reduces the reproductive rates of mosquitoes that don’t carry wAlbB. Hatch rate rose to 85.9% when infected mosquitoes were treated with tetracycline, indicating that the reduced hatch rate was a result of Wolbachia infection. wAlbB’s cost to fitness could potentially be the demise of its prospective role as a means to prevent malaria transmission, because infected mosquitoes could be outcompeted by wild mosquitoes. The research team noted that reduced hatch rate is not observed in A. aegypti mosquitoes infected with the same strain, and proposed that the reduced rate might be due to using mice to feed the mosquitoes. This is because mice are not a natural host for A. stephensi, and reduced hatch rates have been observed during a study in which Aedes mosquitoes infected with a Wolbachia strain were fed with nonhuman blood in comparison to human blood.

Wolbachia certainly has potential for use as an antimalarial tool, but its impact on reproductive success might prevent it from being implemented in malaria control strategies. Regardless of the success of wAlbB, studies on Wolbachia could be extremely useful because of their potential to be applied to other bacteria that are being considered for malaria control operations. Knowledge accrued for the several years since Wolbachia has been studied could be used as a foundation for researchers engineering a GM bacterium. Using genetic engineering, scientists can attempt to make a bacterium that is optimized in its effectiveness in preventing malaria transmission, as it can be changed to maximize antimalarial factors and minimize drawbacks to fitness. After a mosquito takes a blood meal, numbers of bacteria increase by factors of 100 to 1000, so if scientists were to insert a GM bacterium that combats malaria into mosquito midguts, it should also replicate after a blood meal. At first, the researchers modified e. coli bacteria, but the lab e. coli didn’t survive in the mosquitoes, so they instead chose to use AS1, a bacterium that multiplies in mosquito ovaries, and engineer it to produce antimalarial proteins. AS1 had negligible effects on fitness and feeding behavior for both A. gambaie and A. stephensi mosquitoes, both before and after being edited to fight malaria. To make the bacterium counter Plasmodium survival in host mosquitoes, researchers used a combination of different anti-Plasmodium proteins with varying killing mechanisms to prevent the parasite from developing resistance. The mosquitoes infected with the GM bacteria with several antimalarial effector molecules had 92% less Plasmodium oocysts, which means that the bacteria was immensely successful at preventing the growth of the malaria parasite in the infected mosquitoes.

Along with anti-Plasmodium factors, the researchers gave the bacteria a gene that codes for eGFP or mCherry, which both produce a fluorescent protein that researchers could use ro keep track of the AS1 bacteria inside the mosquitoes. The AS1-GFP bacteria were fed to mosquitoes in a sugar meal and managed to survive and reproduce despite the already existing microbiome, and 24 hours after a blood meal was taken, the bacterial population had multiplied in quantity by a factor of more than 200. AS1-GFP not only survived in adult mosquitoes, but could also be spread vertically and horizontally, meaning that offspring or mates of mosquitoes with AS1-GFP were also colonized by the bacteria. When males infected with the GM bacteria were mated with wt females, the AS1-GFP bacteria was found in the female mosquitoes, and in infected female mosquitoes, AS1-GFP clung to laid eggs, multiplied in the water, and were then eaten by the hatched larvae. To test the longevity and infectiousness of AS1-GFP and ASI-mCherry, researchers set up a cage with 95% wt mosquitoes and 5% virgin female and male mosquitoes fed with the GM bacteria, and found that all the offspring of the infected mosquitoes carried the GM bacteria and continued to pass it on for the following generations.

This GM bacteria holds immense potential, though there has not been any major developments on this strain published since 2017, when the initial study was published. As further research continues, it is wise to look to other mechanisms for disease control in order to have a wide range of options, which can serve as backups or even be used alongside other methods. 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 A. 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 populations. 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 entails 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. CRISPR/Cas9 is a modern genetic engineering tool that can be used to find specific gene sequences and delete, add to, or modify them in groundbreaking precision. 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.

Ifegenia could be used to control population size and produce mass amounts of male mosquitoes, which could be widely used in malaria control strategies. Another strategy under investigation is the use of genetic engineering to make mosquitoes better at fighting the malaria parasite. Since Plasmodium is a parasite, the immune system of every mosquito that carries it tries to attack it, so a stronger immune system leads to lower parasitic survival rates, and therefore less cases of malaria in humans. Whether or not Plasmodium can survive and replicate inside of a mosquito depends on a variety of genetic factors, including the activity of agonists. Agonists control pathways that the development of Plasmodium depends on, so to understand how specific agonists affect Plasmodium infection, researchers investigated the effects of silencing the FREP1 (fibrinogen-related protein 1) gene on infection rates and fitness in A. gambiae mosquitoes, a primary vector for malaria in humans.

Transgenic mosquitoes were created by putting synthesized gRNA (guide RNA) and Cas9 protein into mosquito embryos, along with a reporter gene. The reporter gene acts as an indicator for successful genome editing, as it causes GM mosquitoes to fluoresce blue under certain conditions. The gRNAs helped CRISPR/Cas9 locate the FREP1 gene, which was selected because of its effect on Plasmodium growth in the mosquito midgut.

To test the impact of the FREP1 inactivation on susceptibility to Plasmodium infection, researchers fed both the mutant and wild-type (wt) groups a culture with an unnaturally high concentration of Plasmodium gametocytes. Eight days after infection, they measured the intensity of the infection with the amount of parasitic oocysts that developed. As a result, the wt mosquitoes developed a median of 91 oocysts, while the mutant population developed a median of just 17 oocysts. This test was then repeated with a tenfold lower concentration of gametocytes to reflect natural levels, with the median oocyst count at 2.0-2.5 in the wt groups and at 0.0 in the mutant groups.

Clearly, the silencing of the FREP1 gene was favorable for decreasing Plasmodium susceptibility, but it had unintended side effects. The FREP1 gene not only regulates Plasmodium infection rates, but also plays a role in blood feeding and digestion, so its complete inactivation had adverse effects on the transgenic mosquitoes’ fitness. In one test, both mutants and wt mosquitoes were prevented from feeding for 3-5 hours before being introduced to anesthetized mice, and while 90-95% of wt mosquitoes fed, only 68% of the mutants fed. Similarly, when the same trial was conducted with human blood-containing membrane feeders, the amount of the mosquitoes that took a blood meal was 73% for the wt group and just 28% for the mutant group. Other aspects of fitness were compromised in the mutant group, including the amount of eggs laid and percentage hatched and life expectancy after a blood meal was taken. The figure below was taken from the study.

In this graphic, the transgenic group is labeled FREP1-KOs for FREP1 Knockouts.

One proposal mentioned in the study for the impact of genetic modification on fitness was that inactivating the FREP1 gene possibly reduced absorption of nutrients, affected midgut immune response against stress, or that unintended changes were made by CRISPR in non-target sites, because although this novel technology is incredible, it is not perfect, and has a tendency to make mistakes.

This study showcased the role of the FREP1 gene in Plasmodium infection rates on Anopheles mosquitoes, along with displaying how CRISPR/Cas9 can be used to identify and change parts of the genome to affect resistance to the parasite. This is certainly a valuable tool for gaining understanding about organisms’ genomes, which can be used in further studies on GM Anopheles mosquitoes. However, the compromised fitness of the transgenic mosquitoes is discouraging for this species of transgenic mosquito, and other studies have found more promising mechanisms of enhancing the immune response of anopheline mosquitoes without high fitness costs, such as changes to the IMD pathway.

Unlike vertebrates, which have vast and adaptive responses to infection that can be combined for specific immune targets in countless ways, the immune response of insects is limited to the genes in their DNA to fight off pathogens, which is a finite and less adaptive system. One way insects fight off infection is with pattern recognition receptors (PRRs), which are proteins that search for molecular patterns that match known pathogens. These pathogen-associated molecular patterns (PAMPs) are present in pathogens, but not the host organism’s cells, so when PRRs find PAMPs, they bind to them, signaling the presence of a pathogen to the rest of the cell. This activates immune pathways that lead to the production of molecules that fight pathogens, defending the cell from dangerous invaders. One such PRR in mosquitoes is AgDscam, which has many forms, and exactly what forms are produced is determined by the IMD pathway. This is of particular interest because some forms of AgDscam are extremely efficient at fighting Plasmodium infection, and can therefore be used to combat the spread of malaria.

To examine the effect of the IMD pathway on AgDscam production, researchers used a special microscope to observe the effects of activating or inhibiting the IMD pathway on the position of AgDscam and P. falciparum ookinetes and found that the number of times AgDscam interacted with the ookinetes was 6.4 times greater when the IMD pathway was activated than when it was inhibited.

The IMD (immune deficiency) pathway in mosquitoes plays a major role in killing off Plasmodium falciparum before it can multiply and colonize in mosquitoes’ salivary glands and be spread to human hosts. This pathway, along with the Toll pathway that specializes in inhibiting P. berghei, which causes malaria in rodents, control the production of molecules that prevent Plasmodium development, which they do by activating or inactivating the production of splicing factors. 

Splicing factors are proteins that help choose what kinds of proteins the cell will make by influencing the spliceosome, something that picks what parts of pre-mRNA will be deleted and what parts will turn into mRNA, which gets turned into protein. Pre-mRNA is made up of introns and exons, and introns don’t carry instructions for making proteins, so they are removed from the pre-mRNA with help from splicing factors. Splicing factors aid in picking which exons will be cut out and which will be joined together, and the final string of mRNA of all the connected exons determines the type of protein that will be produced. AgDscam’s ability to fight Plasmodium infection depends on how it’s spliced, so what exons are chosen and connected from the pre-mRNA. The researchers hypothesized that the best splice forms of AgDscam at preventing Plasmodium development are controlled by the Rel2 transcription factor in the IMD pathway, which regulates the activity of splicing factors that control how different forms of AgDscam are cut. To test this hypothesis, they found nine AgDscam splicing factors with activity that responded to infection and observed the results of inactivating the IMD pathway by silencing Rel2. As predicted by their hypothesis, the activity of the nine splicing factors was reversed when Rel2 was silenced.

Two of the nine splicing factors examined by the researchers are Caper and IRSF1, and to gather evidence that they create various splice forms of AgDscam, some of which are more effective than others at fighting Plasmodium, researchers silenced both splicing factors and found that AgDscam was spliced differently than under normal circumstances. When Caper was silenced, AgDscam resembled the alternative splicing of when the IMD pathway was inhibited, which includes fewer forms of AgDscam known to fight Plasmodium. When IRSF1 was silenced, there was actually an increase in the production of antimalarial forms of AgDscam. The research team applied this knowledge with actual mosquitoes and found further evidence; silencing Caper increased the medium numbers of P. falciparum oocysts in mosquitoes’ midguts by 174%, with a median oocyst count of 54 compared to 31 in the control, and silencing IRSF1 resulted in a median of just 6 oocysts, less than one fifth of the control.

To gather more support that Caper and IRSF1 play a role in Plasmodium success, the researchers also measured the amounts of their transcript produced before and after injecting mosquitoes with P. falciparum. They did this 24 hours after the mosquito had taken an infected blood meal, and found that quantities of Caper transcripts multiplied by a factor of 1.8, and IRSF1 transcripts were cut by a factor of 2.1. To confirm that different forms of AgDscam have different effects on Plasmodium resistance and investigate which splice forms are most effective, the researchers genetically modified A. stephensi mosquitoes to produce short and long forms of AgDscam, Pf-S for short and Pf-L for long. They then used the Cp-1 (carboxypeptidase 1) promoter to create an abundance of the AgDscam splice forms each time the mosquito took a blood meal. The researchers fed a parasite culture to GM and wt mosquitoes and found that compared to the wt mosquitoes, the Pf-S mosquitoes had 1.5 times fewer oocysts and the Pf-L mosquitoes had 2.4 fewer oocysts. These experiments fed the mosquitoes abnormally high levels of parasites, so they were repeated with lower concentrations to mimic natural levels and while the Pf-S mosquitoes did not exhibit significant resistance when compared to the wt mosquitoes, the median oocyst count in the Pf-L mosquitoes went from 4.5 to 0, making them almost entirely resistant to P. falciparum.

AgDscam plays a major role in mosquitoes’ ability to prevent Plasmodium development, and its splice forms impact parasitic success in different ways, with the Pf-L form being one of the most effective. AgDscam is a PRR, which recognizes pathogenic patterns (PAMPs) on Plasmodium parasites and signals presence of the foreign invader to the cell, activating immune pathways that prevent parasitic development. It can be spliced in different ways, notably by splicing factors Caper and IRSF1, which are regulated by Rel2, a transcription factor in the IMD pathway. AgDscam has been shown to drastically decrease the amount of oocysts that form on mosquitoes’ midguts, indicating its significance in immune response against malaria and the potential it holds to reduce cases in human populations as a result.

To test the role of Rel2 in the immune response to Plasmodium, researchers created three types of transgenic mosquitoes with over-expression of Rel2, then collected data on their resistance to Plasmodium, which they labelled Cp-Rel2, Vg-Rel2, and Hyb, a hybrid of the two. When a blood meal is taken, it can induce Cp (carboxypeptidase) or Vg (vitellogenin) promoters, which initiate the production of Rel2. In Cp-Rel2, the highest concentration of transcript occurred 6-12 hours after a blood meal, while the parasite was in the mosquitoes’ midgut lumen, and in Vg-Rel2, highest concentration occurred 12-24 hours after blood meal, when the parasite was located near the basal region of the midgut tissue. Experimental results indicated that Cp-Rel2’s more immediate response is favorable to the elimination of Plasmodium, as the Vg-Rel2 strain experienced less Plasmodium resistance than the Hybrid and Cp-Rel2 lines. The location of the transgene affected resistance to Plasmodium, but since having multiple copies of the transgene did not differ from having just one, researchers were able to experiment with putting the gene in random places and use a system to find the best combination.

Overall, the experiment was a novel success, as infection rates were significantly lower in the GM mosquitoes, especially in the Cyp and Hyb types. The near-complete resistance to Plasmodium by the GM mosquitoes has huge implications for fighting the painfully high numbers of cases and deaths from malaria every day. However, disease resistance is irrelevant if the anti-Plasmodium transgenes cannot persist in wild populations due to fitness costs, so a group of researchers conducted a variety of experiments comparing the fitness, mating preference, and longevity of resistance of the wt and three lines of GM mosquitoes.

In biology, fitness is a measure of an organism’s reproductive success, which is essential when considering the introduction of GM mosquitoes to natural environments. If the impacts of genetic engineering negatively impacts fitness, then the GM mosquitoes will have less offspring, which means that GM traits are less likely to spread in a wild population. Of course, the entire point of creating transgenic mosquitoes that are more resistant to Plasmodium infection than wild mosquitoes is to reduce rates of malaria transmission, so fitness costs are not optimal. Researchers conducted an experiment to see if there were any evident fitness costs to the GM mosquitoes by crossing them with wild mosquitoes for five generations and treated them with consistent conditions to avoid confounding variables. Four of the five lines they examined showed no fitness costs, measured by survival and egg-laying and hatching rates and sex ratio, though they did find an elongation of pupation time in GM mosquitoes, which could lead to a slight disadvantage. They also tested the strength of Plasmodium resistance after several generations, culturing two GM lines, one that acts in the midgut and one that acts in the fatbody, for more than 50 generations, and both maintained their resistance to the malaria parasite.

Researchers then chose to examine the competitiveness of transgenic mosquitoes in comparison to wt mosquitoes. They selected the two genes that were most effective at inhibiting Plasmodium colonization in the midgut tissue, CpRel215 and CpDsPfs3, along with one gene that acts in the fatbody, VgRel21, and put each variant in an environment with 50% GM and 50% wt mosquitoes. With each subsequent generation, they measured the portion of mosquitoes with the antimalarial transgene, and found that by the first generation, the percentage of GM mosquitoes was 90%, a ratio that persevered in all nine following generations. This showcases the competitiveness of the GM lines, as Hardy-Weinberg equilibrium predicts a prevalence of just 75% for the GM trait without an advantage. 

The advantage in competitiveness of the GM lines is likely due to a phenomenon in the mating preference of male mosquitoes. When CpRel215 males were given the choice between WT and GM females, they preferred to mate with WT mosquitoes, measured by the percentage of females that did or did not lay eggs when exposed to the GM males. This tendency to mate with mosquitoes that don’t already carry the transgene accelerates its spread, and the inverse mating behavior is observed in WT males, which preferred GM females to WT females, furthering the spread of the anti-Plasmodium transgene. What, then, causes this mating preference?

The reason that the CpRel215 and CpDsPfs3 transgenes were selected for these experiments is that they affect Anopheles mosquitoes’ immune pathways in a way that inhibits Plasmodium oocyst development, which is desirable for decreasing malaria transmission rates. However, they have a wider impact than just Plasmodium resistance; they also fight bacteria, and that change to the mosquito microbiota can have a wide range of effects, including alterations to mosquitoes’ mating preference. To see if the 90% GM prevalence found in the experiment that monitored the percentage of GM mosquitoes for ten generations was caused by mating changes made because of the shift in bacterial colonies in the mosquitoes, researchers crossed GM and wt mosquitoes in two separate groups, treating one group with antibiotics to remove the effect of the altered microbiota caused by the transgene. As a result, GM prevalence in the first generation was about 75%, as predicted by Hardy-Weinberg equilibrium. The following generations had increasing levels of transgenic mosquitoes in comparison to wt mosquitoes, presumably because the original microbiota reestablished itself in subsequent populations. These findings support the hypothesis that the change in microbiota created by genetic modification impacted mating preferences in a genetically advantageous way, though lab mosquitoes having different bacterial compositions than wild mosquitoes, so these experiments should be replicated in natural conditions to further validate these conclusions.

The GM transgenes created by editing aspects of the IMD pathway are incredibly promising for global initiatives to lower the impact of malaria, especially because of their ability to spread into wild populations. The ability of a GM trait to integrate into wild populations is crucial, and there is a lot of ongoing research regarding gene drives, which are a powerful tool that can promote the rapid spread of a modified gene.

Homing endonuclease genes (HEGs) are naturally-occurring genes that produce proteins that cut certain pieces of DNA. When the DNA is cut, the cell tries to fix it by copying information from the homologous chromosome, which contains genetic information for the gene that was snipped. This homing strategy can be used to increase the spread of a specific gene, which is desirable for GM traits that lead to the decrease of malaria transmission. Basically what can happen is when a GM and wt mosquito mate without a gene drive, fewer and fewer of their descendants will have the GM trait, because it is mixing with natural populations that don’t carry the GM allele. With a gene drive, though, the HEG drive cuts the wt parent’s DNA in the same place on the chromosome that codes for Plasmodium control factor(s). Since the DNA is damaged, the cell copies the alleles from the GM parent, and as a result, both chromosomes code for the GM traits, not just the chromosome from the GM parent.

Improving mosquitoes’ ability to fight off Plasmodium infection is one way to reduce cases of malaria, but another option is reducing vector populations in general; inhibiting the ability of A. gambiae mosquitoes to reproduce will decrease the rate of malaria transmission. By altering a recessive gene that is necessary for female fertility, affected A. gambiae mosquitoes won’t be able to reproduce, decreasing the amount of carriers of Plasmodium and therefore rates of infection. However, homing must occur before gametes are formed, because otherwise the gene drive would change the other parent’s alleles and the offspring would be homozygous for the GM trait, meaning they would be infertile and unable to spread the gene. Heterozygous mosquitoes can carry and spread the gene without expressing it, therefore not compromising their fertility.

The researchers referenced a fertility index based off of a species of fruit fly to see if they could identify similar genes in A. gambiae, then used CRISPR or another gene editing tool called TALEN to disable the three fertility genes they found. They also included the fluorescent GFP marker for GM identification, which they use to monitor offspring. The intensity of GFP indicated if the offspring were heterozygous or homozygous recessive, so scientists were able to compare fertility, and found that all the female mosquitoes with two modified genes were sterile, while the mosquitoes with just one GM gene reproduced as normal.

To test the effectiveness of the gene drive, scientists monitored the success of homing, that process in which genes were cut and copied from another chromosome so both sets contained the GM information. In this test, when the natural homologous chromosome was cut and received a copy of the information from the GM chromosome, it also received RFP, which produces a red fluorescent protein that was used for identification. The gene drive was correlated with significant rates of inheritance of the allele marked with RFP, ranging from 94.4-100% when heterozygous GM mosquitoes were crossed with wt mosquitoes.

Researchers also created a population with equal numbers of wt and GM mosquitoes, and found that the percentage of mosquitoes carrying the GM allele rose from 50 to 75.1% over four generations, displaying its affinity to spread through populations.

Evidently, gene drives are powerful tools that enable the application of promising GM traits. Even if a gene was identified that could completely prevent Plasmodium infection, it would be useless if it could not be integrated into wild populations, so it’s very exciting to see what methods exist to spread new genes. There are a lot of pieces in the puzzle of genetic engineering, but if they work together effectively, they could save countless lives. As research on antimalarial tools continues, more discoveries and applications will be found, and one day these solutions might be applied and drastically reduce the countless lives taken prematurely by an ancient disease.

Did you really read all of that? If you did, what you just read was a comprehensive summary of everything I learned writing my research project. My final submission for the Honors project was very similar, but didn’t really connect the papers because I was supposed to do an annotated bibliography for each paper individually. None of this information is new to my dedicated readers, but it might be nice to see it all together, especially as it’s been many months since I started this project.

Thank you so much for sticking around for this massive project! I’m so glad that I did it, and I’m glad that you all seemed to have enjoyed it, too. I’ve learned a lot over the course of this project, and I’m a little nervous about how much it may have impacted what I want to do with my career. Last semester, I figured I’d want to focus on agriculture, but disease is just so incredibly fascinating to me! The problem with studying disease, though, is that means I’d almost certainly have to take a ton of different classes outside of my major, and I don’t really find anatomy and physiology interesting. I get that it’s relevant, but I just want to learn about the little guys! I don’t know; I’d probably be happy either way. We shall see. Stay tuned to learn with me!

Citations are in chronological order of reference.

“Fact Sheet about Malaria.” World Health Organization, World Health Organization, 4 Dec. 2023, http://www.who.int/news-room/fact-sheets/detail/malaria. 

Institute of Medicine (US) Committee on the Economics of Antimalarial Drugs; Arrow KJ, Panosian C, Gelband H, editors. Saving Lives, Buying Time: Economics of Malaria Drugs in an Age of Resistance. Washington (DC): National Academies Press (US); 2004. 5, A Brief History of Malaria. Available from: https://www.ncbi.nlm.nih.gov/books/NBK215638/

Roser, Max, and Hannah Ritchie. “Malaria.” Our World in Data, 29 Feb. 2024, ourworldindata.org/malaria. 

“Life Cycle of Anopheles Species Mosquitoes.” Centers for Disease Control and Prevention, Mosquitoes, 24 Aug. 2023, http://www.cdc.gov/mosquitoes/about/life-cycles/anopheles.html. 

Lovett, Brian, et al. “Transgenic Metarhizium Rapidly Kills Mosquitoes in a Malaria-Endemic Region of Burkina Faso | Science.” Transgenic Metarhizium Rapidly Kills Mosquitoes in a Malaria-Endemic Region of Burkina Faso, Science, 31 May 2019, science.sciencemag.org/content/364/6443/894.

Guowu Bian et al., Wolbachia Invades Anopheles stephensi Populations and Induces Refractoriness to Plasmodium Infection.Science340,748-751(2013).DOI:10.1126/science.1236192

Driving Mosquito Refractoriness to Plasmodium Falciparum with Engineered Symbiotic Bacteria | Science, Science, 29 Sept. 2017, http://www.science.org/doi/10.1126/science.aan5478.

Smidler, Andrea L., et al. “A Confinable Female-Lethal Population Suppression System in the Malaria Vector, Anopheles Gambiae.” Science Advances, vol. 9, no. 27, American Association for the Advancement of Science, July 2023, https://doi.org/10.1126/sciadv.ade8903.

Dong, Yuemei, Maria L. Simões, et al. “CRISPR/Cas9 -Mediated Gene Knockout of Anopheles Gambiae FREP1 Suppresses Malaria Parasite Infection.” PLOS Pathogens, Public Library of Science, 8 Mar. 2018, journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1006898#ppat.1006898.s003.

Dong, Yuemei, et al. “Anopheles NF-ΚB-Regulated Splicing Factors Direct Pathogen-Specific Repertoires of the Hypervariable Pattern Recognition Receptor AgDscam.” Cell Host & Microbe, U.S. National Library of Medicine, 18 Oct. 2012, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614911/.

Y. Dong, S. Das, C. Cirimotich, J. A. Souza-Neto, K. J. McLean, G. Dimopoulos, Engineered anopheles immunity to Plasmodium infection. PLOS Pathog. (2011).

Pike, A., Dong, Y., Dizaji, N. B., Gacita, A., Mongodin, E. F., & Dimopoulos, G. (2017). Changes in the microbiota cause genetically modified Anopheles to spread in a population. Science, 357(6358), 1396-1399. https://doi.org/10.1126/science.aak9691

Hammond, A., Galizi, R., Kyrou, K. et al. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34, 78–83 (2016). https://doi.org/10.1038/nbt.3439