To prevent the transmission of malaria, insecticides have been used to kill anopheline mosquitoes 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 mosquitoes 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, and is 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 (wild type) 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 GM mosquitoes with resistance to malaria, such as those with edits made to the IMD pathway as previously discussed, could be selected and given features that make them less susceptible to death by the fungal spores, which would eliminate wild mosquitoes but not transgenic ones, which would decrease the transmission of malaria but maintain populations of mosquitoes, which are a part of the food chain that predators rely on.

I had no idea that there were types of fungi that only harm mosquitoes, but it’s super interesting and could hopefully be implemented in the near future! The proposition of combining the use of the GM fungus with GM mosquitoes wasn’t mentioned in the original article this post is based on, but I think it would be a really great solution if done effectively.

Something that annoyed me while writing this post was that no tables were available for figure one, which I would have used to find precise values and compare experimental results. Instead, I had to just include the graph, because I couldn’t just estimate what the values appeared to be from the figure.

I’m thankful that I prepared a series of these article summaries over Spring break, as I’ve had a busy semester and I can’t imagine how I would have fit these in. Unfortunately, this is the last of the articles I’d prepared, and I hadn’t transcribed my notes until this weekend, which took an embarrassingly long time. I’m definitely not looking forward to both reading and writing about the remaining articles, but we’re at the home stretch! Only two more to go! Hopefully I’ll be able to manage everything even with finals looming ahead, because I need to turn it in (wish me good luck guys). Stay tuned to learn with me!

“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.