Tiny paper cups filled with water and floating mosquito larvae occupied space in Aseel Rawashdeh's bedroom for months as she monitored the juvenile insects for changes in their behavior and structure. The 17-year-old was running an experiment: Would her inexpensive, eco-friendly concoction of baker's yeast and essential oils kill the wiggling larvae?
The young scientist—now a senior at L.C. Anderson High School in Austin, Texas—had done the research needed to make an informed hypothesis. If all went according to plan, the Aedes mosquito species, known for their transmission of yellow fever, dengue and other viruses, would die after digesting her homemade larvicide, while beneficial mosquito species like Toxorhynchites rutilus, which feed on Aedes larvae and other insects, would be spared.
A larvicide that targets disease-spreading mosquitoes but not beneficial species is a delicate balance entomologists have spent decades trying to understand.
"There's not a single agent or single silver bullet that will eliminate them all. So that's why we investigate various tools to help us against them," explains Anita Schiller, director of the Harris County Precinct 4 Biological Control Initiative (HCP4-BCI) in Houston, Texas. Schiller and her team work on researching and developing multiple biocontrol agents that are effective against mosquitoes but won't disrupt the environment. "In the pragmatic world, you have to know your enemy before effectively reducing them or eliminating them. And that's where the details, the weeds, are in mosquito control," Schiller explains. "You really must know the biology of your target."
Worldwide, over one million people die from mosquito-borne diseases like West Nile, dengue, Zika, yellow fever, malaria, and lymphatic filariasis every year. By targeting mosquito larvae before they grow into pupae and then into flying adults, Rawashdeh wanted to prevent the transmission of mosquito-borne diseases at the root source.
After Rawashdeh learned about the socio-economic and political impacts mosquito-borne diseases have on marginalized groups around the globe through her debate team at school, she began an extensive literature review on the subject. "It's almost like a puzzle," she says. "I have an idea of how I want to contribute a solution to a current issue. But then it's a question of how do I get there? How do I get to that end goal?"
In her research, Rawashdeh came across a 2014 study published in PLOS One about how entomologists at Louisiana State University created a bait using yeast. The bait would kill cellulose-digesting protozoa in the termite gut, leading to the termite's death. "It was the first time the yeast capsule was used for insecticides, and I was pumped up, like what if I could apply this to something else? What if I could apply it to mosquito larvae?" Rawashdeh gushes while explaining her research. Since some mosquitoes in the wild feed on yeast already, hiding a larvicide like essential oils within the yeast, she thought, could target specific species.
Rawashdeh knew from her readings that essential oils could act as a larvicide. Adding the yeast encapsulation made the oils more effective than if they were added to water with mosquito larvae on their own.
"We know these essential oils will kill mosquitoes, but can we eliminate target impacts by just focusing on the mosquito and making them eat the essential oils? The vast majority of mosquitoes, especially those that are a public health concern, feed on yeasts and algae matter in the water as juveniles," Schiller says.
Rawashdeh's experiment was novel in her method of encapsulating the essential oils into yeast, so that disease-spreading Aedes mosquitoes would ingest it while other organisms in the environment would not.
A paper published in Plant Metabolism and Chemodiversity in 2021 found that using essential oils derived from Piper species of plants as a larvicide was effective at targeting Aedes mosquitoes that had become resistant to pyrethroids. Pyrethroids are a type of pesticide used to control adult mosquitoes. Essential oils, however, encapsulate many compounds, and it is unknown which compounds in oils are causing mortality in larvae.
"Essential oils absolutely can be toxic to insects," says Lindsay Baxter, an entomologist specializing in vector-borne diseases at Cornell University. "They are a direct extract from a plant with all sorts of active molecules that can protect the plant from fungus or bacteria, or predators like mammal predators. We just don't exactly know why."
As soon as Rawashdeh confirmed that a yeast encapsulation could work with essential oils, she began to reach out to universities across the country to obtain various mosquito eggs for use in her experiments. In the summer of 2020, she received eggs from the Rockefeller strain of Aedes aegypti from Michael A. Riehle's entomology Lab at the University of Arizona. After countless emails sent across the country, Rawashdeh found Schiller, one of a few researchers in the United States who works closely with Toxorhynchites rutilus, or the mosquito assassin. These mosquitoes are dubbed "mosquito assassins" because they voraciously prey on other container-breeding mosquito species during their juvenile stage. When mosquito assassins grow into flying adults, they exclusively feed on flowers and do not require a blood meal. Schiller has worked to release mosquito assassins to help control other container-breeding mosquitoes that bite and take a blood meal from mammals in Harris County.
With mosquito eggs on hand, Rawashdeh conducted bioassays—tests where organisms are exposed to various concentrations of toxicants—in her bedroom to see if the larvicide she prepared in lab spaces and with equipment she rented from the University of Texas-Austin and Texas A&M University would work to kill Aedes mosquitoes. One by one, Rawashdeh used a small pipette to move the larvae into paper cups.
To create the homemade larvicide, Rawashdeh mixed essential oils with yeast and water and placed the medium into a shaking incubator, letting it soak for 10 hours at 45 degrees Celsius. "I mainly tested the cheapest essential oils like peppermint, eucalyptus, orange and garlic. Those were just the most common ones and were reported to have some larvicidal activity in the literature," Rawashdeh says. While peppermint and eucalyptus were tested, Rawashdeh found in initial experiments that these oils did not work as well as the cinnamon, orange and garlic oils.
Rawashdeh tested the larvae in a set of controls—with essential oils alone, with the yeast alone, and with the bacterium Bacillus thuringiensis subspecies israelensis (Bti). Bti is another widely used larvicide. For her experiment, she sprinkled the powdery larvicide on the water of cups containing Aedes larvae.
The results were promising. After the larvae fed on the bait, the essential oils inside caused neurotoxicity and mid-gut and tracheal damage, among other morphological changes not yet recorded in literature in larvae belonging to the Aedes mosquito species. Rawashdeh noted that some larvae died while others failed to grow into pupae.
She also tested her larvicide on beneficial species like water fleas and mosquito assassins. When water fleas ate the larvicide, there were no morphological changes to their bellies or mortality, but their swim speed was slightly lethargic. She found that the larvicide did not kill mosquito assassins because larvae of the giant, metallic, sapphire-colored mosquitoes did not consume it at all.
The yeast and essential oil-based larvicide costs less than $10 per pound to produce and has a toxicity concentration of LD50, meaning it is effective at killing less than 50 percent or half of the mosquito larvae in a concentration of 50 milligrams per liter or less than 90 percent of mosquito larvae in a concentration of 90 milligrams per liter.
"I found out that this is the first-ever accessible larvicide not to affect Toxorhynchites rutilus,” says Rawashdeh. “And it's difficult not to affect that species because it is so anatomically similar to the disease-causing species. So just with the targeted effect of the yeast, I was able to avoid any effect on those whereas if I were to apply the essential oil directly to water, that would have caused some abrasive effect to the cuticle [of the larvae]."
Rawashdeh's innovation won sixth place in this year's Regeneron Science Talent Search, the country's most prestigious and oldest science and math competition for promising young scientists in their senior year. First place was awarded to Christine Ye, a senior at Eastlake High School in Sammamish, Washington, for her research on gravitational waves from collisions caused by neutron stars. Other award-winning projects included a short-range, distance sensing radar that can help the visually impaired and a computational tool used to reveal how ribosomes pause and move along a cell's mRNA transcript to produce proteins.
Molly Scheel, a vector biologist at the Indiana University School of Medicine, stresses that cost-effective, environmentally-safe insecticides that don't pose threats to ecosystems or humans are needed. "[Rawashdeh’s] attention to keeping costs down is something we should all adopt in our research programs," she says.
Essential oils may also be an answer to growing insecticide resistance. Larvicides made from natural substances like the bacterium Bti, for instance, have been used for three decades and cost about $1.50 per pound in the U.S. Yet, mosquito vector experts are seeing less than desirable results.
"Bti is a bacterium that disrupts the mosquito's gut, and we've seen some resistance to that," Baxter says. "And then there are other species of mosquitoes that don't seem to react to it at all. So that can even be species-specific and not even mosquito specific."
While Rawashdeh's larvicide still needs to undergo field studies and further testing on other mosquito species like Culex and Anopheles, experts think her project has potential.
"Her study outcomes can steer future entomologists towards what is best suited for further investigations," says Schiller. "Also, from the Biological Control Initiative’s standpoint, since Aseel's study demonstrated no harmful impacts on mosquito assassin larvae, we can look into utilizing both in a synergistic approach."
Last summer, Rawashdeh interned as a Mosquito Habitat Mapper with NASA's STEM Enhancement in Earth Science (SEES) program, using machine learning models to track mosquitoes and dengue fever outbreaks in Brazil. Now she has her sights set on possibly studying tropical diseases or mosquito-borne diseases, perhaps at MIT, Rice University or the University of Austin, Texas.
"It was great to see a young woman advancing mosquito control and such a thoughtful project with a thoughtful approach," says Scheel. "I think she'll find that many bright young women are coming up through the ranks in the vector control field, and I hope she'll be one of them someday."