SEES 2021 - Larvicidal “Trojan-Horse”: Experimentally Developing a Novel Low-Cost & Eco-Friendly Mosquito Vector Control Treatment - Mission Mosquito

Aseel, a SEES 2021 Earth System Explorer/Mosquito Mapper shares this research.

This summer, I am delighted to have a unique opportunity to dig deeper and learn more about mosquitoes, trends in larval habitats, and environmental satellite data. It's giving me a chance to expand my knowledge about mosquitoes. Last year, I researched and developed a low-cost and environmentally sustainable mosquito larvicide for use in large-scale vector control.

Because vaccines and specific treatments for most MBDs are not widely available, vector control, or the preemptive reduction of disease vector populations, is the primary and only universal means of MBD mitigation. Current methods focus heavily on synthetic larvicides, leading to widespread resistance among mosquito populations and increased non-target toxicity to ecosystems. Existing biological alternatives, such as Bacillus thuringiensis israelensis (Bti) larvicides and genetically modified mosquitoes, are cost-prohibitive for large-scale use in endemic regions, which are usually in developing nations.

In my research, I developed three biodegradable and inexpensive larvicides by loading different essential oils (EOs) into Saccharomyces cerevisiae cells, or baker’s yeast, using a simple procedure. S. cerevisiae was chosen due to the high mechanical strength of its cell wall, which stabilizes the volatile EO, as well as mosquito larvae’s preference for consuming yeast. This yeast microcarrier method, initially used in the food industry in the 1980s, has expanded to various fields such as pharmaceuticals, vitamins, and, more recently, termite treatments.

I tested the efficacy and toxicity of these larvicides in three different procedures: larvicidal bioassays utilizing Aedes aegypti larvae (compared to the efficacy of bioassays with EOs alone), ecotoxicity bioassays utilizing freshwater invertebrate Daphnia magna and microalga Raphidocelis subcapitata, and Toxorhynchites rutilus (Tox/Toxos) bioassays. Toxos are a species of non-hematophagous mosquitoes whose larvae consume large amounts of harmful vector mosquito larvae, making them an ideal biocontrol agent for Aedes aegypti. The negative control consisted of freeze-dried yeast and the positive control was a commercial Bti larvicide (Bacillus thuringiensis subspecies israelensis (Bti) bacteria is found in soil, and kills larvae but is safe for other organisms).

There were four main results:

1. EO-yeast microcapsules exhibited high larvicidal activity at significantly low concentrations compared to the essential oils alone. The LD50 values for cinnamon, garlic, and orange loaded yeasts were 23.7mg/L, 30.8mg/L, and 34.2mg/L, respectively. Additionally, the loaded yeasts significantly inhibited pupation in surviving larvae exposed to the treatments. In fact, 100% of cinnamon loaded-yeast larvae did not pupate at concentrations tested, indicating 100% efficacy starting at 20ppm!
2. 7 different categories of morphological/behavioral alterations were observed under a simple light microscope, including melanization, neuroexcitation, neural inhibition, midgut and tracheal damage, the expulsion of malpighian tubules, and cuticle blebbing. The likely cause of each alteration was investigated further by literature review. Three of these were previously unreported in the literature, suggesting the discovery of new target sites and physiological responses in this research. This exploitation of multiple target sites may help prevent the rapid selection of resistance.
3. All three treatments showed no significant lethal effects in ecotoxicity assessments on D. magna and R. subcapitata (confirmed with two-way ANOVA tests for each) suggesting the larvicides exhibit low non-target toxicity.
4. Finally, all three of the yeast larvicides were not toxic to Tx. rultius larvae, most likely because Tox larvae do not consume yeast (visibly confirmed with empty midguts). The positive control, Bti, killed all Tox larvae. This is particularly exciting because it is the first larvicide to show no lethal effects on Tox larvae, confirming the larvicide’s unique potential in widespread integrated vector control.

I am incredibly grateful to now be a part of the Mosquito Habitat Mappers team in the STEM Enhancement in Earth Systems (SEES) program. Within just the first three weeks, I have learned so much about many innovative research projects using AI, environmental satellite data, disease data, and more to accurately track and predict mosquito populations. I am excited for our individual projects, where we will be able to work with peers and mentors to further contribute to our understanding of mosquito habitats!

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Visual Aids:

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CZ (-LY or -O)= Cinnamomum zeylanicum (Loaded Yeast/ Oil) --- type of cinnamon essential oil

CS (-LY or -O)= Citrus sinensis (Loaded Yeast/Oil) --- type of sweet orange essential oil

AS (-LY or -O)= Allium sativum (Loaded Yeast/Oil) --- type of garlic essential oil

Treated larvae:

^ Cuticle blebbing, likely caused by interference with chitin synthesis (has not been confirmed in previous research). Blebbing occurs during cell death,  where the cytoskeleton breaks up and causes the membrane to bulge outward, sometimes separating from the cell.

^ This was a surviving larva, indicating Malpighian tube expulsion is an immune response to foreign insults. However, this has not been confirmed in previous studies.

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^ Expulsion of the peritrophic membrane, indicating midgut damage, likely leading to the hindrance of pupation. 

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Additional information:

This work was presented at the Austin Energy Regional Science Fest (AERSF), the Austin Geological Society (UT Austin) poster session, the Texas Science and Engineering Fair (TXSEF), and the International Science and Engineering Fair (ISEF).

Aseel is a  high school student in Austin, TX who is working on a research project this summer using the GLOBE Observer Mosquito Habitat Mapper. Her virtual internship is part of a collaboration between the Institute for Global Environmental Strategies (IGES) and the NASA  Texas Space Grant Consortium (TSGC) to extend the TSGC Summer Enhancement in Earth Science (SEES) internship for US high school (http://www.tsgc.utexas.edu/sees-internship/). She shares her experience this summer in this guest blog post.

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