Loading...

Mosquitoes are the major transmitting agents of various diseases, including malaria, yellow fever, and dengue fever. Such mosquito-borne diseases lead to global as well as local problems, leading to many deaths and negative economic impacts. Though mosquito control methods exist, the efficacy remains unsatisfactory. Many researchers worldwide have been therefore trying to use a more promising solution, namely the attractive targeted sugar baits (ATSBs) containing sugar and chemical toxins, to attract and kill mosquitos. However, limitations exist, disease-carrying female mosquitos mainly feed on blood instead of sugar, and therefore cannot be effectively attracted by ATSBs.

This year, AIS-China has made optimization on the traditional ATSBs. We've introduced (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a compound proven to enhance attractiveness of ATSBs to blood-feeding mosquitoes.

Additionally, we have integrated small hairpin RNAs (shRNAs) that are specifically designed to target and silence vital mosquito survival genes like 5-HTR1 and Rbfox1. Once digested, shRNAs can activate the RNA interference (RNAi) mechanism and lead to the death of mosquitoes.

The fusion of ATSBs with RNAi techniques has given rise to Moskilla, a novel mosquito control method that is not only eco-friendly and safe but also effective and specific to mosquito populations. Moskilla will provide a promising new solution for the prevention and control of vector borne diseases.

There are very few winners in the climate crisis, but scientists are pretty sure there's going to be at least one: Mosquitoes.

CNN

∙ Global Issue

In summer, along with the rising temperature, people's daily lives and sleep are troubled by active mosquitoes, and the demand for mosquito repellent products is also increasing. However, people rarely realize that mosquitoes are the deadliest animals in the world through transmitting diseases.

Nowadays, mosquitoes have highly affected the human race through transmitting diseases. According to researches, dengue fever, transmitted by mosquitoes, is prevalent all over the world: about 400 million people are infected and 21,000 deaths are caused by it annually (CDC, 2018).

Figure 2. Global distribution of Dengue Fever in 2018

However, diseases that mosquitoes transmit is not only dengue, but also malaria and many others. According to the statistics of the World Health Organization, more than 700,000 deaths are caused by mosquito-borne diseases annually. Taking all mosquito-borne diseases into account, mosquito-borne diseases contribute to 17% deaths among all infectious diseases . (WHO, 2020; Junhao W. et al., 2023)

Furthermore, mosquito-borne diseases cause significant economic damage, costing an estimated $12 billion annually. Lymphatic filariasis alone incurs $5.8 billion, with projected losses for 2021-2030 at $13.8 billion. Dengue, Rift Valley fever, and Zika virus each impose costs ranging from millions to billions, contributing to acute and long-term complications, thus affecting the gross domestic product (GDP) of a country. These statistics reflect the urgent need to combat mosquito threats. (Nagavalli C. et al, 2023)

Figure 3. Significant damages caused by mosquitoes

∙ Local Issue

China has always been heavily impacted by mosquito-borne diseases. China was once strongly affected by malaria, reporting over 24 million cases in the early 1970s (WHO, 2024). In 2010, the government launched the National Malaria Elimination Programme, effectively mitigated the problem. Starting from 2017, China reported zero malaria cases, making itself a malaria-free country recognized by the WHO.

However, while malaria is eliminated, dengue remains its significance in China. In 2023 alone, over 5,000 cases of Dengue Fever were reported in China (National Disease Control and Prevention Administration, 2023). In the post-COVID era, the transmission of various mosquito-borne diseases, especially Dengue Fever, is likely to rise in China, posing new challenges in disease control. Novel prevention and control methods need to be developed to improve the current situation.

Figure 4. Dengue prevalance in China 2005-2020

∙ Significance of mosquitoes control

Mosquito-borne diseases pose serious threats to health of the humanity. However, many mosquito-borne diseases are preventable. Through strategies eliminating or controlling the mosquito population and the contact between humans and mosquitos, the spread of these infectious diseases could be restricted. Therefore, the authorities are now endorsing mosquito control, encouraging people to actively kill mosquitoes. For instance, the Centers of Disease Control and Prevention (CDC) encourages individuals and community directors to kill mosquitoes, and provides guidance for mosquito control.

Figure 5. CDC's guidance for mosquito control

Several mosquito control methods are developed, including biological, chemical, and physical ones, using mosquito-feeding organisms, chemical insecticides, and mechanical devices, respectively.

However, these methods have concerning efficacy, and may potentially lead to ecological disturbances and negative health impacts. Attractive targeted sugar baits (ATSBs) emerge as a promising novel solution, incorporating the use of both chemical and physical means. It effectively reduces mosquito populations, including those resistant to insecticides, by using mosquitoes' sugar-feeding behavior against them.

However, since the attractants in ATSBs can rarely attract blood-feeding mosquitoes--the main culprit for mosquito-borne diseases, improvements are still needed. Currently, ATSBs are not widely used in the market.

You can learn more about current mosquito control methods from the interactive diagram below!

Certain mosquito-feeding organisms are exploited as a method for mosquito control. However, such a method may bring ecological disturbances and the effectiveness is limited as organisms can only work in fixed conditions.

Mosquitofish, Gambusia Affinins, is one of the most classic examples for the biological control of mosquitoes. A mature mosquitofish can consume about 100 larvae per day for two to three years under various temperatures and salinities (ABILITY Magazine, 2017). The ease in raising mosquitofish grants such a method a huge advantage. However, considering the aggressiveness of mosquitofish, the local aquatic systems may be disturbed once mosquitofish were introduced, which tend to kill local organisms (Brand et al., 2018). Also, the effectiveness of this method is limited to conditions where water is sufficient.

Several devices for mosquito control are invented for personal usage, including bug zappers and electric swatters. Nonetheless, such devices cannot effectively attract mosquitoes, and bear the risk of harming non-target organisms.

Physical mosquito control measures are widely applied. Bug zappers and electric mosquito swatters with light bulbs and electrified wire nets are the most common ones used in the public. With the ultraviolet (UV) light emitted by the lightbulbs within these devices, certain insects will be attracted and direct themselves toward the device. As they are approaching the light source, they will touch the power grid first and be killed by high voltage electricity. With the lack of chemicals and the ease of use of such devices, these are the most widely used mosquito control methods. Unfortunately, such a method is less than effective since mosquitoes are not sensitive to UV light and therefore can hardly be attracted by it. Instead, thousands of non-target insects are killed because of their positive phototaxis. (How Bug Zappers Work, 2001)

While chemical insecticides are also widely used for killing mosquitoes given the immediate effects and the ease of use, long-term exposure to these insecticides can bring irreversible impacts to both humans and other animals. Also, it may lead to insecticide-resistance in the mosquito population.

Chemical methods are also a popular mosquito control option. The main components of adulticides include organophosphate, sumithrin, permethrin etc. Though different insecticides may differ subtly, most of them target the receptors in insects’ nervous systems, whose misfunction will then lead to the death of the impacted insects (Uragayala et al., 2015). While immediate effects can be achieved by using insecticides, other beneficial insects may also be affected apart from mosquitoes. Also, some negative influences on both humans and other animals due to the use of insecticides have been reported. For instance, long-term exposure to insecticides can lead to defects in olfaction, and a higher risk of development of Parkinson’s disease (Mendoza et al., 2023). Besides, it may lead to insecticide-resistance in the mosquito population.

Raising and releasing genetically modified mosquitoes is a top-to-bottom mosquito control method. However, since such a method is still being investigated, the possible ecological and health consequences are still not fully discovered, thus making it currently inaccessible to the market.

Top-to-bottom methods are also used for mosquito control. For instance, genetically modified male mosquitos are raised in the laboratory released to mate with female mosquitoes, leading to the situation where only non-blood-feeding male offsprings can be produced (CDC, 2024). With only non-blooding-feeding male offsprings, diseases can hardly be transmitted as the main way of mosquito-borne disease transmission is through the exchange of blood. Compared with other techniques, this measure is targeted only on mosquitos, minimizing negative impacts on non-target organisms. Nonetheless, this measure is still being studied in the laboratory without being implemented. Therefore, considering the undiscovered possible ecological and health impacts, such method is still inaccessible for large-scale usage.

Attractive targeted sugar baits (ATSBs) incorporate the use of chemical and physical means, acting as a promising means of mosquito control considering its cost-effectiveness. It effectively reduces mosquito populations, including those resistant to insecticides, by using mosquitoes' sugar-feeding behavior against them.

Attractive targeted sugar baits (ATSBs) are introduced as a novel mosquito control method combining chemical and physical elements. Specifically, ATSBs are the mixture of sugar suspension containing poison (Njoroge et al., 2023). Since carbohydrates are a kind of essential nutrient for insects' development, mating, and reproduction, sugars in ATSB can attract mosquitoes (Fiorenzano et al., 2017). After the ingestion of sugar baits in the ATSBs, mosquitoes can be killed. With their cost-effectiveness and less ethical concerns, ATSBs can be more easily approved by the authorities and used by the public (Rochlin et al., 2022). Additionally, experiments under various settings all prove a remarkable success in reducing the mosquito population (Rochlin et al., 2022). Disappointingly, not all the blood-feeding mosquitoes are attracted by ATSBs, and mosquitoes will not die within a short time (Maia et al., 2018). ATSBs may take up to 10 days to reach a mortality rate close to 100%. Therefore, within this range of time, escaped mosquitoes can still transmit the diseases (Rochlin et al., 2022). To solve this problem, improvements on ATSBs need to be made.

∙ HMBPP attracts blood seeking mosquitoes

(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), known as an isoprenoid precursor, has been revealed also a phagostimulant for blood-feeding mosquitoes. It was first found that the parasite Plasmodium falciparum produces HMBPP during malaria infection to indirectly increase the release of carbon dioxide, aldehydes, and monoterpenes in red blood cells. It enhances attraction and feeding behaviors of mosquito vectors. (Emami et al., 2017) Based on its phagostimulatory effect, the addition of 10 uM HMBPP to plant-based solution in combination with chemical toxins has been shown to have potential as an environmentally friendly ATSB for mosquito control (Viktoria E. S. et al., 2021).

This inspired us to use HMBPP to attract those blood-feeding mosquitoes. However, HMBPP can not yet be chemically synthesized. Therefore, we have decided to choose E. coli as the chasis cell producing HMBPP, as it has the same natural MEP pathway as Plasmodium. Also, E. coli is a type strain which can be easily engineered to increase the yield of HMBPP. (Zhou et al., 2017)

Figure 6. Compared to sugar baits, blood-seeking mosquitoes prefer HMBPP

∙ RNAi yeast for mosquito control

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that operates through the sequence-specific degradation of target mRNA transcripts, mediated by small RNA molecules (sRNA). Given the increase of reported chemical insecticide resistance and the rising concern for negative effects of broad spectrum toxic mosquitocides on non-target organisms, there is an urgent need for the development of novel, environmentally-friendly mosquitoicide. To this end, RNAi approaches are pursued as a promising strategy for mosquito control.

Whereas, both application in ATSBs and large-scale production require the stability and efficient uptake by mosquitoes. In this regard, dried and inactivated yeast short hairpin RNA (shRNA) pellets has been identified as an optimal approach to deliver shRNA mosquitocide. This method has demonstrated the highest efficacy in inducing mortality across various mosquito species, while simultaneously achieving the silence of targeted genes. (Molly D. S., 2019)

For the purpose of gene silencing, genes that are pivotal to mosquito fecundity, behavioral patterns, survival, and vector competence are prioritized as targets. In our study, we have selected the 5-HTR1, Rbfox1, Shaker and Irx genes, which encode serotonin receptors, evolutionarily conserved RNA binding proteins, an evolutionarily conserved subunit of voltage-gated potassium channels, and Iroquois-class homeodomain-containing proteins respectively. Previous research indicated that silencing either gene leads to the death of mosquitoes, with negligible impacts on non-target organisms. (Keshava et al., 2021; Keshava et al., 2023; Corey et al., 2023; Keshave et al., 2021)

Figure 7. RNAi mechanism activated in the body of mosquitoes consuming targeted shRNAs

Now, let us introduce a novel solution, Moskilla, which is an optimized ATSB containing two main components: HMBPP addition sugar bait to attract mosquitoes and targeted shRNA to kill mosquitoes. Our objective is to develop an eco-friendly, safe, effective, and highly specific method for mosquito control. Moskilla is mainly composed of the following 2 components.

HMBPP addition sugar bait: Except traditional sugar bait, HMBPP producing E. coli is also added. The presence of HMBPP and sugar in Moskilla can optimize its attractiveness to both sugar- and blood-feeding mosquitoes. To reduce the risk of E. coli leakage, the engineered E. coli has been constructed to a nutrient-deficient strain, thereby limiting its survival outside our controlled application context.

shRNA mosquitocide: shRNA mosquitocide of Moskilla is encapsulated within dried, inactivated yeast cell pellets that express the targeted shRNA sequences. This formulation is designed to initiate the RNA interference (RNAi) mechanism upon ingestion, effectively silencing vital mosquito survival genes, such as 5-HTR1, Rbfox1, Shaker and Irx. Our shRNA sequences are rigorously designed to minimize off-target effects, thereby significantly reducing the toxicity to other non-targeted organisms compared to conventional chemical mosquitocides. This innovation renders Moskilla a more sustainable, safe, and highly targeted approach to mosquito control.

Figure 8. Composition of Moskilla