Every time a mosquito bites a person, multiple feasts take place. As in most animals, a meal provides nutrients to the mosquito as well as to the troop of bacteria that inhabits its gut.
Occasionally, the mosquito may also ingest a parasite: a Plasmodium protozoan responsible for malaria in humans, or an arbovirus, which can cause dengue, chikungunya, Zika, or yellow fever.
The pathogen is just along for the ride, but those first hours after being ingested are crucial for its survival. The parasite develops in the insect’s midgut lumen, and any interference during this period may hamper its future survival and transmission.
When the mosquito ingests blood, the number of bacteria in its midgut increases dramatically. “That’s easy to understand because they use the nutrients of the blood to multiply,” noted Marcelo Jacobs-Lorena, a malaria researcher at the Johns Hopkins Bloomberg School of Public Health. “You have a few parasites surrounded by a huge number of bacteria.” These bacteria become promising targets for intervening in the development of parasites during their most vulnerable stages, either by competing for resources or directly attacking the threat.
In the ongoing battle against mosquito-borne diseases that kill more than a million people each year, scientists like Jacobs-Lorena increasingly turn to mosquito microbiota as allies in disease prevention (1). Delving into the guts and other bacteria-inhabited parts of mosquitoes is beginning to bear fruit. The performance of these bacteria in laboratory and semi-field experiments is promising. Releasing mosquitoes armed with a virus-fighting bacterium in cities affected by dengue significantly decreased its incidence in those locations. While these interventions don’t offer a silver bullet against mosquito-borne diseases, they appear to be a game-changer in the fight against those deadly parasites.
From engineered bacteria to natural killers
The ideal bacterium to fight a midgut parasite in mosquitoes must have several qualities. First, it must easily spread among populations without regular interventions, for instance, via transmission from mother to offspring. It must be cultivable in the lab and amenable to genetic modifications. It’s also critical that the bacterium is well adapted to the conditions of the mosquito’s gut, said Jacobs-Lorena. “For that reason, all the bacteria that have been successfully used for this purpose...were originally isolated from mosquitoes.”
Guido Favia, a molecular entomologist and parasitologist at the University of Camerino, found many of these properties in Asaia, a symbiotic bacteria genus that lives in the gut, salivary glands, and reproductive organs of Anopheles mosquitoes, which are malaria vectors. Its presence in the gut and salivary glands make it attractive for fighting Plasmodium since both sites are key for the parasite’s development and transmission. Asaia is also easy to cultivate and transform. “It was sort of a lucky combination,” Favia said.
When we are talking about mosquito control or insect vector control, we say that anything should be done in a view of an integrated program.
- Guido Favia, University of Camerino
Favia came across Asaia by chance. He and his colleagues were doing 16S rRNA gene amplification from Anopheles mosquitoes. Asaia consistently showed up in these analyses (2). Based on these findings, Favia and his team proposed Asaia as a candidate bacterium for paratransgenesis, which consists of genetically modifying a symbiotic organism to deliver effector molecules against a pathogen. Today, thanks to the efforts of Favia’s and other teams, there are various transgenic strains of Asaia that can block malaria transmission in Anopheles (3).
Jacobs-Lorena and his colleagues have also engineered mosquito gut bacteria to inhibit Plasmodium development in the animal’s midguts (4). They used a Serratia strain that they fortuitously found in the ovaries of Anopheles female mosquitoes. The recombinant strain persisted for multiple subsequent generations.
“We have a bacterium that not only can spread but can also kill the parasite,” said Jacobs-Lorena. “But then we faced another very big barrier. If we wanted to implement this strategy to contain malaria, we would have to release genetically modified organisms in nature.”
The regulatory and ethical barriers around the release of mosquitoes carrying genetically modified bacteria like Serratia and Asaia have thus prevented this approach from moving forward.
In recent years, though, scientists have come across strains that are natural killers of Plasmodium. Favia’s team, for example, found an Asaia strain that naturally activates Anopheles mosquitoes’ immune genes against the malaria parasite (5). A team led by Sibao Wang, a molecular biologist at the Shanghai Institute of Plant Physiology and Ecology, also encountered a Serratia strain that expresses a lipase gene with antimalarial activity (6). Jacobs-Lorena and Wang plan to test the potential of this strain as a malarial control method in a semi-field condition.
Fortuitously, a group of researchers at the biopharma company GSK in Madrid found another bacterium that naturally inhibits the malaria parasite. While working on next generation malaria medicines, they observed that one of their mosquito colonies lost susceptibility to Plasmodium without an obvious explanation. The team hypothesized that the insects’ symbiotic bacteria was involved in this resistance and isolated a few candidates. They contacted Jacobs-Lorena to delve further into the mystery.
Jacobs-Lorena’s team took up the challenge. They found that one of the candidates, a Delftia tsuruhatensis strain, secretes harmane, a small hydrophobic molecule that penetrates the mosquito’s cuticle and inhibits parasite development (7). The promising results led the teams to test the effects of Delftia in semi-field conditions at a unique containment facility in Burkina Faso known as the MosquitoSphere. “We are trying to mimic what is going on outside,” said Etienne Bilgo, a medical entomologist at the Institut de Recherche en Sciences de la Santé and coauthor of the Delftia study. The facility has compartments covered with mosquito-proof nets, but it’s otherwise open to the environment.
To test whether Delftia can populate the guts of mosquitoes caught in the field and inhibit the local circulating parasites, Bilgo and his colleagues conducted several experiments inside the MosquitoSphere. For instance, the team soaked cotton balls with a Delftia suspension in sugar and placed them in clay pots inside the chambers. “Then, we released mosquitoes and we let them feed on the cotton balls. The following morning, we captured them, and we brought them to the lab,” Bilgo explained.
After the overnight feeding, Delftia colonized 75 percent of the mosquitoes. Then, the team challenged the mosquitoes with Plasmodium obtained from blood samples of local patients with malaria. The Delftia strain successfully inhibited parasite development.
Bilgo, who has worked with different approaches in the fight against malaria, said that these are very interesting results. “First of all, Delftia is not a GMO bacteria, and it’s naturally occurring bacteria in mosquitos,” he said, adding that its performance in the lab and in these semi-field conditions is really promising. The biggest challenge, though, Bilgo acknowledged, will be to deploy this in the field in real-life conditions.
According to Jacobs-Lorena the next step is to identify possible sites for release. “The ideal site, in my mind,...would be an island...where there is malaria,” he said. “Another desirable feature would be, if there is malaria, to have a baseline to see how many cases per year occur before introducing the bacteria, and then you have a comparison to see how effective it is in stopping parasite transmission.” But, he acknowledged, these studies are not yet scheduled. “We are talking about the future.”
Wolbachia against dengue
Bilgo is excited about the results achieved by the World Mosquito Program when targeting the mosquito vectors of dengue and other viruses. Researchers at the program, a not-for-profit group of companies owned by Monash University, infect Aedes aegypti mosquitoes with the bacterium Wolbachia pipientis. The release of infected mosquitoes in a few cities around the globe in recent years is already impacting the incidence of arboviral diseases in those places. “This is giving a lot of hope for other biological control tools,” Bilgo said.
Until a few decades ago, W. pipientis was considered an insect symbiont of little clinical importance. It is an intracellular bacterium that occurs naturally in many species of mosquitoes, yet it is absent in A. aegypti (8). However, when researchers infected A. aegypti mosquitoes with a Wolbachia strain isolated from fruit flies, it primed the animals’ innate immune systems, stimulating the expression of immune effector genes and protecting the insects from viruses such as dengue and Chikungunya (9). Nonrandomized trials in field sites in Australia and Vietnam confirmed the potential of Wolbachia for dengue control (10).
Next, researchers planned the first controlled trial specifically designed to measure the public health outcomes of releasing mosquitoes carrying Wolbachia. In 2017, a team led by Katie Anders, a public health researcher at Monash University and director of impact assessment at the World Mosquito Program, conducted a randomized epidemiological study in Yogyakarta City in Indonesia. Anders and her colleagues divided the 26 km2 area — about a quarter the size of Disney World in Florida — into 24 adjacent clusters and released Wolbachia-infected mosquitoes in 12 of them, randomly chosen. The intervention resulted in a 77 percent reduction in dengue incidence and an 86 percent reduction in dengue hospitalizations in the clusters receiving the mosquito deployments (11).
Following these positive outcomes, Anders and her colleagues intervened in all 24 areas in Yogyakarta City at the request of the local community. “That was an agreement upfront that if there was evidence that this was effective in preventing dengue, then we would fill in the rest [of the clusters] because then it would be inequitable or unethical to leave the city untreated,” she said.
Anders further explained the importance of community engagement in every project conducted by the World Mosquito Program. In every site or city where the team starts a project envisioning the release of infected mosquito populations, they ensure a long enough lead-in time to engage with the local community to explain the methodology and its goals. Depending on the location, they do this by organizing public events or going door by door, Anders explained. The team only releases mosquitoes in those places where the public acceptance of the intervention is higher than 90 percent.
Today, the main challenge ahead for this program is how to scale it up in new locations. “Wolbachia [intervention is] most cost-effective as you deploy it over larger areas,” said Anders. “The cost per person is higher if you’re just deploying it over a small area because you have all this setup and infrastructure.”
“We’ve already deployed across cities of about one million people, but what about five million people in the real urban metropolis in Latin American countries or in the Philippines?” Anders said. The team aims to continue innovating to improve the method. They plan to design facilities that can produce millions of mosquitoes per week. In this realm, “Brazil is the first country really just to take that leap,” said Anders.
The World Mosquito Program has a joint venture with the René Rachou / Fiocruz Institute with funding from the Brazilian government. Luciano Moreira, a public health scientist at the René Rachou Institute, leads the project. He came back to Brazil in 2010 after completing a postdoctoral fellowship at Monash University, and started a local project using Wolbachia to fight mosquito-borne diseases. It started as a small project with a very small team, he said, but it slowly expanded and continues to do so. “We’re getting bigger and bigger,” he noted.
“The main bottleneck now is how to produce enough mosquitoes to be able to release in many cities at once,” Moreira explained. As part of their collaboration with the World Mosquito Program, they are planning to build a big, automated facility to produce from 50 to 100 million eggs per week. “We’ll be the biggest facility of Wolbachia in the world,” he said.
Wolbachia releases in Brazil and other countries promise not only to reduce the incidence of dengue but also that of other arboviruses. In the laboratory, Wolbachia limits various infections transmitted by A. egypti, including chikunguya, yellow fever, and Zika (9,12,13). “It’s harder to measure efficacy against those viruses in the field because they’re much more sporadic,” said Anders. Yet, a few studies point towards protection against some of these viruses in field conditions (14,15). This suggests that the effects of Wolbachia interventions are panviral or at least multiviral, said Anders.
Every effort matters
The promising results of using Wolbachia and other bacteria like Asaia, Serratia, and Delftia in combating mosquito-borne diseases are further supported by evidence of their safety. There is no evidence that any of these strains circulate in humans (16). Wolbachia-infected mosquitoes do not transfer the bacteria to the humans they bite or to their predators (17). Mosquitoes do not release Delftia while feeding (7). Furthermore, an independent risk assessment of the World Mosquito Program Wolbachia deployments revealed negligible risk (18). According to Jacobs-Lorena, researchers are also studying the potential threat of some of these bacteria if they are encountered by other insects that are not common hosts, such as bees. While these experiments are still ongoing, so far, there is no evidence of risk, he said.
Despite its success and safety, this novel approach will not replace the other existing methods of disease prevention and treatment. “You cannot choose; you have to use every tool,” said Jacobs-Lorena. “It’s not a silver bullet.”
“It’s also not an outbreak response,” added Anders. Public health researchers still need other tools to react to outbreaks in addition to proactive Wolbachia approaches. “It’s very effective,...but it complements the other interventions,” she concluded.
Correction: March 26, 2024: An earlier version of the story referred to Katie Anders as the director of the World Mosquito Program. She is actually the director of impact assessment at the World Mosquito Program. The text has been corrected.
Correction: April 1, 2024: An earlier version of the story indicated that the researchers at GSK were working on transmission-blocking vaccines, but they were actually working on next generation malaria medicines. The text has been updated.
References
- Caraballo, H. and King, K. Emergency department management of mosquito-borne illness: malaria, dengue, and West Nile virus. Emerg Med Pract 16, 1-23 (2014).
- Favia, G. et al. Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector. Proc Natl Acad Sci USA 59, 9047-51 (2007).
- Shane, J. L. et al. Blood meal-induced inhibition of vector-borne disease by transgenic microbiota. Nat Commun 9, 4127 (2018).
- Wang, S. et al. Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria. Science 357, 1399-1402 (2017).
- Cappelli, A. et al. Asaia Activates Immune Genes in Mosquito Eliciting an Anti-Plasmodium Response: Implications in Malaria Control. Front Genet 10, 836 (2019).
- Gao, H. et al. A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase. Nat Microbiol 6, 806-817 (2021).
- Huang, W. et al. Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes. Science 381, 533-540 (2023).
- Gloria-Soria, A. et al. Lack of Evidence for Natural Wolbachia Infections in Aedes aegypti (Diptera: Culicidae). J Med Entomol 55, 1354-1356 (2018).
- Moreira, L. et al. A Wolbachia Symbiont in Aedes aegypti Limits Infection with Dengue, Chikungunya, and Plasmodium. Cell 139, 1268-78 (2009).
- Nguyen, T. H. et al. Field evaluation of the establishment potential of wmelpop Wolbachia in Australia and Vietnam for dengue control. Parasit Vectors 8, 563 (2015).
- Utarini, A. et al. Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue. N Engl J Med 384, 2177-2186 (2021).
- Van den Hurk, A. F. et al. Impact of Wolbachia on Infection with Chikungunya and Yellow Fever Viruses in the Mosquito Vector Aedes aegypti. PLoS Negl Trop Dis 6, e1892 (2012).
- Aliota, M. T. et al. The wMel strain of Wolbachia Reduces Transmission of Zika virus by Aedes aegypti. Sci Rep 6, 28792 (2016).
- Ribeiro dos Santos, G. et al. Estimating the effect of the wMel release programme on the incidence of dengue and chikungunya in Rio de Janeiro, Brazil: a spatiotemporal modelling study. Lancet Infect Dis 22, 1587-1595 (2022).
- Moledo Gesto, J. S. et al. Reduced competence to arboviruses following the sustainable invasion of Wolbachia into native Aedes aegypti from Southeastern Brazil. Sci Rep 11, 10039 (2021).
- Epis, S. et al. Do mosquito-associated bacteria of the genus Asaia circulate in humans?Eur J Clin Microbiol Infect Dis 31, 1137-40 (2012).
- Popovici, J. et al. Assessing key safety concerns of a Wolbachia-based strategy to control dengue transmission by Aedes mosquitoes. Mem Inst Oswaldo Cruz 105, 957-64 (2010).
- Murphy, B. et al. Risk Analysis on the Australian release of Aedes aegypti (L.) (Diptera: Culicidae) containing Wolbachia. CSIRO (2010).
