Mosquitos, ticks, worms—these all are "middle men" when it comes to disease spread. They can carry and transmit bacteria, viruses and other pathogens to us that cause dengue fever, Lyme disease and other vector-borne infections. As one approach to help reduce and even eliminate these diseases in people, researchers funded by the National Institutes of Health are studying the basic mechanisms that let the infection-causing organisms flourish inside their hosts.

A "Mind-Blowing" Bacterium

One such bacterium that scientists are interested in is called Wolbachia.

"It's probably the coolest bacterium in the world," says NIH's Irene Eckstrand.

Wolbachia infects more than a million species, including insects, shrimp, spiders, mite and tiny worms. It lives and reproduces in its host's cells, primarily reproductive ones. The bacterium can manipulate these cells in ways that boost its own survival and reproductive success.

Seth Bordenstein, a Vanderbilt University scientist who studies Wolbachia, says the bacterium alters the reproductive life of its insect and other hosts in four "mind-blowing ways":

1. It kills infected males.
2. It turns genetic males into females by shutting down certain hormones.
3. It allows infected females to reproduce asexually.
4. It promotes the survival of embryos from infected females only.

Ecology and Evolution

"Aside from stimulating the imagination, Wolbachia serves as a tool for studying the evolution and ecology of infectious diseases," notes Eckstrand, who co-manages an NIH-National Science Foundation program that's dedicated to this topic.

When Bordenstein, whose research is supported through the program, started studying Wolbachia about a decade ago, scientists thought that the bacterium and ones like it didn't frequently acquire new genetic machinery. The theory was that living inside other cells isolated the bacterial genomes, inhibiting the exchange of genetic elements with other bacteria that enables evolution.

Since that time, the theory—and Wolbachia itself—have evolved. Bordenstein discovered that Wolbachia can move to different cells and to new hosts, some of which are infected with other types of bacteria as well as different Wolbachia strains. This co-infection allows the bacterium to acquire new genetic elements.

Scientists also have learned that Wolbachia harbors a bacteria-infecting virus, called a bacteriophage, that can introduce other genetic elements. Bordenstein found that Wolbachia's interaction with the bacteriophage and its exchange of genetic elements through co-infections creates a cycle of evolution with implications for how diseases spread.

Disease Control

While Wolbachia doesn't directly infect mammals, it is the root cause of several serious mammalian diseases. It infects parasitic worms that, by way of mosquitos, can cause heartworm in our pets. Other types of Wolbachia-infected worms hitch rides to ultimately reach human hosts, where they can trigger severe inflammatory responses that lead to river blindness and elephantiasis. In an ironic twist, researchers are actually using Wolbachia to help fight these infections.

One strategy that Bordenstein is beginning to explore focuses on bacteriophage enzymes that can wipe out Wolbachia. Some organisms, including the parasitic worms, actually need the bacterium to reproduce. By eliminating the Wolbachia infection, the enzymes could render the worms sterile and unable to further spread disease.

Scientists also are harnessing Wolbachia's wanton ways to control the spread of dengue virus, which is transmitted to humans by mosquitoes. Researchers have discovered that Wolbachia-infected mosquitoes can't replicate the dengue virus. Releasing more Wolbachia into the mosquito population or finding and introducing the Wolbachia genes that interfere with replication are promising new avenues for reducing the spread of dengue. The approach could potentially apply to other vector-borne diseases like sleeping sickness, which is transmitted by the tsetse fly.

According to Bordenstein, studying Wolbachia has yielded some surprising new insights on microbial evolution that could help us understand, treat and prevent certain infectious diseases. "It's what gets me up every day and keeps me excited about doing this work," he says.

Wolbachia (wohl-bach-ee-uh) is a common type of bacteria found in insects. Approximately 6 in 10 of all types of insects, including butterflies, bees, and beetles, around the world have Wolbachia. Wolbachia bacteria cannot make people or animals (for example, fish, birds, pets) sick.

In the United States, the use of mosquitoes with Wolbachia is regulated by the U.S. Environmental Protection Agency (EPA). Prior to release of mosquitoes with Wolbachia into an area, EPA must grant an Experimental Use Permit (EUP).

Aedes aegypti mosquitoes spread viruses including dengue, Zika, and chikungunya. Aedes species of mosquitoes are common throughout many areas of the United States. EPA has registered mosquitoes with Wolbachia to evaluate how effective they are in reducing numbers of Ae. aegypti mosquitoes, not other types of mosquitoes.

Mosquitoes with Wolbachia are not genetically modified.

How mosquitoes with Wolbachia are used to control Ae. aegypti mosquitoes

• Wolbachia bacteria are not found in Ae. aegypti mosquitoes.
• Scientists introduced Wolbachia into Ae. aegypti mosquito eggs.
• When male Ae. aegypti mosquitoes with Wolbachia mate with wild female mosquitoes that do not have Wolbachia, the eggs will not hatch.
• Male mosquitoes with Wolbachia are released regularly into an area by mosquito control professionals.
• Male mosquitoes with Wolbachia mate with wild female mosquitoes.
• Because the eggs don’t hatch, the number of Ae. aegypti mosquitoes decreases.

Did You Know

• Only female mosquitoes bite. They need a blood meal to produce eggs.
• Male mosquitoes do not bite. They feed on nectar from flowers.

How scientists “make” mosquitoes with Wolbachia

• First, Wolbachia bacteria are introduced into male and female Ae. aegypti mosquito eggs.
• From these eggs, new mosquitoes with Wolbachia are bred.
• Mosquitoes are mass-produced in a factory.
• Male and female mosquitoes with Wolbachia are sorted. Only males are kept.
• Females are not released but may be used for breeding in the laboratory.

Effectiveness of mosquitoes with Wolbachia in reducing numbers of mosquitoes

• Communities in Texas and California that have released mosquitoes with Wolbachia report a significant drop in the number of Ae. aegypti mosquitoes. Mosquitoes with Wolbachia have been successfully used in Singapore, Thailand, Mexico, and Australia. Puerto Rico is releasing mosquitoes with Wolbachia in Ponce.
• When mosquitoes with Wolbachia stop being released into an area, the Ae. aegypti mosquito population will slowly return to “normal levels.”
• Mosquitoes with Wolbachia will only work to reduce numbers of target mosquito species, not other types of mosquitoes. Most communities have many types of mosquitoes.

Mosquitoes with Wolbachia and integrated mosquito management

Using mosquitoes with Wolbachia may be more effective if used along with other mosquito control methods as part of an integrated mosquito management (IMM) approach, including:

• Educating the community about how they can control mosquitoes in and around their homes
• Conducting mosquito surveillance (tracking and monitoring the number of mosquitoes, types of mosquitoes in an area)
• Removing standing water where mosquitoes lay eggs
• Using larvicides and insecticides to control mosquito larvae, pupae, and adult mosquitoes
• Monitoring how effective mosquito programs are at reducing numbers of mosquitoes.

Release of mosquitoes with Wolbachia is not intended to stop an outbreak. However, releasing mosquitoes with Wolbachia over several months can reduce the number of a specific mosquito species, such as Ae. aegypti. We do know that the best way to control mosquitoes is to start before an outbreak happens.

Mosquitoes with Wolbachia and the environment

• Wolbachia are very common bacteria found in insects throughout the world.
• Approximately 6 in 10 of all insects around the world have Wolbachia.
• Once an insect dies, the Wolbachia will also die.
• The type of Wolbachia used in these mosquitoes are same types of Wolbachia found throughout the world in many types of insects. Every day, insects with Wolbachia are around us. They die and decompose in the environment.
• There is no data to suggest that Wolbachia bacteria cause any harm to people or animals or the environment.
• The EPA determined that mosquitoes with Wolbachia are not likely to harm the environment.

Mosquitoes with Wolbachia do not harm animals

Wolbachia bacteria cannot make people or animals (for example, fish, birds, pets) sick.

Permits are required for release of mosquitoes with Wolbachia in a community

• Release of mosquitoes with Wolbachia requires an EUP from the EPA.
• Release of mosquitoes with Wolbachia requires approval from the EPA and local authorities.

Researchers established a bacterial infection in mosquitoes that helps fight the parasites that cause malaria. The infected insects could be a significant tool for malaria control.

Malaria is caused by a single-cell parasite called Plasmodium. The parasite infects female mosquitoes when they feed on the blood of an infected person. Once in the mosquito’s midgut, the parasites multiply and migrate to the salivary glands, ready to infect a new person when the mosquito next bites.

Malaria remains one of the most common infectious diseases in the world. It kills hundreds of thousands each year, mostly young children in sub-Saharan Africa. Treating bed nets and indoor walls with insecticides is the main prevention strategy in developing countries. However, the mosquitoes that transmit malaria are slowly becoming resistant to these chemicals, creating an urgent need for new approaches.

Wolbachia is a naturally occurring bacterium that was previously found to block development of Plasmodium parasites in mosquitoes. Wolbachia can be transmitted by an infected female insect to her offspring. Uninfected females that mate with infected males rarely produce viable eggs — a reproductive dead end that gives infected females a reproductive advantage and helps the bacteria spread quickly. Wolbachia were successfully used in a field trial to control dengue, another mosquito-borne disease. However, the bacteria don’t pass consistently from a mother to her offspring in Anopheles mosquitoes, which spread malaria.

A team led by Dr. Zhiyong Xi at Michigan State University set out to establish a stable, inherited Wolbachia infection that could block Plasmodium growth in Anopheles. They focused on Anopheles stephensi, the primary malaria carrier in the Middle East and South Asia. Their work was funded in part by NIH’s National Institute of Allergy and Infectious Diseases (NIAID). Results appeared on May 10, 2013, in Science.

The researchers injected a strain of Wolbachia derived from another type of mosquito into A. stephensi embryos. Once matured, the adult females mated with uninfected male mosquitoes to create a stable Wolbachia infection that persisted for 34 generations (the end of the study period). Uninfected females rarely produced viable eggs with infected males.

To see how well the infected mosquitoes could invade an uninfected A. stephensi population, the researchers tested groups of insects in the laboratory. When infected females comprised as little as 5% of the population, all the mosquitoes became infected with Wolbachia within 8 generations.

The researchers found that Wolbachia infection reduced the number of malaria parasites in both the mosquito midgut and salivary glands. They hypothesize that Wolbachia infection causes the formation of unstable compounds known as reactive oxygen species, which inhibit parasite development.

This study highlights the potential of using Wolbachia in malaria control. “Wolbachia-based malaria control strategy has been discussed for the last 2 decades,” Xi says. “Our work is the first to demonstrate Wolbachia can be stably established in a key malaria vector, the mosquito species Anopheles stephensi, which opens the door to use Wolbachia for malaria control.”

— by Harrison Wein, Ph.D.

What is the purpose of MosquitoMate’s technology and how does it differ from conventional mosquito control?

The purpose of MosquitoMate’s technology is to reduce local populations of Aedes aegypti and Aedes albopictus mosquitoes. These species are invasive mosquitoes in the United States, and can transmit diseases such as dengue, Zika, and chikungunya to humans; therefore, mosquito control is important for protecting human health. Additionally, the use of modified mosquitoes could reduce the use of chemical pesticides for mosquito control.

How does this technology result in mosquito population decline?

MosquitoMate had developed two mosquito products that rely on the same underlying technology. Wolbachia pipientis is a bacterium that is present naturally in about 60 percent of insect species, including many mosquito species. Naturally occurring strains of Wolbachia are used to infect captive male Aedes mosquitoes, which are then released into the wild. When a Wolbachia infected male mosquito mates with a wild female mosquito who carries a different strain of Wolbachia or who is not infected with Wolbachia, neither male nor female offspring survive due to a phenomenon called cytoplasmic incompatibility. With cont nued releases of infected males, the native mosquito population in the release area ultimately declines.

Where can MosquitoMate’s technology be used?

ZAP Males, used for suppression of Aedes albopictus, are registered for commercial use in the following states: California, Connecticut, Washington DC, Delaware, Illinois, Indiana, Kentucky, Massachusetts, Maine, Maryland, Missouri, New Hampshire, New Jersey, Nevada, New York, Ohio, Pennsylvania, Rhode Island, Tennessee, Vermont, and West Virginia.

Commercial use of WB1 Males, used for suppression of Aedes aegypti, is currently under review. The product was previously tested under an Experimental Use Permit (EUP), which expired at the end of 2021.

Will this technology adversely affect human health or the environment?

No. Like all pesticides, MosquitoMate’s technology is regulated under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). MosquitoMate's technology will only be used in male mosquitoes. The mosquito gender separation process used for Wolbachia male mosquitoes for release is highly efficient. Since male mosquitoes do not bite and do not feed on blood, they do not significantly contribute to the exposure of humans to the Wolbachia strains. The expected accidental release rate of one Wolbachia infected female for every 250,000 infected males is considered negligible exposure to humans resulting in a negligible human health risk. Therefore, when used according to the label, these mosquitoes meet FIFRA standards of not causing unreasonable adverse effects to humans or the environment.

Additionally, the Wolbachia bacterium is a ubiquitous microbe that humans and the environment have been exposed to. It is estimated to be present in about 60 percent of insects, some crustaceans, and nematodes.

What protection measures are in place as a part of MosquitoMate's FIFRA Section 3 registration?

In evaluating a pesticide registration application, EPA assesses a variety of studies to determine the likelihood of adverse effects (i.e., risk) to occur from exposures elicited by the use of the product before a registration decision is made. Risk assessments are developed to evaluate how the active ingredient might affect a range of non-target organisms, including humans and terrestrial and aquatic wildlife (plants and animals).

Based on the results of these assessments, EPA evaluates and approves language for each pesticide label to ensure the directions for use and safety measures are appropriate to mitigate any potential risk and ensure the efficacy of the product. In this way, the pesticide label communicates essential limitations and mitigations that are necessary for public and environmental safety. The mosquito specific protections for ZAP Males are as follows:

• Survivability of shipped ZAP Males must be recorded at the time of delivery and the day of product expiration;
• Bi-annual reports must be made to EPA which include any incidences where batches of ZAP Males exceed the established gender ratio or any decrease in ZAP Males fitness and viability;
• Any batches of ZAP Males that do not pass the standards of the approved quality control measures must be halted;
• Monthly monitoring for ZAP-infected females must be conducted within each of the geographic regions where ZAP Males are approved for use; and
• Any occurrence of ZAP-infected females in the environment must reported to EPA within 30 days of detection.