Precision genetic methos are enabling more efficient, environmentally friendly pest control methods for agricultural use as well as stopping the spread of disease.
By Caroline Seydel
In June 2024, the St. Louis-based pest control company Agragene released genetically modified fruit flies on berry farms in California and Oregon, moving the technology out of the laboratory and into contained field testing. The trial marked a milestone for a next generation biocontrol technology called the precision-guided sterile insect technique, or pgSIT.
“The spotted wing drosophila (SWD) is the number 1 problem for any kind of strawberry, blueberry, raspberry, blackberry grower,” said Bryan Witherbee, president and CEO of Agragene. The flies have developed resistance to conventional chemical pesticides, and fruit growers suffer enormous economic losses due to the pest. “Growers are crying out for new tools,” Witherbee said.
Hope is on the horizon, not only for farmers battling SWD and other agricultural pests but also for public health agencies struggling to control disease vectors. Several companies, including Agragene, are bringing biological pest control into the CRISPR era with pgSIT and other molecular tools that can specifically target the pest without killing beneficial insects, polluting the water or blanketing communities with toxic airborne chemicals. San Diego–based Synvect is applying pgSIT to disease-causing mosquitoes. Meanwhile, Oxitec, which has already successfully commercialized its “Friendly” genetic modification platform in mosquitoes, is turning its attention to crop pests.
Bringing SIT into the genome era
Though the details differ, all of these companies are pursuing the same general strategy: applying genetic methods to a decades old tactic called the sterile insect technique or SIT.
SIT has been used since the 1950s to control agricultural and disease-causing pests. The traditional method is to rear the insects in the lab, separate them by sex, sterilize the males with radiation and then release the sterile males into the field to mate with wild females. These matings produce no offspring, reducing the pest’s numbers. Although SIT has achieved tremendous success in control ling some insects, it hasn’t caught on widely for mosquitoes, in part due to the laborious process of separating males from females. Also, irradiation can make the male insects less competitive in finding a mate. “When you irradiate the insect, you’re shooting it with x-rays all over—it’s not tissue specific,” said Stephanie Gamez, R&D director at Agragene. “That lowers the fitness of your insect.”
Agragene, a spinout from Omar Akbari’s lab at the University of California, San Diego, uses pgSIT to streamline both the sterilization and sex-separation processes. Lab researchers start by breeding two separate lines of fruit flies, one that carries a gene encoding the cutting enzyme Cas9 and the other carrying sequences for guide RNAs specific to two genes targeted for disruption. One of these targeted genes is necessary for female survival, the other for male fertility. When the two lines are mated, Cas9 forms complexes with the guide RNAs and disrupts the target genes, causing all the surviving eggs to develop into sterile males. This eliminates the need to painstakingly pick out and kill the female offspring and allows the eggs to be shipped to the field site before they hatch.
The females lay their eggs into a block of fly food, which is then shipped to the farm. Those eggs hatch and grow into larvae, then pupae, sustained by the nutrients in the box, until they finally metamorphose into sterile adult male flies. “All that happens within our shipping box for the most part,” Witherbee said. “It’s a simplified process and it’s less damaging on their health.” So far, Agragene’s studies have indicated that male SWD sterilized in this way retain much more reproductive fitness than flies sterilized by radiation. The company is now collecting data from controlled field trials to prepare an application to the appropriate regulatory agencies, including the US Department of Agriculture (USDA) and US Environmental Protection Agency (EPA), which will want to see that the flies don’t pose a risk to humans, crops or the environment. Although pgSIT uses genetically modified insects, it doesn’t involve the introduction of any new genes into the wild population—unlike earlier ‘gene drive’ methods that use selfish genetic elements inserted into genomes and passed on to progeny to impede disease transmission or reduce insect population numbers. “They’re not going to transfer genetic material, and they’re not going to proliferate themselves,” said Witherbee. “They live for around 20 days and then they die. And also, there’s no ability for female flies to be resistant to our sterile males.” They’re also susceptible to chemical pesticides, so growers who used the pgSIT flies would need to time their chemical pesticide spraying appropriately to reduce the amount they have to spray. “Right now, they’re spraying multiple times a season,” Gamez said. “The strategy would be to release the sterile males early, to get the population at a lower level, and then the grower would come in and spray later if needed."
Agragene box in a cherry orchard.
Fighting pests with “Friendly” fire
UK-based Oxitec pioneered the commercialization of genetically engineered insects, beginning with the Aedes aegypti mosquito, which spreads viruses including dengue, Zika and chikungunya. Now, the company is are moving on to other species, including major crop pests.
Oxitec’s Friendly system doesn’t use CRISPR to disrupt necessary genes, but rather relies on a tetracycline-suppressible lethal gene spliced into the genome of the target insect. “We introduce a self-limiting gene which produces a nontoxic protein called tTAV which, in itself, isn't harmful to the insect," explained Niel Morrison, chief strategy officer in Oxitec. “But we set up the genetic system in such a way that, in the absence of the chemical antidote, it forms a positive loop in females: tTAV expression is so high that it saturates the gene expression machinery of the cell, meaning other genes cannot produce enough of the proteins needed for natural function of the cell.” To rear the insects in the lab, researchers supply them with that antidote, tetracycline, but when the males are released into the field, their female offspring cannot survive. That’s a key difference between Oxitec’s platform and pgSIT: with pgSIT, the engineered males are sterile and produce no offspring, whereas Oxitec’s males produce offspring that cannot survive without tetracycline.
In May 2024, the Djibouti Friendly Mosquito Program released Oxitec's Friendly Anopheles gambiae mosquitoes in Djibouti City as part of a project to fight malaria. The program, created in 2022, is a partnership between Djibouti’s National Malaria Control Program, the public health not-for-profit Association Mutualis and Oxitec. These are the first genetically modified mosquitoes ever released in East Africa, and only the second such release on the entire continent. Before 2012, Djibouti had all but eliminated malaria, averaging fewer than 50 cases per year among a population of around 1 million people. Since the arrival of the malaria-transmitting mosquito Anopheles stephensi, in 2013, cases have soared to 73,000 in 2020. If the Friendly mosquitoes perform well in the pilot studies, other African countries could adopt the technology to help control the spread of malaria.
So far, the only Friendly insects available for sale are A. aegypti mosquitoes, which have been sold in Brazil since 2022. However, Oxitec is also testing the platform in various agricultural pests, including fall armyworm, which destroys crops such as corn; medfly, which targets fruit crops; and diamondback moth, which damages Brassica crops. “The genetics are largely applicable across virtu ally every important pest type we would want to target,” said Grey Frandsen, CEO of Oxitec. “The gene technology is now, in essence, perfected.” As of 2024, Friendly fall armyworm has been approved for commercial use in Brazil and Paraguay.
Automating the sex-sorting process
Synvect, another spinout from the Akbari lab, is applying pgSIT to disease-causing mosquitoes. Using the same principle that works for Agragene in fruit flies, Synvect creates boxes of mosquito eggs that will hatch into sterile males ready to mate with local females. “You can deploy it in your backyard,” explained Nikolay Kandul, cofounder and CEO of Synvect. “You just put that egg pod in water, close the container, and in two weeks only sterile males will emerge.” From the end user’s point of view, this is very similar to Oxitec’s Friendly product: a just-add-water box that releases a protective cloud of sterile mosquitoes into the local neighborhood. But, Kandul said, the Synvect’s method doesn’t reduce the fitness of the male mosquitoes as much as Oxitec’s method.
Also, the method is expected to scale well. “Every female we mate in the lab will give us hundreds of eggs,” Kandul says. “We can also desiccate the eggs and store them, and we can deliver them globally. We have global logistics that make it a very promising system.”
Synvect is working with the EPA to secure an experimental use-permit to begin field trials with its product, which it calls “next-generation SIT,” or ngSIT. The company has adapted the technology both in A. aegypti mosquitoes and in A. gambiae, the species that transmits malaria. ngSIT combines precision-guided SIT with a genetically encoded sex-sorting tool called SEPARATOR. “The presence of SEPARATOR enables sex-sorting and efficient genetic crossing of both strains for ngSIT egg production,” Kandul says.
SEPARATOR involves differentially expressed fluorescent proteins, allowing for easy visual identification of male mosquitoes. “Basically, it leverages sex-specific alternative splicing,” explained Akbari, who is cofounder of Synvect. It turns out that male and female mosquitoes have sex-specific introns, so by inserting a sex-specific intron into the gene encoding green fluorescent protein (GFP) and then adding it to the mosquito genome, it’s possible to create a line of insects in which only the males express GFP. “You can position it anywhere in the genome, and only the males will make GFP,” said Akbari. “The females do not produce visible GFP because they can’t splice out that intron properly.” Because the sex-specific intron splicing occurs early in development and relies on multiple protein pathways, the system is stable across generations. Rather than requiring males to be manually plucked out at the pupa stage, SEPARATOR allows the mosquito larvae to be sorted using a device called a COPAS machine (complex object parametric analyzer and sorter), similar to a flow cytometer. For mosquito control districts that are currently using traditional, radiation-based SIT, Akbari said that incorporating SEPARATOR into their mosquito lines could dramatically improve cost-efficiency for the production of sterile males. “A facility could produce twice as many males due to removal of females very easily in the production process, thus saving more resources for male rearing,” he says. SEPARATOR components are considered inert fluorescent markers and would not need to be regulated as a biopesticide, according to Synvect and confirmed by the EPA.
Tackling the most difficult species
Despite the variety of pests upon which these biocontrol methods are being used, there remain plenty of species that resist genetic modification as it’s currently performed. Jason Rasgon, an entomologist and Huck Chair of Disease Epidemiology and Biotechnology at Pennsylvania State University, is developing new methods that will open up some formerly intractable species to human intervention.
“It’s a general problem across organisms,” he said. “If you want to make heritable germline edits, you need to be able to edit the germline of the organism you’re interested in.” In animals, this means embryonic microinjection: using a needle to introduce new genetic mate rial into the cells of the developing embryo. The method’s success depends on several factors, Rasgon explained, such as the ability to collect eggs at the right stage of development and the ability to physically pierce them.
Rasgon developed a way to sidestep the embryonic microinjection step altogether, introducing genetic modifications to the egg while it is still inside the mother. “What we’ve done is identified small peptide ligands that will bind to receptors on the developing ovaries,” Rasgon explained. The researchers attached CRISPR components to these pep tide ligands that seek out the ovaries and then injected them into the female bug. “When you inject the mother, it’s like injecting all of her eggs simultaneously,” Rasgon said. “It’s very easy, in most cases, to inject the adult female as opposed to the embryos. For embryo injection, the setup is pretty expensive, and it takes time to learn how to use it. Whereas to inject the adults, we can train undergraduates how to do that in 15 minutes.”
Rasgon’s lab has shown that the method, called ReMOT Control (for Receptor-Mediated Ovary Transduction of Cargo), works in a variety of species, including mosquitoes, beetles, ticks and moths. His focus is on tool development rather than pest control applications, and he has shared the ReMOT Control system with labs around the world working on different arthropods. “The unofficial slogan of the project is ‘any species, any gene, any lab’,” Rasgon said. “We want anybody to be able to apply these tools to whatever their system of interest is.”
In April 2024, a team at the Federal University of Rio de Janeiro in Brazil, led by Helena Araujo, collaborated with Rasgon to apply the method to triatomine bugs. Nicknamed “kissing bugs,” triatomines are a threat to people because they feed on blood and can transmit a parasite called Trypanosoma cruzi that causes Chagas disease. Nobody has successfully genetically modified them before, because their eggs have a hard outer coating that makes them difficult to inject. Araujo’s team used ReMOT Control to change the bugs’ eye and body color, providing proof of concept that the method works and opening the door to future genetic biocontrol of triatomines and Chagas disease.
How are genetically modified insects regulated?
In 2020, Brazil’s national biosafety commission, CTNBio, approved Oxitec’s Friendly A. aegypti mosquitoes for full commercial release throughout the country, after more than a decade of field trials and pilot pro jects. The commission assessed the product’s safety to both the environment and human health. That same year, the EPA granted Oxitec an Experimental Use Permit (EUP) to release the mosquitoes in pilot studies in the United States.
After the EPA has approved a product for use, though, each state may conduct its own review process, and Oxitec’s reception in the United States has been mixed. The company performed field trials of Friendly A. aegypti mosquitoes in Florida over three consecutive seasons, from 2021 to 2023. In California, however, the mosquitoes have not received the go-ahead for release. The two states provide a study in contrasts about how genetically engineered insects could find a place among existing pest control strategies.
“It really all started when we had our first outbreak of dengue fever, in 2009–2010,” said Andrea Leal, executive director of the Florida Keys Mosquito Control District (FKMCD). The disease had not been seen in Florida since the 1930s, but that season, they logged nearly 100 cases.
The problem the FKMCD faced was that traditionally, their focus had been on control ling nuisance mosquitoes, which are susceptible to different control strategies than the A. aegypti mosquitoes that carry dengue virus. Aedes aegypti are resistant to most of the chemical pesticides that the FKMCD uses, Leal said, and the only sure way to kill them is with larvicide—a labor-intensive approach, as these mosquitoes can hide out underneath people’s homes and breed in a vessel as small as a flower pot collecting water in a backyard. When Leal heard about Oxitec’s technology, she reached out.
It took a decade to determine which federal agency would regulate the insects. Eventually, the EPA claimed responsibility, and in 2020 it granted approval to begin small-scale trials. In the meantime, the FKMCD had held public information meetings to involve the community in the decision. In a 2016 public referendum, 31 out of 33 FKMCD precincts voted to begin trials with genetically modified (GM) mosquitoes. Although anti-GM activists did launch a petition to stop the trial, Oxitec said they had plenty of volunteers to place the insect release boxes on their property. “We had local businesses, local politicians, elected officials, all come out in support,” Frandsen said. Three seasons of pilot studies have shown that the Friendly mosquitoes successfully reduced the A. aegypti population in south Florida. Now, the EUP has expired, so it will be up to the EPA to evaluate the data from the field trials and decide whether to approve the product for registration. If it is approved, Leal said, the FKMCD will consider incorporating Oxitec’s mosquitoes into their toolkit, if the price is right. “We have to do a strong cost benefit analysis,” she said.
In 2022, Oxitec applied to the California Department of Pesticide Regulation (CDPR) for a research authorization to conduct field trials and perform a rigorous review to look at California-specific conditions. A year later, the CDPR had still not issued a decision, and Oxitec withdrew the application. However, the lack of action does not necessarily indicate hostility toward genetic biocontrol methods but, rather, that genetically modified insects require a completely new regulatory pathway. California’s environmental standards exceed those of the federal government in some cases, and the agency must ensure that any new pesticide complies with state regulations. “Anytime we have new technologies coming in, we’re now having to ask slightly different questions,” explained Karen Morrison, chief deputy director of the CDPR. “We want to be able to provide the answer to, ‘What do we need to be able to evaluate that?’” In October 2023, the agency hosted a workshop entitled “Novel, Non-Chemical Technologies for Pest and Vector Management—Engineered and Sterile Insects and Related Technologies,” which included presentations by Oxitec, Agragene, Synvect and Verily (formerly Google Life Sciences), which is marketing a Wolbachia-based sterile insect approach. The CDPR will use the information to develop a framework for evaluating the safety of new biocontrol products. “We’re reviewing products on the merits of those products as they’re coming in,” Morrison said. “A lot of what we’re thinking about in developing this broader framework is how we can apply that same concept of reviewing products based on the information we have, in a more organized way, to the new technologies coming out.”
Caroline Seydel
Los Angeles, CA, USA.
Published online: 23 October 2024
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