Researchers are refining methods for applying gene-editing technology CRISPR/Cas9 in species of fruit flies, which could soon lead to useful advances in management of fruit fly pests like the sterile insect technique. In an effort to develop a reliable and detailed approach to genetically modify tephritid fruit flies, a research team led by Daniel Paulo, Ph.D., a researcher in the Department of Plant and Environmental Protection Sciences at the University of Hawaiʻi at Mānoa, knocked out the gene responsible for the transportation of red pigment in the eyes of the melon fly (Zeugodacus cucurbitae), left, and oriental fruit fly (Bactrocera dorsalis), right, which led to a generation of flies with white eyes in both species. The team chose this trait to test their approach because it leads to an easy-to-see change in the flies. (Photos by Daniel Paulo, Ph.D.)

By Carolyn Bernhardt

More than 4,000 known species of fruit flies in the family Tephritidae are found in the wild. And, of those, roughly 250 species destroy crops by laying their eggs inside fruits and vegetables. When the larvae hatch, they eat the crop, rendering it useless for the agricultural market. Scattered across countless agricultural contexts worldwide, these little buzzkills are one of the most destructive pests around.

For the most part, farmers manage tephritid fruit flies with insecticides—an approach with mixed results and many downsides. And, right now, a method called the sterile insect technique (SIT) is the most effective, species-specific, and environmentally friendly alternative to control pest species of the tephritid fruit flies found in nature. A study published in October 2022 in the Journal of Economic Entomology showcases advances in genetic-engineering techniques that could one day make SIT even more effective.

Sterile Insect Technique: Effective, But Not Always Easy

The SIT involves scientists raising the target fruit fly species in the lab, sterilizing the flies by exposing them to a certain amount of radiation while they are still pupae, and releasing large amounts of the newly sterile flies into nature. When the released, sterile flies mate with wild flies, they produce infertile eggs that never hatch and, over time, this reduces—and hopefully eliminates—the population of flies.

“But research shows that if you only release sterile males—instead of sterile males and females at the same time—you can more efficiently accomplish management,” says Daniel Paulo, PhD, a researcher in the Department of Plant and Environmental Protection Sciences at the University of Hawaiʻi at Mānoa. Male flies mate more often than females and don’t damage fruits in the field because they don’t lay eggs. Male-focused SIT programs also guarantee sterile males are using their energy to mate with wild-type females, not with females who were also sterilized.

So, scientists target male flies with sterilization efforts by using a classic genetic method called genetic sexing, which helps create sex-specific, visible physical characteristics or markers (called phenotypes) among fruit flies. These phenotypes make it possible for scientists to tell the two sexes apart more easily and parse the males and females before sterilizing. A colony of such flies is called genetic sexing strain (GSS).

A GSS requires a phenotype scientists can create by inducing a genetic change, called a mutation, that leads to a visible characteristic. Next, they link the normal, non-mutated visual marker to the male sex.

Just like in humans, the sex of a tephritid fruit fly is defined by sex chromosomes: XX for females and XY for males. Because only males have a Y chromosome, scientists can insert the original normal version of the marker there, resulting in a population where female flies display a mutant phenotype and males a normal one. In other words, the normal appearance of these flies is “rescued” only in the male sex. One example of genetic sexing scientists often use is making the female fruit flies white in the pupal stage rather than their normal brown color. This approach is made possible by the fact that the brown color pupae have has been linked to the male-specific Y chromosome.

The white pupae trait sometimes shows up in individual female fruit flies spontaneously, but exposing flies to certain levels of radiation in the lab can also cause the trait to emerge. “You’re lucky if you find a fly carrying a suitable genetic marker for developing a GSS,” Paulo says. “On the other hand, radiation mutagenesis is not target-specific, meaning that other genomic regions are likely affected [when you use this technique].” And that can damage the wrong genes, which likely reduces how well the male flies can mate in the wild or pass their trait on to offspring. As a result, it is not always certain that the desired trait will appear consistently enough throughout the females in a population for scientists to fully rely upon it to parse the sexes.

Using CRISPR/Cas9 genetic editing, a research team developed a stable mutant line of the melon fly (Zeugodacus cucurbitae) with white pupae. (Photo by Daniel Paulo, Ph.D.)

How Genetic Editing Could Improve the SIT Process

Paulo says the gene-editing technology CRISPR/Cas9 helps resolve this inconsistency, allowing researchers to develop new, more reliable GSSs by inserting specific genetic mutations directly into the gene of interest in these flies. And an efficient genome editing approach would allow scientists to recreate mutant phenotypes in just a few generations without the downsides of using radiation.

Using CRISPR/Cas9 to genetically tweak tephritid fruit flies is not entirely new. In fact, as Paulo and colleagues reviewed in their article, the CRISPR technology has been routinely used to study genetics of these flies since its first demonstration in the Mediterranean fruit fly (Ceratitis capitata)  back in 2017. But scientists all seem execute the approach differently. “If you look at the literature, everyone is using different protocols and methods,” Paulo says. “For instance, papers report different optimal concentrations of CRISPR components, even for the same species. For someone less experienced in the field starting their own experiments, that can be very overwhelming.”

This discrepancy motivated Paulo and a team of collaborators to describe a reliable and detailed approach as a framework for researchers who are interested in implementing CRISPR in their own model fruit fly. He and the team revisited outcomes from the last five years of CRISPR research, which includes their own, to build a streamlined, unified protocol.

A Proof of Concept, Illustrated in Two Fruit Fly Species

The team outlined its proposed protocol in a report published in October 2022 in the Journal of Economic Entomologyand demonstrated how effectively and efficiently it works. The researchers used it to disrupt the eye pigmentation gene in two major pest tephritid fruit fly species: the melon fly (Zeugodacus cucurbitae) and the oriental fruit fly (Bactrocera dorsalis). Nearly 67 percent of the surviving embryos on which the team tested the protocol developed as white-eyed flies in the very first generation born with CRISPR-manipulated DNA.

Using CRISPR/Cas9 genetic editing, a research team developed melon flies (Zeugodacus cucurbitae) with white eyes, a useful mutation for testing the gene-editing protocol because the classic genetic marker is easy to see. (Photo by Daniel Paulo, Ph.D.)

Paulo says the white pupae phenotype is still more suitable for sex separation, but the white eye trait was a useful mutation for testing their protocol because the classic genetic marker is easy to see, which helps scientists score how well their mutations are working. And he says having mutants in the first generation right after the microinjections to tweak their DNA is “always a good thing,” because it shows results right away without having to wait for the flies to reproduce. “And it means you can speed up the process of establishing a mutant colony,” he says.

The research team chose the melon fly as its model to demonstrate the efficiency of the protocol because scientists had not yet reported any successful CRISPR-based genome modifications for the species. In the study, the researchers also showed that the protocol can work on distantly related species by developing the same white-eyed mutant in the oriental fruit fly. They also used their protocol to recreate the white pupae phenotype in the melon fly, illustrating how the approach can help researchers isolate suitable visual phenotypes for the development of new GSSs in other tephritid species.

Both the CRISPR protocol that Paulo’s team tested and the radiation-based approach to creating white pupae work because they “knock out” the white pupae gene, a key gene related to the normal brown pigment in pupae. In both cases, scientists are disrupting the normal function of this gene to create a visible, abnormal (mutant) trait. So, the next challenge Paulo’s team faces is devising an equally efficient CRISPR-based “knock-in” method to link a non-mutated functional copy of the white pupae gene to the male-specific Y chromosome, which would rescue the normal brown pupal color only in males. That way, researchers can even more firmly link the trait to the male sex.

Three sets of melon fly (Zeugodacus cucurbitae) pupae illustrate the changes resulting from CRISPR/Cas9 genetic editing. In the top row, wild-type, unaltered melon fly pupae display a range of normal brown pupae coloration. In the middle row are white pupae mutants obtained in the first generation after using CRISPR to knock out an important gene related to the normal pigmentation in pupal stage of fruit flies. These individuals are called “mosaics,” because only some of their cells harbor loss-of-function mutation in the targeted gene. In the bottom row are white pupae mutants of the melon fly developed after breeding mosaic flies from the first generation. (Photo by Daniel Paulo, Ph.D.)

However, Paulo says, this process would require inserting a much larger piece of DNA into a difficult-to-reach region of the genome. But if researchers can establish an effective protocol for knocking in a trait in tephritid fruit flies, they could eventually create a process to use CRISPR to specifically knock in a trait that makes the flies sterile.

So, Paulo thinks of CRISPR as a key to the genome, and he’s excited to see where it takes him and his colleagues next. “With that key working,” he says, “you can open other doors and see what other interesting genes are doing and whether there are other genetic targets that could be useful to control these flies.”

Carolyn Bernhardt, M.A., is a freelance science writer and editor based in Portland, Oregon. Email: carolynbernhardt11@gmail.com.