A collaboration between Yale and Vanderbilt University has shed light on how a common bacterium could bring about new methods of pest control and disease prevention.

Wolbachia has been known for decades to cause sterility in male insects, but its mechanism of action has eluded scientists. Papers by Yale and Vanderbilt researchers published March 1 in Nature Microbiology and Nature approached this issue through biochemistry, molecular biology and genetics, ultimately implicating two genes in causing cytoplasmic incompatibility.

“I think forever, now, these two papers will be seen as the foundational pioneering papers for the bacterial genes that cause [cytoplasmic incompatibility], a trait that has never been genetically dissected from the bacterial side before,” said Seth Bordenstein, corresponding author of the Vanderbilt study.

John Beckmann, co-first author of the Yale study and a co-author of the Vanderbilt study, said the Yale researchers tested what they believed to be a two-gene operon found in the bacterium with the hypothesis that Wolbachia infection functions as a toxin-antidote system, since infected female insects can produce normal offspring even with infected males that are otherwise considered sterile.

By inserting this operon first into yeast and then into fruit flies, the Yale researchers were able to show cell toxicity and the potential for rescue function. Using biochemical assays, they were also able to demonstrate that cytoplasmic incompatibility occurs through a deubiquitinase enzyme, or DUB, Beckmann said.

The Vanderbilt study identified two candidate genes through comparative genomics and transcriptomics by looking for genes that were conserved through multiple strains of Wolbachia. By cloning various combinations of these candidate genes into plasmids and inserting them into fruit flies, the researchers both observed the known effects of Wolbachia infection and provided evidence to support the Yale team’s findings.

“In an age where replication in science is actually quite rare, the fact that we’ve come largely to the same result is a strong endorsement of the science,” Bordenstein said.

However, Beckmann said the Yale team proposed a toxin-antidote mechanism for cytoplasmic incompatibility and subsequent rescue, a pathway which he said was lacking from the Vanderbilt paper.

Bordenstein said the Vanderbilt team has different opinions on how to characterize the genes both teams identified. He said he does not see evidence to call the two genes an operon or a toxic-antidote system, although more work is needed in the area.

Bordenstein said the two studies were “a really good testament to the collaborative nature of science.” The two groups had been in contact for the past year, sharing data, writing their papers on the same timeline, submitting to the same journals and applying for a joint patent.

The main flaw in the Yale study was the research team’s inability to show that a female fly possessing the two Wolbachia genes could produce a viable embryo when fertilized by an infected male, Yale study co-first author Judith Ronau said. The methodology they used could have just been “finicky,” she added.

Beckmann said these results do not counter the team’s hypothesis but rather leave questions to be addressed in future studies.

“In the future, those are the question that need to be addressed mechanistically: How does rescue happen and what are the genes regulating rescue?” Beckmann said.

Scott O’Neill, co-author of a commentary that ran alongside the two studies in Nature, called the study a step forward in understanding the mechanism of Wolbachia infection.

Still, he said the study had some limitations. For instance, the study does not directly test the teams’ hypotheses, since there is currently no way to genetically transform Wolbachia, O’Neill said in an email.

But according to Bordenstein, the aggregate findings about Wolbachia have been heralded as “holy grails in the field that have now been addressed.” And implications of the research stretch beyond purely scientific curiosity and into the realms of agriculture and epidemiology.

“The implications of the research are pretty tantalizing,” Ronau said. “It can be used as a means of preventing the spread of insect-borne diseases, or it could save billions of dollars of crop damage, potentially.”

Wolbachia has already been used to create sterile males as a means of insect population control, Beckmann said. He explained that male insects can be reared and infected with Wolbachia to create hordes of sterile males, which can then outcompete wild-type males for mates, producing sterile eggs that die upon fertilization.

“The reason that this system is so important is because when you engineer these sterile insects, that’s a species-specific mechanism,” he said, contrasting the system with pesticides that indiscriminately kill many species.

Additionally, Bordenstein said Wolbachia infection makes mosquitoes resistant to both Dengue and Zika viruses, meaning they would be unable to transmit the viruses to humans.

O’Neill, who is also the director of the Eliminate Dengue Program, said that his program is already releasing male mosquitos made sterile through Wolbachia infection into five countries to control Dengue, Zika and Chikungunya viruses. However, he said the papers are not immediately useful for his program.

Beckmann’s team is already conducting future research on the mechanism. Ronau said she is looking into the structure of the other, non-DUB protein the researchers identified as well as trying to obtain structural data of how the two proteins bind to one another.

Bordenstein said it is hard for him to say the teams overlooked something in their research, with the exception of not being able to knock out the two implicated genes in Wolbachia to show that knockout bacteria would not cause male sterility upon infection. However, technology is not yet advanced enough to accomplish this.

Wolbachia infects approximately two-thirds of insect species worldwide, according to a study in the Federation of European Microbiological Societies Microbiology Letters journal.