ABOVE: The cotton bollworm (H. armigera) is one of the species discovered to have parasitoid killing factor genes.

tI’s a scene straight out of your worst nightmare: dozens of tiny, milky larvae wriggle out of their still-living caterpillar host, leaving behind the scarred, gaping holes from whence they emerged. The larvae belong to a type of wasp called parasitoids, whose young dine on the flesh of hosts their parents pick out for them.  

But research published Thursday (July 29) in Science suggests that not all caterpillars infected with parasitoid wasps will meet the same grisly end. The study identified a new family of proteins—parasitoid killing factors (PKFs)—that kill parasitoid larvae. PKF genes were found in several large double-stranded DNA viruses that infect lepidopteran insects (moths and butterflies), but also within the genomes of several Lepidoptera species themselves, suggesting that the genes have been swapped between viruses and infected hosts over the course of evolutionary history. 

“I thought it was very well done,” says Stockholm University evolutionary biologist Naomi Keehnen, who was not involved with the study. “It’s very, very nice research.”

Studies of host-pathogen interactions often reveal evidence of evolutionary arms races, as organisms hone their strategies for virulence and defense. Virologist Jean-Michel Drezen of the University of Tours in France says viruses usually only encode genes that are absolutely necessary for viral replication and that are involved in adaptation to the host. In this case, however, the viral PKF genes adapted to the virus’s potential parasitoid competitors. “This is quite new in virology,” he says. Drezen has collaborated with some of the paper’s authors in the past but was not involved in the current study. 

Insect virologist Madoka Nakai of Tokyo University of Agriculture and Technology and her team first identified the PKFs in northern armyworm (Mythimna separata) caterpillars—a lepidopteran species—infected with entomopoxvirus. The researchers saw that when the caterpillars were infected with the virus, they were protected from certain parasitoid species. This wasn’t the first time researchers had noted such a connection; in the 1970s, University of California, Davis, entomologist Harry Kaya noticed that virally-infected caterpillars were protected from parasitoids, but didn’t understand the mechanism of protection. 

To find that mechanism, Nakai’s group, working with researchers led by Salvador Herrero at the University of Valencia and Martin Erlandson of the University of Saskatchewan, exposed parasitoids to plasma from the virus-infected caterpillars that had been stripped of any virus particles. The wasp larvae died. By comparing the plasma proteins present in healthy versus virus-infected M. separata larvaethe researchers identified a 28-kDa protein—a PKF—that was only present in the infected insects. Using Edman sequencing—which identifies the sequence of amino acids in a peptide—they worked backward to match the amino acid sequence of the protein to sequences in the viral genomes. In addition to the PKF in the entomopoxvirus, they also found homologs in the genomes of ascoviruses and baculoviruses, which also infect insects. Some viruses had up to five PKFs in their genomes.

See Parasite’s Genes Persist in Host Genomes

While visiting Herrero’s lab in Spain, Nakai presented her group’s discovery of the viral PKFs. While discussing the data with her over coffee, Herrero says he remembers thinking he should do a quick scan through his moth genomic databases, just to see if there was anything similar to PKF genes directly encoded in the moth genome.

Sure enough, they identified PKF genes in the moths as well, and the phylogenetic analysis suggested that horizontal gene transfer of PKFs between the DNA viruses and the insects happened multiple times throughout the evolutionary history of Lepidoptera. 

Nakai says they don’t know yet whether the genes originally moved from virus to host or vice-versa, but the end result of the gene transfer is clear: “The common enemy is the parasitoid,” she says, “so they eliminate the common enemy.”

Previously, “it was expected that virally-infected larvae weren’t appropriate hosts for the development of parasitoids because of resource competition,” says Drezen, “But we did not expect that there was a specific mechanism to kill parasitoid wasps by the viruses, so I was quite surprised to read this paper.”

“It’s very clear that even though we thought that it was very basic, the immune system of insects is not as basic as we think,” says Keehnen. “And we still don’t really know exactly how it works—so I think finding a whole new gene family that’s involved with [antiparasitoid] responses shows that we really need to study more.” The researchers found that PKFs induce apoptosis in susceptible parasitoids. 

Moving forward, the group plans to look for PKFs in other lepidopterans. They are also using CRISPR-Cas9 to make PKF knockout Lepidopterans in order to more clearly understand the roles of each PKF gene, says Nakai. An additional, more conceptual question raised by their research is why ascoviruses, which carry PKFs, are transmitted to Lepidopteran hosts by the very parasitoid wasps they can kill. “It’s a little bit strange,” says Nakai. “Why would ascovirus kill its vector?” 

Further down the road, there may be agricultural applications for the discovery. According to Herrero, harnessing the power of “natural enemies” like parasitoids to manage agricultural pests has been common practice in both conventional and organic farming. Learning more about the PKFs may explain why some pests are resistant to parasitoid killing, and may help inform the design of better natural enemy solutions in the future. 

University of Georgia entomologist Michael Strand, who was not involved in the study, notes that the PKFs are able to kill their insect target, but not their insect host, and the study shows that the PKFs only target certain subfamilies of parasitoids, suggesting that the genes are extremely specific in what they can target. In the future, he says, people may be able to manipulate PKFs to “make them boutique killers” of certain pests. That kind of specificity could be leveraged to avoid using “broad-spectrum, ‘I kill everything I touch’” pesticide strategies. 

The study shows that “the insects that survived the virus had the opportunity to acquire that gene that gives protection against the parasitoid,” says Herrero—demonstrating that in this instance, at least, “what doesn’t kill you makes you stronger.”