Evolutionary biologists Hassan Salem and Aileen Berasategui wondered what to make of a white, waxy material that builds up on juvenile tortoise leaf beetles (Chelymorpha alternans). The most common hypothesis posited that it was some kind of secretion, similar to what scale insects produce, but “it was showing up where it really shouldn’t,” says Salem, of the Max Planck Institute for Biology Tübingen in Germany.
Curious, they and their colleagues decided to investigate. “It was so surprising when we put the white substance on a petri dish, and then it grew,” says the University of Tübingen’s Berasategui. That test, which they performed in 2020, revealed the substance was microbial, but they didn’t yet know what kind of microbe it was, or whether it affected the beetles.
A subsequent analysis by the team, published today (August 19) in Current Biology, reveals the substance to be the fungus Fusarium oxysporum and documents its complex symbiotic relationship with the beetle. During the beetles’ vulnerable pupal stage, the fungus somehow wards off insect predators. Then, when adult beetles emerge from their pupal cases as adults, they disperse the fungus to their mutual host plant species, such as sweet potato plants (Ipomoea batatas). An added intricacy in this multispecies dynamic is that the fungal symbiont causes wilting disease in the host plant.
“I think the study is really interesting in that it describes a symbiont that is beneficial for one host but pathogenic towards another host,” says Kim Hoang, evolutionary biologist at the University of Oxford who wasn’t involved in the research but who assisted one of the journal’s peer-reviewers in providing commentary on the paper.
Using scanning electron microscopy and genetic sequencing, the researchers first identified the culprit: the filamentous fungus, F. oxysporum, a well-known plant pathogen. The fungus is detectable on the beetle throughout its lifecycle, suggesting a lifelong symbiotic relationship, the authors write. But the researchers focused their analysis on the pupal stage because they observed a 1,000-fold increase in fungal growth shortly after pupation began. The team members hypothesized that the fungus may be essential in deterring predators such as ants over the beetles’ six-day pupation period, as they don’t spin protective cocoons like some other insects.
To test this, the researchers measured the survival rates of 98 beetle pupae under different conditions in their natural habitat—the understory of the Panamanian rainforest. Half were cleaned of the fungus and placed either in sealed cages that prevented predatory insects from entering or in exposed cages. Beetles that had not been cleaned of the fungus were also placed in one of the two cage types. All of the beetles in the sealed cages survived, but those in the exposed cages were not so lucky. Those with their protective fungus intact did ok, however, with 88 percent surviving over the four-day test period, compared with just 43 percent of those lacking the fungus.
“So the anti-predation or defensive function for the pupa was clear,” Salem says of the fungus. It’s not yet understood how the fungus specifically deters beetle predators, but surveying the fungus’s genome, the researchers found gene clusters responsible for producing metabolites known to have insecticidal properties.
Because of F. oxysporum’s known pathogenic properties against plants, the researchers then asked if this particular beetle-loving strain had maintained that destructive function, even while establishing its protective symbiosis with the beetle, Berasategui says. The results indicate that the fungus leads a double life, she says, one as the beetle’s defender and another as the host plant’s attacker—but it likely needs the beetle to hitch a ride.
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Berasategui and colleagues found that exposing sweet potato plants, native hosts for the tortoise beetle, to the fungus consistently induced wilting disease over a three-week period. Furthermore, when they confined 10 fungus-free plants with two beetles that had just emerged from their pupal cases, the beetles spread the pathogen to nearly 80 percent of the plants’ foliage over a four-week period.
It may seem counterproductive for a beetle to spread a toxin that can kill the host plant that the insect lives on throughout its lifecycle. But the researchers say the story may be more complex. One hypothesis is that the beetles may prefer plants weakened with fungus-induced wilt because the disease subdues the plant’s defenses against their herbivory. “Yes, it completes its entire lifecycle on the plants but can also easily move on to new plants after this one wilts,” Salem points out.
One result that initially surprised the researchers was how small the genome of this strain of F. oxysporum is compared to most others within this species group, says Salem. Other strains have massive genomes to help them deploy different strategies to colonize different host plants, he explains. “But then if you have a beetle that is actually vectoring you from one plant to the other, maybe you don’t need these accessory gene sets.”
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Hoang says that one of the next steps could be to further explore the specific workings behind the defense mechanisms. She commends the study authors’ methodology of combining genomic studies, bench work, and field experiments. “They used different approaches to tell a more complete story about the symbiosis,” Hoang says. “They not only focused on the host and the symbiont, they also looked at how their interactions impact other members of the ecological community,” she says. “I think that’s definitely something that more researchers should do.”
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