The Culprit of a Mysterious Superworm Epidemic Finally Identified

ABOVE: Using cryoelectron microscopy, researchers identified the pathogen responsible for an agricultural epidemic in farmed superworms. ©istock, sumnersgraphicsinc

In March 2022, Judit Pénzes, a virologist at Rutgers University, was contacted by a farmer in Utah who was worried about the wide-scale die-offs happening in his superworm populations. Superworms, the larvae of the Zophobas morio beetle, are a protein-rich food source for captive reptiles, birds, and amphibians, and in the face of a growing world population, a potential alternative protein source for humans.¹ They also chow down on polystyrene, offering an innovative solution to humanity’s increasingly dire plastic waste problem.² But for the last few years, these small but mighty larvae have been threatened by a deadly disease sweeping across the nation.

The frustrated farmer’s once-healthy superworms were exhibiting distressed, uncontrollable wiggling and blackening followed by stiffening, liquefaction, and death. He wasn’t the only one who had observed these bizarre deaths—for years, insect-rearing facilities throughout the United States had been struggling to determine the cause of this superworm apocalypse. “I went down the rabbit hole to try and figure out what's going on,” said Pénzes, who started browsing insect forums and social media posts on the topic dating back to 2019. People put forth humidity, temperature, and even a fungus in the superworms’ food as a potential culprit, but Pénzes had another agent in mind. “No one really thought of it being a virus, which was strange at the time because to me, who has seen several viral infections taking place in farmed insects, that was my absolute first guess,” she recalled.

Now, with the help of cryogenic electron microscopy (cryo-EM), Pénzes and her colleagues identified the cause of the ongoing agricultural pandemic: a parvovirus that they named the Z. morio black wasting virus (ZmBWV).³ In characterizing the virus, the researchers identified a prophylactic approach to protect superworm populations. They published their findings in Cell. 

“Apart from the cool detective story that it was, there was also lots of very exciting structural biology in there,” said Joost Snijder, a structural virologist at Utrecht University who was not involved in the study.

Insect-borne viruses that plague humans, like dengue, are the focus of much research, but over the years, Pénzes has established herself as an expert in the lesser-known viruses that primarily harm the insects themselves. Before joining virologist Jason Kaelber’s team at Rutgers University, she worked as a postdoctoral researcher in an insect diagnostic lab at the Armand-Frappier Health Biotechnology Research Center.  “When insect farms had problems with their insects dying from unexplainable reasons, then they would reach out to us and would send samples,” said Pénzes. “This farmer got my information, that I am the person to contact if you have an insect problem.”  She agreed to look at the farmer’s superworms. “A week later, we found about four pounds of very dead, oozing, stinky, superworm larvae in our mailroom,” said Pénzes.   

Although she had a hunch that a virus might be to blame for the mass die-offs, she didn’t know where to start. “Beetle viruses are really under-investigated,” said Pénzes. So, she ground up the dead worms and ran the superworm slurry through a sucrose cushion, a density gradient that separates the liquid contents. Piled up in the lowest rungs of the gradient were her first clues: evidence of viral capsids. 

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“A week later, these samples were already in our cryoelectron microscope, and that's when we first got the first glimpse of what this virus actually looks like and what kind of virus it may be,” said Pénzes.

Using cryo-EM, the researchers captured a near-atomic-resolution three-dimensional structure of ZmBWV’s capsid protein and its genome contents. After querying the Protein Data Bank for known proteins with similar structures, it became clear that they were looking at a virus belonging to the Densovirinae subfamily of the Parvoviridae. 

“To infer at the genus level what kind of pathogen you're looking at, this is very impressive to get this just from the EM maps,” said Snijder. 

Pénzes and her team took a somewhat unusual approach by using cryo-EM as the primary diagnostic tool; sequencing-based techniques are still the most common approach for detecting pathogens.⁴ Although cryo-EM infrastructure is harder to come by and poses technical constraints, Pénzes noted that EM can help scientists quickly identify a virus at the genus level and, unlike metagenomics, it is not reliant on reference databases that are restricted to known sequences. "It's really cool that you can get that far from the cryo-EM maps, and for an EM expert like Jason that was a logical first thing to do, though, I think in most virology labs, epidemiological labs and most public health institutions, next-generation sequencing will, for a very long time, still remain the go to tool," said Snijder.

To make sure they correctly identified the culprit behind the epidemic, the researchers collected additional samples from other superworm breeders and local pet stores. “Whenever I went to a pet store, I always opened up the superworm containers to see whether they had infected ones,” said Pénzes. The symptomatic superworm larvae from these sites tested positive on a diagnostic polymerase chain reaction (PCR) test that they developed for ZmBWV. Additionally, healthy larvae that they infected with the newly identified virus went on to develop the deadly disease. 

When Pénzes visited a superworm farm, she noticed that a handful of the breeder’s mealworms—a closely-related species of darkling beetle—had died in a manner that looked suspiciously similar to the virus-stricken superworms. A curious Pénzes returned to the laboratory with a sampling of asymptomatic mealworms and tested them for the virus. “To my great surprise, I found the virus at the very same yield as we find them in the blackened superworm carcasses,” said Pénzes. However, unlike the superworms, the mealworms experienced a much lower mortality rate, suggestive of species-specific susceptibilities to the same virus. Further experimentation revealed the presence of ZmBWV-like viruses, leaving Pénzes to wonder whether the mealworms also harbored non-pathogenic variants.

Pénzes isolated and purified the viral variants identified in mealworms and introduced them to healthy superworms. “The superworms didn't get sick from it either, so it was clear that we found the non-virulent variant,” she said. Furthermore, superworm larvae treated with a non-virulent strain were protected from subsequent exposure to the pathogenic variant, highlighting a potential prophylactic approach for preventing the development of ZmBWV-induced illness. 

Pénzes and her team are currently developing a vaccination strategy to deliver non-virulent strains of ZmBWV to beetle larvae. Like many human vaccines, their prophylactic approach wouldn’t necessarily prevent infection, but it would reduce mortality, improving not only the lives of the superworms, but the farmers, too.

Disclosure of Conflicts of Interest: Judit Pénzes and her colleagues have submitted a preliminary patent for developing non-virulent ZmBWV strains into a superworm vaccine.

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