Moran and her team raise thousands of bees for their research in rooftop hives on The University of Texas at Austin campus. [Marsha Miller/University of Texas at Austin.]

Scientists at The University of Texas at Austin have harnessed specially engineered symbiotic honey bee gut bacteria to protect the insects from Varroa mites and deformed wing virus, two key honey bee pathogens that represent major causes of colony collapse. The engineered bacteria live as part of the the honeybee gut microbiome, and effectively act as biological factories that produce double stranded RNA (dsRNA) to alter gene expression and trigger immune responses. The researchers believe their approach could feasibly be scaled up for agricultural use, because the engineered bacteria are easy to grow, inoculating the bees is straightforward, and the engineered bacteria are unlikely to spread beyond bees.

“This is the first time anyone has improved the health of bees by genetically engineering their microbiome,” commented Sean Leonard, a graduate student and first author of the team’s study, which is published in Science. “It has direct implications for bee health,” added Nancy Moran, a professor of integrative biology and the primary investigator on the study, which is titled, “Engineered symbionts activate honey bee immunity and limit pathogens.”

Honey bees (Apis mellifera) are essential crop pollinators worldwide, but are threatened by colony losses linked to the spread of parasites and pathogens, the authors explained. Figures suggest that honey bees contribute nearly $20 billion each year to the value of U.S. crop production, and they play an huge role in global food production. Without honey bees, dozens of crops, from almonds to berries to broccoli, would either vanish or produce significantly less food.

Worryingly, an increasing number of honey bee colonies in the U.S. have seen a serious dwindling of the adult bees, and the results of one national survey suggested that beekeepers lost nearly 40% of their honey bee colonies last winter, the highest rate reported since the survey began 13 years ago.

Two of the major honey bee pathogens are Varroa mites and deformed wing virus, which often come together. As the mites feed on the bees they can spread the virus, but they also weaken the bees and make them more vulnerable to other pathogens in the environment. “Recently, high honey bee colony mortality, attributed largely to synergistic interactions between parasitic mites (Varroa destructor) and RNA viruses, has become a critical problem for agriculture and the maintenance of natural biodiversity,” the scientists wrote. Yet despite the global importance of honey bees, studying their biology is difficult because of their unusual social structure and reproductive biology. New genetic tools and methods for tackling pathogens are vital to help protect honey bees, the team noted.

A Varroa mite, a common pest that can weaken bees and make them more susceptible to pathogens, feeds on a honey bee. [Alex Wild/University of Texas at Austin]
One potential strategy could harness the insects’ own immune system. Like humans, honey bees’ gut harbors an ecosystem of bacteria, or microbiome. Like humans, honey bees can also mount an antiviral defense mechanism called RNA interference (RNAi), which helps the body fight off RNA virus pathogens. Invading RNA viruses produce dsRNAs that the bee cells detect, triggering an RNAi immune response. “Honey bees possess the molecular machinery for RNA interference (RNAi), a eurkaryotic antiviral immune system in which double-stranded RNA (dsRNA) triggers degradation of other RNAs with similar sequences,” the authors stated. “You usually only get signs of these molecules when an RNA virus is replicating,” Moran said. “It’s a signal that this might be an evil thing and you should attack it.”

RNAi responses can be induced by either injecting or feeding dsRNA to the bees, and this approach has been used to inhibit bee genes and to block replication of RNA viruses, including deformed wing virus, the researchers continued. dsDNA given to the bees can also be transmitted to their parasites, and induce a parasite RNAi response. This approach has been used against the Varroa mite, by using dsDNAs that block key parasite genes.

However, there are a number of drawbacks to the general approach of administering dsRNAs and, as the investigators wrote, “… use of dsRNA for sustained manipulation of been gene expression or control of bee pests has proven difficult.”

As an alternative approach, the team engineered strains of the symbiotic bee gut bacterium, Snodgrassela alvi, to carry plasmids that would continually produce dsRNA with specific sequences that would could change specified honey bee gene expression, protect them against the deformed wing virus, and result in death of Varroa mites. To promote a helpful RNAi response to viruses in bees— and trigger a lethal RNAi response in the mites—the team introduced modified bacteria to hundreds of bees in a laboratory setting. Sprayed with a sugar water solution containing the bacteria, the bees groomed one another and ingested the solution.

The investigators first confirmed that the engineered S. alvi could churn out dsRNA during colonization of the bees’ microbiome, and also demonstrated that constant production of dsRNA would trigger the activation of bee immune pathway genes. Another set of tests demonstrated that bacteria producing dsRNA with specific sequences that could lead to the inhibition of targeted host genes.

The scientists then evaluated their approach in bees given either oral innoculations or injections of S. alvi engineered to produce dsRNA that targeted part of the deformed wing virus genome. The results showed that compared with control bees, virus-infected bees treated with the strain of bacteria targeting the virus were 36.5% more likely to survive to day 10.

Compared with control bees, the bees treated with the strain of bacteria targeting the deformed wing virus were 36.5% more likely to survive to day 10. Meanwhile, Varroa mites feeding on another set of bees treated with the mite-targeting strain of bacteria were about 70% more likely to die by day 10 than mites feeding on control bees. [University of Texas at Austin.]
In a final set of tests the scientsits evaluated whether symbiont-produced dsRNA could protect bees against the Varroa mites. To do this they exploited the fact that when the Varroa parasitize honey bees, the mites ingest dsRNA in the fat tissue on which they feed, and this triggers their own RNAi response. In order to turn this against the mites, the team engineered S. alvi to produce dsDNA (pDS-VAR) that would target essential mite genes. Their experiments showed that Varroa mites feeding on bees treated with the mite-targeting strain of bacteria were about 70% more likely to die by day 10 than mites feeding on control bees. “Mites that fed on bees colonized with pDS-VAR bacteria died more quickly than mites fed on control bees,” the team stated.

The reported experiments were carried out under strict biocontainment protocols required for genetic engineering, Moran said. However, even in the absence of such protocols, the risk of the engineered bacteria escaping into the wild and infecting other insects should be very low. The bee symbiotic gut bacteria used are highly specialized to live in the bee gut. They also don’t survive for long in the environment, and are protective against a virus that only infects bees. Nevertheless, further research will be needed to determine the effectiveness and safety of the treatments in agricultural settings. More tests will also be needed to determine whether engineered symbiotic bacteria can improve whole hive health. “Ongoing within-hive transmission could increase the effectiveness of this treatment by promoting the persistence and spread of engineered strains to new bees,” the scientists suggested.

Researchers could also use the technology as a tool for studying bee genetics. The engineered bacteria can knock down specific bee genes, giving scientists new insights into the bee genome function, and possibly enabling new breeding strategies to produce more robust bee colonies. “Symbiont-mediated RNAi provides a new tool to study bee biology and to improve resilience against current and future challenges to honey bee health,” the authors stated.

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