RNA interference (RNAi) for targeted control of invasive zebra mussels

Researchers are developing RNA interference (RNAi)-based biocontrol methods to specifically target and inhibit invasive zebra mussels, aiming to protect native mussel populations and mitigate the impact on military operations and freshwater ecosystems.

Zebra mussels (Dreissena polymorpha) are highly invasive in freshwater ecosystems and can encumber military operations by encrusting underwater infrastructure and clogging pipes. They also spread easily among water bodies and therefore require enhanced biosecurity actions that take time, are expensive, and may slow military operations. Additionally, zebra mussels impact the health, reproduction, and recruitment of native mussels by attaching to their shells. This detrimental activity is causing severe declines in already threatened native mussel populations, many of which are likely managed for on U.S. Department of Defense (DOD) lands. 

Eradication efforts using copper sulfate or other chemical methods are often unsuccessful, and chemical controls generally lack specificity and impact many other species. To address the need for effective and specific inhibition of zebra mussels, MAISRC researchers are working to develop RNA interference (RNAi)-based biocontrol. This project aims to establish a proof-of-concept that RNAi techniques can alter expression of essential genes in invasive zebra mussels to ultimately control zebra mussels with high specificity. The work will establish primary cell culture models for zebra mussels, identify target gene sequences and microbial delivery vectors that are best suited for zebra mussel biocontrol, establish quantitative molecular and phenotypic assays for testing RNAi efficacy in isolated cells and in live mussels, define dosing and vector production requirements, and screen for off-target effects on native freshwater mussel species and other invertebrates. Apart from the benefits of specific biocontrol of zebra mussels, RNAi technology has the potential to become an effective, species-specific tool to manage invasive species more broadly.

Project manager: Daryl Gohl

Funded by: U.S. Department of Defense’s Strategic Environmental Research and Development Program (SERDP)

Project timeline: Mar. 2024 - Feb. 2028

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Phase I

Project manager: Daryl Gohl

Funded by: Environment and Natural Resources Trust Fund as recommended by the Legislative-Citizen Commission on Minnesota Resources

Project timeline: Jan. 2021 - June 2023

This project aimed to test whether RNA interference (RNAi) can be used to manipulate the expression of genes in zebra mussels through the delivery of double-stranded interfering RNA (dsRNA) produced by bacteria which are fed to zebra mussels.

The major objectives of this project were to:
1) Produce bacterial strains expressing dsRNA targeting zebra mussel genes involved in critical processes and verify the successful production of dsRNA.
2) Establish phenotypic assays and carry out RNA interference screens for genes affecting zebra mussel feeding success, survival, reproductive function, shell growth, and byssal thread attachment.

Results:
Due to the difficulty of cultivating zebra mussels in captivity throughout their entire life cycle, genetic biocontrol methods that rely on producing heritable genetic changes in zebra mussels (such as gene drives) are currently not viable approaches. MAISRC researchers began this project to test an approach for manipulating zebra mussel gene expression with RNAi. They established a number of phenotypic assays to investigate the effects of dsRNA feeding. The researchers constructed several dozen bacterial strains that expressed dsRNA targeting zebra mussel genes and tested them in a repetitive reattachment assay. 

No reproducible effects on zebra mussel reattachment were observed in these experiments, suggesting that either the nature of the RNAi trigger (dsRNA versus small hairpin RNA) or the delivery mechanism (feeding bacteria) was not able to produce an attachment defect in zebra mussels. Future work will focus on testing additional RNAi triggers, additional delivery mechanisms (transfection into tissue culture cells, injection, or algal delivery), additional phenotypic tests, and more extensive molecular testing of transcript knock-down. The preliminary data from this project and the phenotypic assays established by the researchers have led to additional funding from the U.S. Department of Defense’s Strategic Environmental Research and Development Program (SERDP). This funding will enable continued progress on efforts to develop genetic biocontrol tools for this damaging invasive species.