Developing RNA Interference to Control Zebra Mussels

Science Center Objects

We aim to develop a control tool for eliminating zebra mussels that exploits natural gene regulation mechanisms (RNA-induced gene silencing; RNAi) to be specific to zebra mussels without non-target effects. In addition to the nuisance they pose, Pimentel et al. estimated that zebra mussels cost the U.S. economy over $1 billion annually. Zebra mussels are among the International Union for Conservation of Nature Global Invasive Species Database’s 100 world’s worst invasive alien species and are an emerging invasive species in Minnesota, found in over 300 waters to date. Furthermore, zebra mussels undergo a microscopic veliger stage where they are free-swimming throughout the water column for up to a month. This enables them to be easily spread into new waterways, making each currently infested lake a potential source for the next invasion.

There are a few chemicals available for controlling zebra mussels, but their use can affect the ecosystems when applied. One such chemical is EarthTecQZ. This is a copper-based compound, and it is also used to control algae as well as zebra mussels. At the recommended levels, it is not harmful to fish and other animals, and it has not been found to contaminate drinking water. It will kill other macroinvertebrates however, and when it is applied to areas with a lot of algae, that can result in dissolved oxygen crashes as the algae degrades which can kill many fish and other aquatic organisms not affected by the copper directly. Another chemical that is used for zebra mussel control is potash. Potash is not registered like EarthTecQZ is, but generally gets approved for use on waivers, because it is regarded as generally benign and commonly used for fertilizer. While potash does not seem to harm fish or other vertebrates, it is toxic to native mussels along with zebra mussels and it can persist in the system for a long time. Another available zebra mussel treatment is zequanox. Zequanox is an attenuated bacteria treatment, and is more selective for zebra mussels than either EarthTecQZ or potash. However, zequanox is mostly used for treating pipes rather than open water applications, because it is prohibitively expensive to treat a large area.

While these treatments have been tested well against fish and some other vertebrate species for lethality, sub-lethal effects and how they affect the microbiome are not as rigorously tested. As molecular tools are becoming more powerful and allowing broader-scale ecological studies, it is becoming apparent that changes to the microbiome can have significant and long-lasting impacts to an ecosystem’s resilience to disease and future invasions. The use of a genetic control allows us to specifically target only zebra mussels with no impact to non-target species.

There are a couple options that can be considered for genetic control. One strategy is to use a gene drive to propagate a negative trait through a population, such as female sterility. Using a CRISPR mechanism or a selfish genetic element to carry defective alleles to all offspring with super-Mendelian inheritance, a few genetically modified individuals could be released to mate with wild type animals and change the genetic makeup of a population so that it is no longer viable. Another strategy is to use RNA interference (RNAi) to selectively turn off critical genes of the target organism. This strategy takes advantage of the natural gene regulation mechanism of RNA-induced gene silencing. We target unique genetic sequences of zebra mussels that do not occur in other species for critical genes provide the instructions (silencing RNA) to turn off those genes.  Because the target sequences do not occur in other species, it makes the control tool very specific. Both strategies have advantages and disadvantages. Gene drives will require less application effort, because it involves releasing a few animals who do the work of spreading the control. Whereas, RNAi controls will require applying the control agent to all areas where the target organism occurs. RNAi control strategy gives managers better control over where and how it is applied though, whereas gene drives will spread wherever genetically modified individuals move to. This could result in accidental escape of a gene drive from a target population to another population of the same organism where locals may not wish for the same control to be applied (i.e. jumping back to the native range).

Methods for deliberate RNA-induced gene silencing were developed in the early 1990’s, and this molecular tool has since been widely used to discover complex gene functions that could not be studied before. Over 3,000 peer-reviewed journal articles have been published using RNAi since that discovery, and the researchers responsible for it were awarded the Nobel Prize in 2006. Only recently, has its potential for controlling harmful pests and invasive species begun to be explored. There are a number of trans-genic crops and sprays in development and being tested that use RNAi to confer resistance to agricultural pests such as corn root worm and striped flea beetle. This project could lead to the first application of this technology by natural resource managers to extirpate an invasive species in an area.

  • Zebra mussels have invaded over 300 waters in Minnesota so far
  • Zebra mussels are very easy to spread unknowingly by recreationists
  • Control tools for zebra mussels are limited by cost and ecosystem impact
  • Genetic control tools can target zebra mussels with greater specificity
  • RNAi microparticles will be cheap to manufacture and safe to deploy
  • RNAi microparticles will facilitate treatment in areas previously prohibited
  • Unlike CRISPR gene drives, there is no risk of this control escaping back to native populations

Objectives:

Design silencing RNA molecules for critical zebra mussel genes that will induce mortality.

Clone silencing RNAs into double-stranded RNA expressing bacteria that zebra mussels can feed on.

Evaluate gene expression knockdown efficiency.

Evaluate zebra mussel lethality.

 

Funding:

U.S. Geological Survey and Minnesota Environmental and Natural Resources Trust Fund