This project focuses on a novel method of biocontrol for common carp which will complement existing technologies by introducing a synthetic species-like barrier to reproduction. Researchers will use programmable transcription activators to drive lethal embryonic overexpression of endogenous genes in hybrid embryos.
Applications for this synthetic incompatibility could include population control of pest or invasive species or be utilized to prevent the spread of transgenes from genetically modified aquatic organisms to sexually reproducing wild populations. The method involves altering the genetics of males in the invasive species before releasing them among the population, leading to sterile offspring and the eventual control of the species overall. In order to make this method usable, this study aims to develop this technology further in zebrafish, from which the system can be applied to other invasive fish species and eventually other vertebrate pests. This control method has promise to be very species-specific, broadly applicable, and cost-effective.
Progress
As of late 2021, several rounds of carp spawning were performed in the MAISRC Containment Lab. Success of each successive attempt increased, with the first attempt resulting in poor spawning, the second resulting in premature, but successful spawning (the early morning spawning was unexpected and prevented an attempt of transgenesis), and the third attempt resulting in verified transgenic carp. While carp transgenesis was successful for this off-cycle spawning, the efficiency of transgenesis was hurt by repeated breakage of our borosilicate glass needles. There are several options for more durable needles. The next grade up in needle durability (aluminosilicate) can still be pulled on our needle puller and moving to quartz needles will require new a new laser needle puller. We plan to experiment with aluminosilicate needles to see if they fare better for injecting the tough carp eggs.
Our carp work is following in the footsteps of the first proof-of-concept work in a model fish species. In zebrafish, we reported the successful experimental confirmation of lethal over- or ectopic expression driven by dCas9-based PTAs in the past reporting period. We are currently at Generation 2 of the strain creation plan below. With all of our components validated in zebrafish, the last step is to go through this pipeline to make the full EGI genotype and test it with controlled matings.
We have created an agent-based simulation model for carp genetic biocontrol and used the model to ‘compete’ several different biocontrol approaches head-to-head. We completed a public opinion survey via two separate surveying efforts: (i) email to MAISRC listserv during Fall 2018 and (ii) at the Driven to Discovery building during the 2019 MN State Fair. In total, we collected over 1300 responses and are close to publishing a manuscript describing the results. As an example of what we learned, Minnesotans are more likely to support biocontrol than chemical control, but not as likely as physical methods (trapping, netting, etc). Of the biocontrol methods (predator release, pathogen release, genetic biocontrol), genetic biocontrol was favored. The perceptions about the relative efficacy of different methods more strongly correlated with comfort levels than did perceptions of risks.
Phase III
In Phase I of this project, researchers developed protocols for year-round carp spawning and carp transgenesis and created initial transgenic carp for the Engineered Genetic Incompatibility (EGI) biocontrol strategy. The success of that project positioned the group as a global leader in carp genetic biocontrol, which will be used to leverage the advance of EGI and several alternative genetic biocontrol methods forward in the next phase. In addition to this, researchers will attempt to develop time-saving strategies to bypass the long generation time in carp to hasten multi-generational engineering/breeding efforts. The team will accomplish these goals by leveraging the lab’s experience in genetic design and construction of engineered systems and our carp transgenesis pipeline to create gene-drive, female-lethal, daughterless, and EGI carp. Researchers will accomplish these goals by leveraging the lab’s experience in genetic design and construction of engineered systems and carp transgenesis pipeline to create gene-drive, female-lethal, daughterless, and EGI carp. The team will test implantation of immature primordial germ cells and embryonic stem cells into the gametes of sterile adult fish as a means to bypass the 12- to 18-month developmental time for juvenile carp. Lastly, researchers will perform a formal Technology Readiness Assessment with stakeholders within the newly-formed Genetic Control for Aquatic and Reef Pests (gCARP) international research consortium.
Developing genetic biocontrol technologies for carp and other aquatic invasive is a many-year process that will involve collaboration across many levels of government, academia, industry, and the watershed managers. No lab or field trials of biocontrol agents will occur during this Phase, but we have the potential to construct viable biocontrol agents by the end of Phase.
Phase II
The overall goal of this project is to demonstrate a novel genetic approach for controlling aquatic invasive species using invasive common carp as proof-of-concept. This project will:
- Develop state-of-the-art carp transgenesis capabilities in the laboratory so researchers have year-round access to young carp embryos
- Begin transitioning the genetic biocontrol strategy – a species-like barrier to reproduction which has already been tested in simple laboratory organisms – into carp
- Use computer modeling to predict the efficacy of the approach
- Engage the public to better understand the attitudes and opinions regarding the use of genetically engineered organisms as part of an integrated pest management plan
If this project is successful, it could lead to implementation of this technology in other aquatic invasive species, including Asian carp and zebra mussels.