Don't move a mussel: Environmental DNA (eDNA) analysis for detecting aquatic invasive species

Angela Strecker, Portland State University & Western Washington University
Crysta Gantz, Portland State University
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Summary

Aquatic plants and animals are often difficult to find or catch, especially if they are not abundant. When an invasive species first arrives in an area, it is usually in low numbers. If an invasive species is found early, before it becomes a problem, there is a much greater chance of preventing its impact and spread. The usual methods that we use to find or catch aquatic animals are sometimes not as effective in the very first stages of invasion, though. This is where a new technique called environmental DNA or "eDNA" can help. Organisms continually shed DNA into the environment and this so-called eDNA is found throughout the water. This lab engages students in collecting eDNA and matching it to a specific species.

With a few sampling materials and some simple steps, the students can search for an invasive species using eDNA, consisting of a field sampling component and a laboratory analysis component. In the first part of the lab, the students will filter water from a water body of interest using a filter funnel attached to a flask. A pump creates a vacuum that draws water through the filtration apparatus, and then cells containing DNA stick to the filter. In the second lab module, the students will learn how to extract DNA from the cells (e.g., DNA extraction) and look at polymerase chain reaction (PCR) results.

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Learning Goals

This module teaches students how to use environmental DNA to detect rare or invasive species in aquatic habitats, specifically to find invasive zebra and quagga mussels. This is highly relevant in the Pacific Northwest, as we are one of the few places in the US that are still free of these harmful species. If these species were to invade, it would have significant consequences for the local economy, industries, and recreation. Many local and regional governments and NGOs already invest in invasive species prevention efforts; this approach can be an effective complement to their efforts. The module combines both field sampling (i.e., lakes, rivers) and laboratory analyses.

The learning objectives for this set of lecture modules and lab exercises are:
- understand the challenges of quantifying species in low abundances in the environment;

- learn field sampling techniques to capture environmental DNA from a water body, including how to report data collection efforts; and

- learn lab protocols to extract DNA from samples and analyze with PCR in order to determine the presence or absence of the invasive species.

Context for Use

These labs could be part of a sophomore or junior biology or environmental science class. The activities need to be spread over two lab periods, requiring at least two hours each. We also present an option for quantitative PCR as an alternative method. This module was taught in a field methods class, with an emphasis on sampling techniques. Lectures prior to the module involved understanding the different techniques that can be used to sample for organisms in water, as well as some of their benefits and drawbacks. The lecture following the module discussed the students' understanding of some of the limitations of the eDNA approach and how it could be improved. Depending on the type of class, it would probably be best taught after more traditional sampling techniques.

Description and Teaching Materials

pre-lab lecture.pptx (PowerPoint 2007 (.pptx) 11.3MB Sep23 19) Pre-lab lecture. A general lecture on what invasive species are, some of the costs associated with invasion, how environmental DNA is used as an early detection tool, and why early detections of invasive species are important. Lecture provides context for some of the additional resources in the module (maps, species), as well as the difficulty of eradicating species once they are established. Additional emphasis can be put on examining the patterns of spread of zebra and quagga mussels, and the importance of protecting entire watersheds from invasive species, where spread can be rapid in waterways.

eDNA Student Handout (Microsoft Word 2007 (.docx) 591kB Sep23 19) eDNA student handout. The handout is provided to students to describe the field sampling protocols (part I), the laboratory protocols (part II), relevant background, and associated assignments (eg. reading, pre-lab quiz, field notebook). Includes lists of materials needed.

Optional qPCR Handout (Microsoft Word 2007 (.docx) 7.9MB Aug28 19) Optional qPCR handout. Provides basic background about what quantitative PCR (qPCR) is and how it is used for eDNA applications.

pre-lab quiz.docx (Microsoft Word 2007 (.docx) 24kB Sep23 19) Pre-lab quiz (10 questions, to be completed online). An open-book quiz to help students prepare for both the field trip and lab. Questions were centered on student understanding of the sampling protocols and the assigned reading, which provided context on invasive species, as well as real-world applications and challenges of using eDNA methods.

Teaching Notes and Tips

  • The students were assigned a pre-lab quiz that helped them prepare for both the field trip and lab. Questions were centered on student understanding of the sampling protocols and the assigned reading, which provided context on invasive species, as well as real-world applications and challenges of using eDNA methods.
  • The module could easily be done with another invasive species, so long as the appropriate species-specific primers are used.
  • This module could also be done to detect the presence of an endangered species. Traditional sampling techniques for plants and animals can be harmful for species that are threatened or endangered; thus eDNA can be a valuable alternative sampling method that does no harm to the target species.
  • If performing eDNA to detect an invasive species, it is worth thinking about what the appropriate action would be if there was a positive detection. If there was a positive detection, reporting would probably best be done by the instructor, but it would be a good teachable moment for students to understand how this contributes to environmental stewardship. Different states have different ways to report invasives; it also depends on whether you are testing for an animal or plant. Here are some suggestions:
  • We walked to a nearby river to sample, but sampling could be at any type of water body.
  • With a longer (or additional) lab period, students could do the activities in the order a researcher would do them. That is, the first step would be to perform the (1) DNA extraction, followed by (2) PCR, (3) gel electrophoresis, and (4) visualization. A PCR run for this exercise takes ~ 1 hour and 45 minutes, and gels need to run for ~45 minutes. With a 2-hr lab, we emphasized that there wasn't enough time to demonstrate everything sequentially and that we needed to do the steps out of order. So, in the shortened lab we started with (1) gel electrophoresis with PCR products completed ahead of time, followed by (2) DNA extraction, and (3) visualization.
  • This worked well with a smaller class (<10 students), where the instructor and TA could keep a close eye on everyone for both safety and properly following lab methodology.

Assessment

The module included a quiz, to be completed before the lab, which examines student understanding of concepts in the lab handout. During the lab exercise, I ask students to present their results to each other as they proceed through the lab (e.g., displaying a gel, confirming the appearance of the extraction end-product), which provides a checkpoint to allow students to help other students and to provide additional information if the whole group is missing key concepts. Students recorded observations in a field notebook during the first part of the exercise (I. Environmental DNA (eDNA) Field Sampling Lab). Notebooks were turned in following the field trip to evaluate student understanding of field sampling procedures and how to report data collection efforts. Students were asked to include a general site description, volume sampled, volume filtered, sub-site details, information about the control sample (i.e. water source), sample identification numbers, and any variations from the protocol.

References and Resources