Introduction:

In our Environmental DNA (eDNA) project, we will be researching and obtaining a better understanding of what exactly eDNA is and how it works. Additionally, we will compare eDNA to traditional methods like swabbing because we really want to see if this new way, which has just been discovered in the last hundred years, is more efficient than the traditional methods. There are multiple areas where eDNA could highly improve, like where the animals are in their life stages, and we were very curious to see why. We will be contacting Jorri Harrison in the field and learning information and her opinions on the topic that we are researching. This is an important part of our project because we need to get her view on highly valued facts and opinions on these topics in order  to see both sides and draw a better conclusion. We will then conduct an experiment in which three eDNA samples from three different places in the same creek will be collected. The purpose of this is to see the presence of different species upstream, midstream, and downstream. We will analyze the findings because we want to understand why different species migrate to different locations of the same creek or aquatic biome. The findings of our analysis will help us draw a conclusion.

What is eDNA used for and Define Traditional Methods:

Before talking about anything else, we first need to describe what exactly eDNA (environmental DNA) is and what it is used for. eDNA, the new and more modern way of detecting species in an aquatic environment, happens when animals or organisms drop DNA into any biome that has accumulated in the water (Rees et al., 2014). Scientists can collect and analyze this information to help us understand more. One thing that eDNA can be used for is to understand where the species are in the environment. This is very helpful to endangered and rare species like Bull Trout and Lake Sturgeon (eDNA Metagenomics Inc., 2020). Now, we are able to protect and close off areas that have endangered species. Additionally, eDNA can detect and analyze environmental health risks like Blue-green algae, Swimmers itch, Legionnaires disease (a type of infection), ticks, and others.(eDNA Metagenomics Inc., 2020)  eDNA also has emerging environmental targets like Jefferson Salamander, Blanding Turtle, and Whirling Disease. (eDNA Metagenomics Inc., 2020) Scientists can now also estimate species biomass and discover more rare species. When combined with a PCR primer, eDNA can amplify the target species range within a few hours.

On the other hand, traditional methods are a variety of methods that have been around for longer. Visual, audio, Collon surveys, foot prints, netting, and electric fishing. (eDNA Metagenomics Inc., 2020)  These methods comprise what is known as the traditional methods in this field.

For our research we used various reputable education sources such as biology books, education websites, and interviewed an expert. We were also able to develop a short experiment in attempt to extract eDNA and illustrate our research. The experimental design and method is written below.

Experimental Design:

Control variable: banana

Independent variable: sample (mud or banana)

Dependent variable: extracted DNA

Experimental Method

Part 1: Collecting Samples

Our experiment starts off with acquiring at least 3 small tupperwares. You then have to wash the tupperwares with soap while wearing gloves. Then, rub isopropyl alcohol (99%) on a paper towel and clean the tupperwares with this again. You have to do this crucial step to make sure that there is no contamination whatsoever. 

Start taking your samples by digging 15 centimeters below the water level and plunge the tupperware inside. Make sure to collect multiple and diverse objects in your sample like sediments, rock and leave some water. At the start, we collected mud in our samples but then, as we used coffee-filters for a later step, the mud wasn’t draining through properly. Repeat these steps for each tupperware, taking one sample at the beginning, middle and end of the creek. 

Our purpose of our experiment was to see if more eDNA pools/collects downstream more than upstream. This is because the eDNA might float downstream and collect by the miniature waterfall. We ran into a few problems with this because at the time we took the samples, the creek was frozen solid, so we had to break the ice. 

Part 2: Extracting DNA

After this, we have to extract the DNA (we had a controlled variable which was a banana sample we did at the same time). Firstly, add water and electrolyte salad to the mixing bag. Find a sample and add it to the bag. Mash up the samples into a slurry by pressing the bag. Add the lysis buffer (which breaks open cells) to the samples, mash and mix. Place the filter in a clean cup. The next step is to pour the sample mix into the filter and wait five minutes for the liquid to pass through the filter. Add 9 ml of isopropyl alcohol (cleanser) to the tube provided. Lastly, pour a small amount of liquid into the isopropyl alcohol and watch the DNA precipitate and float up to the surface. 

Part 3: Identifying the DNA (This part wasn't completed. We made an agarose gel to run the DNA, but didn't have time to run it)

Next we have to make a DNA gel which separates DNA bands for purification. You have to move the negatively charged DNA through the agarose gel towards a positive signal. Shorter DNA fragments, called bands, migrate through the gel quicker than larger ones. Add 100 grams of agarose to 150 milliliters of TAE buffer. Heat the solution for 1-3 minutes, stirring occasionally as the solution heats up. Wait until the solution cools down until 55 degrees celsius. To reduce time, you can add the ethidium bromide solution directly to the solution. Once the gel has cooled, place a comb corresponding to the number of needed wells into the gel tray, and slowly pour the gel. Make sure the tray is properly sealed around the outsides so the gel doesn't pour out. Use a pipette to push away any bubbles in the solution. Let the newly poured gel sit at room temperature for 1 hour until completely solid. Then, fill the gel electrophoresis chamber with the same buffer solution you used for preparing the gel. Pour until the gel is covered and make sure that the buffer enters the wells. Connect the power source to the gel box and turn on the machine. When everything transfers to red, cut the machine off.

eDNA Compared to Traditional Methods:

Through multiple references and websites, the majority of geneticists say that when eDNA is compared to traditional methods, it has fewer drawbacks and more benefits. (eDNA Metagenomics Inc., 2020)  Let us define what we are comparing eDNA against. Visual, audio, surveys, footprints, netting, and electric fishing are traditional methods because of their popularity amongst the scientific community. (eDNA Metagenomics Inc., 2020)  With eDNA, a new and more modern method, you can now track more accurately than ever before. This is true because traditional methods, like water filtering, lack in a lot of categories, such as efficiency, detection, and taxa, just to name a few.  (eDNA Metagenomics Inc., 2020) Additionally, surveys show that for almost any species living in an aquatic biome, like micro-organisms, eDNA is exceptional and more accurate.  (eDNA Metagenomics Inc., 2020) Although the more modern methods have some advantages over traditional methods, there are still some areas where traditional methods are better, as we will consider in a later paragraph 

Environmental DNA has more advantages over traditional methods in an eminent range of categories, including cost, sensitivity, detection, and accuracy. One of the categories in which eDNA excels is defense because of its ability to detect invaders in 2-4 hours and defend against non-native invaders.  (eDNA Metagenomics Inc., 2020) Environmental DNA can also detect bacteria and environmental health risks in a very short period of time. On the other hand, traditional sampling protocols are slower and may not be as effective in reporting on a target species. This is because the traditional methods are not fast enough to still have the species DNA still in the water and not in a state of diminishing. This is usually because most of the traditional methods can be a pain to detect because they are extremely slow and have low-density information. (Rees et al., 2014)This will take a painstaking amount of time, and it could take you hours looking for a single individual .(Rees et al., 2014) With many of Alberta’s watershed systems being highly active with species like newts, toads, or frogs, the health risk and unwanted bacteria are consequential. (eDNA Metagenomics Inc., 2020)  Using more modern methods to monitor and watch the species that you are researching proves to be more beneficial because this new way is not as confusing, a time waste, or has low density detection. If used correctly, eDNA can offer almost everything in terms of supplying research for aquatic environments.

The general consensus is that eDNA methods are more accurate and cost-effective than traditional survey methods.(L. Rourke et al., 2021) Additionally, eDNA methods are more sensitive and can detect more species with fewer sampling events. Surveys for almost anything living in an aquatic biome, like macroorganisms, show that eDNA is more accurate. However, they are only approved to conduct research for a few species globally, like Bighead carp, Silver carp, molluscs, and a few species of mammals and amphibians.(eDNA Metagenomics Inc., 2020)  eDNA can detect targets faster and provide results within 2-4 hours, as well as provide quantification on other bacteria and environmental health risks. Sometimes, when monitoring rare species using traditional methods, it can be difficult to detect anything because these methods are extremely hard to understand and have low-density information.(Rees et al., 2014) This will cost you a lot of time and it could take you hours looking for a single individual.(Rees et al., 2014)  If using environmental DNA, multiple samples across the target environment can be taken and analyzed for traces of the DNA that has been shed in the environment.

Disadvantages Of eDNA

eDNA still has some drawbacks compared to traditional methods. One of the main drawbacks is that traditional methods have been around longer. This means that eDNA is only formally approved to conduct research on a few species, like aquatic plants, aquatic mammals, fish, mussels, fungi, and parasites. (eDNA Metagenomics Inc., 2020) The eDNA method is only capable of detecting the presence or absence of a species. (Roussel et al., 2015) Unlike traditional methods, eDNA can’t collect information on the life stages, population, or health of the target species. This is a very important limitation if additional information is needed, for example, about the reproductive success of a species. Another important fact to consider is that a positive signal does not necessarily mean the species is present in the area. (Roussel et al., 2015) This is because the eDNA could have been transported or preserved after an animal’s death. (Roussel et al., 2015) Although eDNA excels at detection, sometimes the detection of aquatic invertebrates is weakly documented. (Rees et al., 2014) This is usually because the effect of species abundance on detection efficiency is not always established. (Roussel et al., 2015)

Comparing eDNA to the Traditional Methods

• WWF is using to track polar bears and other elusive Arctic wildlife
• In Australia, it is used to monitor coral reef. It is used in several other places to track endangered species
• eDNA approach is generally considered to be more cost-efficient than conventional sampling and can often increase species detection,especially when species are rare
• BEST USED WITH OTHER METHODS

Bylemans J, Gleeson DM, Duncan RP, Hardy CM, Furlan EM. (2019). A performance evaluation of targeted eDNA and eDNA metabarcoding analyses for freshwater fishes. Environmental DNA. 1(4), 402–414. https://doi. org/10.1002/edn3.41

eDNA Metagenomics Inc. (2020). Available target species. https://www.e-dna.ca/deliverables

Rees, H. C., Maddison, B. C., Middleditch, D. J., Patmore, J. R. M., Gough, K. C.,  (2014). The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology. Journal of Applied Ecology. 51(5). 1450–1459 doi: 10.1111/1365-2664.12306

Rourke, M. L., Fowler, A. M., Hughes, J. M., Broadhurst, M. K., DiBattista, J. D., Fielder, S., Walburn J.W. & Furlan, E. M. (2022). Environmental DNA (eDNA) as a tool for assessing fish biomass: A review of approaches and future considerations for resource surveys. Environmental DNA, 4(1), 9-33. https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/edn3.185?cookieSet=1

Roussel, J., Paillisson, J., Tréguier, A., & Petit, E. (2015). The downside of eDNA as a survey tool in water bodies. Journal of Applied Ecology, 52(4), 823–826. https://doi.org/10.1111/1365-2664.12428

• Dr.Soares (our amazing mentor)
• Jori Harrison (an expert we interviewed)
• Ms. Madison (our help with citations)
• Dr. Miri (our help with editing)