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Collecting Environmental DNA Samples on the 2022 Spring Ecosystem Monitoring Survey

June 17, 2022

Planning a sea-going research cruise since the pandemic started has been difficult, and at times impossible, so it is exciting to get back out on the ocean again.

 A color image taken on the ship's deck in fair weather at night. At center, three people remove water samples from bottles held in  an open, metal, cylindrical frame about 8 feet high. The bottles are cylindrical, opaque, and look like scuba air tanks.

In the fall of 2019, I went to sea to start collecting samples. In 2020, as COVID spread, federal research cruises were canceled one after another and this research came to a grinding halt. Cruises resumed slowly and we collected more water samples in the summer of 2021. Today I find myself at sea again, which completes my goal to sample in three different seasons.

I have been collecting environmental DNA samples on NOAA Ecosystem Monitoring (EcoMon) surveys since 2019. These samples will help develop eDNA metabarcoding, an innovative way to determine what fish species live in what parts of the ocean without actually seeing any fish.

A color image taken on a sunny, windy day. A casually dressed slender woman with dark hair is at center, standing on a concrete dock area. A large, light colored ship alongside the dock takes up the background. A steel frame is seen on the ship’s deck at right, with dark colored cylinders hanging from it.
I proudly pose in front of the NOAA Ship Henry B. Bigelow. The new Niskin rosette (equipment used to sample seawater at different depths) is in the background, over my left shoulder. Credit:NOAA Fisheries/ John Gallagher

As I write this post, I am aboard the NOAA Ship Henry B. Bigelow with the 2022 Spring EcoMon. The general survey region of EcoMon overlaps with NOAA bottom trawl surveys, which allows us to compare eDNA survey results with bottom trawl survey results. I am grateful to the EcoMon team to accommodate my sampling needs. “Piggybacking” on their surveys not only reduces the operational cost for eDNA work, but also gives me access to other data important to the interpretation of eDNA data, such as temperature, salinity, and ichthyoplankton composition.

How Does eDNA Metabarcoding Work?

Fish shed DNA into the surrounding environment. We “filter and fix” DNA molecules out of the seawater on the ship, then bring the filters back to the laboratory for DNA extraction. From the DNA on the filters, we create many copies of the different versions (representing different species) of a DNA segment.

We contract with the Cold Spring Harbor Laboratory to assemble all the DNA from different species using next-generation sequencing. Our goal is to find out what fish are present in the seawater without using a hook or net. Since eDNA metabarcoding identifies multiple species simultaneously, it works like another kind of fishing net. The unique strength of metabarcoding is that it allows us to identify fish that may be too small, too big, too fast, or too rare to collect with conventional sampling gear.

The EcoMon program has good spatial and temporal sampling coverage, so we also hope to derive information on the number of fish.

Collecting eDNA Samples on a Ship

 A color image taken in a well-lit laboratory space.  A series of clear containers with filters attached is secured to a frame that rests on a countertop.
Filtration apparatus is secured using bungee cords for both calm and rough seas. Credit: NOAA Fisheries

On load-in day, we set up our filtration apparatus and secure everything so rough seas won’t create chaos.

When we get to a station, I first need to decide the depths from which to collect eDNA water samples. We securely attach environmental sensors to the bottom of a metal frame, which holds 24 10-L Niskin bottles arranged as a rosette. As the rosette goes down the water column, we acquire vertical profiles of chlorophyll a, temperature, and other parameters. As the rosette slowly rises back up, computers send commands to open the Niskin bottles so they fill with seawater at various depths.

I check vertical profiles of environmental parameters and chlA color photo taken in a well-lit ship’s laboratory. At right,  a woman is writing notes. She faces a bank of computer monitors. Most are on a counter in front of her. One is perched over the counter, displaying closed-circuit camera feeds from other parts of the ship.
I check vertical profiles of environmental parameters and chlorophyll a to decide eDNA sampling depths. Credit: NOAA Fisheries
Atl text: A color image taken on the ship's deck in fair weather during the day. At center, an open, metal, cylindrical frame about 8 feet high encloses 24 sampling bottles that are arrayed around the inner arc of the frame. The bottles are cylindrical, opaque, and look like scuba air tanks.
The newly purchased Niskin rosette, which holds twenty-four 10 L Niskin bottles, is ready for its maiden voyage. Photo credit: NOAA Fisheries/John Gallagher

After the rosette is back on deck, we transfer seawater from the different depths from the Niskin bottles into our sampling bottles.

A color image taken on the ship's deck in fair weather at night. At center, three people remove water samples from bottles held in  an open, metal, cylindrical frame about 8 feet high. The bottles are cylindrical, opaque, and look like scuba air tanks.
We collect seawater from Niskin bottles to our sampling bottles. From left to right: Socrates Loginidis, Chris Taylor, Yuan Liu. Credit: NOAA Fisheries

We then take the sampling bottles back to one of the ship’s laboratories and start filtration. An air pump creates a low-pressure environment in a flask that sucks seawater through membrane filters to trap the DNA molecules.

A color image taken in a well-lit ship’s laboratory space. On the counter, a dozen plastic sample bottles are aligned neatly with 6 filtration bottles. In the background a woman wearing a mask and protective gloves uses a tube to move sampled water into the filtering system.
Filtration in process. Seawater is filtered in two steps for each sample. I take out a second bottle of seawater from the fridge as the contents of the first bottle is about to finish filtering. Credit: NOAA Fisheries

Lastly, we put the filters into 2 mL sterile tubes and store the tubes in a deep freezer (-80°C/-112°) until we take them back to the lab for DNA extraction.

A color image taken in a well-lit ship’s laboratory space. A hand covered with a safety glove holds a clear plastic vial about 2 inches long with an opaque strip inside.
Success! A membrane filter loaded with materials for DNA extraction is stored in a 2ml Eppendorf microcentrifuge tube. Credit: NOAA Fisheries

eDNA metabarcoding is an extremely sensitive method, so avoiding contamination is important when working in the field. We do not want to introduce fish signals from the environment (surfaces, air, human, etc.) into our samples, or from sample A to sample B. To achieve this, we wash our sampling bottles and filtration apparatus as thoroughly as we can with fresh water between samples. When time allows, we also decontaminate them in a diluted bleach solution to remove any residual DNA.

A color image taken in a well-lit ship’s laboratory space. At center, a young man wearing dark sweat clothes and brightly colored dishwashing gloves holds a plastic bottle over a plastic tub containing water and bleach. His sweatshirt has a large image of an Atlantic cod on its upper front, with  “ Gloucester Biotechnology Academy,” printed beneath it.
Socrates Loginidis, the molecular technician and a proud graduate of Gloucester Biotechnology Academy, washes sampling bottles in the bleach bath. Credit: NOAA Fisheries

The entire process from rosette deployment to completing filtration is very time-consuming. Rosette deployment and retrieval can take from 20 to 50 minutes depending on the water depth at a station. The time to transfer seawater from Niskin bottles to sampling bottles can easily take 30 minutes with two people working. This is because we sample from as many as six different depths, and we collect duplicate samples at each depth. Lastly, filtering a single 2 liter sample can take up to 3 hours if particulates in the sample clog the membrane filter.

When sampling stations are close, meals can be rushed and sleep may be skipped, but every station is another opportunity to help shape the direction of eDNA research. For example, preliminary analysis of our 2019 samples showed us that 2 liters of seawater, compared to 1 or 3 liters, seemed to optimize the ratio of DNA signal over filtration time. In future analyses, I hope to better understand the vertical profiles of fish assemblages and reduce vertical sampling, if that can be backed up by data. We would also like to look into ways to simplify the filtration process. Stay tuned!

A Few Reflections

My Ph.D. training was in microbial oceanography, a field that routinely uses DNA metabarcoding to identify species. Microorganismal DNA is wrapped in cell membranes and is an integral part of the environment, which was probably why we didn’t specifically call it “environmental DNA” back then.

Working with fish, I am overwhelmed by all their Latin species names, appearances, and distributions. By one estimate there are more than 300 marine fish species off New Jersey alone. Fortunately, at the Northeast Fisheries Science Center, I can easily find answers to my questions from fisheries experts such as Dr. Richard McBride and Dr. David Richardson. We work closely to learn from other experts about how my research can help answer fisheries questions.

Today is June 3, 2022. We are heading south to the Mid-Atlantic Bight area after a brief interruption caused by a COVID case. The impact COVID has on surveys is tangible at all levels: planning, executing, and mitigating the situation when cases happen. Truthfully, though, we all cherish the opportunity to work closely together and collect valuable samples, and we are ready to do whatever it takes to continue the work. I will always remember these sea days.

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