Whitney Genetics Lab the Evolution Continues
BY NICHOLAS BERNDT, WHITNEY GENETICS LABORATORY
in our receiving room. Here Mai Yang is centrifuging 50ml sample
tubes, and decanting excess isopropanol on a particularly busy
day. Credit: USFWS
From the first days of Spencer Baird and the United States Fish Commission, the science of fisheries management has evolved greatly over the years. Here at the Whitney Genetics Laboratory, we are constantly evolving as well. Genetic tools for fish management and conservation are rapidly advancing and more people are looking at ways the science of genetics fits into the fish management equation. It is an exciting challenge to be able to tackle these problems head on.
Invasions of non-native species are one of the biggest ecological threats we face today. In recognition of this, federal and state agencies have begun cracking down on illegal movement of fish and water to a degree never seen before, citing violators with ever increasing fines. This is with good reason, as the harm these invasive species inflict is high and persists for years. Tools to combat these invaders are limited for aquatic species. Unlike a terrestrial ecosystem where monitoring cameras can be set up, or an area can be swept over and examined as often and as closely as needed, aquatic organisms can be right in front of our faces, but remain out of sight under a murky veil of water undetected for years. So the question that remains is how do you peel away the veil? Traditional fish capture gear like netting and electrofishing is rather inefficient if you want to find those first pioneering individuals moving into a system. A new tool was needed. Something that could find fish “tracks” we could follow and then lead to the capture of that fish. Enter environmental DNA (eDNA) and the Whitney Genetics Laboratory.
Wisconsin. Credit: USFWS
So what is all the excitement over this eDNA tool? Well to put it simply it’s like when a crime lab compares DNA from suspects to the crime scene. Our suspects are the invasive species we are looking for like silver and bighead carp, and the crime scene is the waterbody of interest. We are now able to lift that veil of secrecy water provides, and find genetic evidence these fish are present.
The Whitney Genetics Laboratory officially took over eDNA monitoring responsibilities from the US Army Corps of Engineers in August of 2013 following a transition plan developed by the Service and the Corps. Though this was only 3 years ago, our eDNA workflow and techniques have advanced at a pace that is quite unbelievable. Advancements like centrifugation collection of samples, development of new quantitative PCR genetic markers, and extraction efficiencies are all helping us process thousands more samples then before, and with a sensitivity that was not possible just 2 years ago.
Complete with 12 thermal cyclers, two Eppendorf 5075 robots, ultra-
cold freezers, hand-loading PCR station, and a data analysis bench.
So what’s new for this sampling season? New for the 2015 sampling season is the use of centrifugation for samples instead of the filtering that has been done since 2009. One sample now consists of five 50-ml centrifuge tubes taken together at a unique location. This is opposed to the old technique of one 2-L sample put through many filters. This is an improvement over the old technique in a few ways. First and foremost, the DNA signal we pick up is just as strong or stronger then when we were filtering. Second, the time savings and logistics are much more streamlined. It is far easier to handle 50-ml tubes then a 2-L bottle in the field, and the elimination of the filtering process greatly reduces the chance for involuntary contamination from one sample to the next. The samples also can be stored on ethanol or isopropanol, eliminating the need to ship samples on dry ice within a certain timeframe. Also new are the addition of a quicker and more efficient DNA extraction kit, and the first full use of our automated liquid handling robots for loading PCR plates. Previous to this year every sample had to be loaded by hand into a 96-well PCR plate. When you take 6600 eDNA samples like last year, and run each sample in octet, that adds up to over 52,800 individual plate wells that had to be loaded by hand with liquid amounts as small as 1µl! It was very precise work that needed extreme focus. The robots now handle this, and we can load several 384 well plates each day. The bottom line is that we are now able to process more samples at a quicker pace with more accuracy than last year.
program and we now recycle 100% of our
nitrile gloves! So far this year we have kept
almost 400 pounds of gloves out of the landfill!
The actual processing of a sample begins in our receiving room where we unpack and check in a box of samples. Once every sample is accounted for, the samples are re-spun in a centrifuge to concentrate any of the sample that may have broken up during shipping. We are looking for a nice dense pellet from our water sample. After centrifugation the sample is decanted of ethanol and set in a sterilized laminar flow hood with it’s cap off to evaporate any remaining ethanol. Ethanol and isopropanol are a strong PCR inhibitors and every last drop must be evaporated in order for the DNA to amplify down the line. After the sample is dried it can now be extracted. DNA extraction is the process by which we purify all of the DNA contained in our 50-ml tube to be ready for PCR and other applications. Ideally, a sample would be taken from a crystal clear body of water free of PCR inhibitors like tannins, humic acid, among others. As you can imagine, some of the water samples we are taking come from sites known for high productivity and lots of organic material. Recent rains can make the problem worse by flushing more sediment into the system. All this other “stuff” can interfere with our downstream eDNA applications. After extraction we now have a purified DNA sample. Next is PCR (Polymerase Chain Reaction). The extract has DNA from all kinds of organisms that live in the water from where the sample was taken. To see if any of that DNA is from an invasive species like a silver or bighead carp, we must use a short chain of species- specific DNA called a primer to “look for” it’s match in our extract, and then copy it many times so our instruments can detect it. The primer is required to serve as a starting point for DNA synthesis. We use a silver carp primer to look for silver carp DNA, bighead carp primer for big head carp, or even a primer that looks for both.
Right now invasive carp monitoring is the bulk of our workload, and this season we are projected to process over 7000 samples. However, as we speak other aquatic invasive species are marching their way across the US hoping to get a foothold somewhere. Monitoring of invasive species is extremely important and helps us get ahead of the front. After primers are developed we can expand our search radius to include many other species. This is only the tip of the ice berg so to speak for eDNA applications. As the only dedicated genetics lab in the Region, the Whitney Genetics Laboratory will also be expanding it’s applications to provide species information in the realm of conservation genetics. Conservation genetics is a broad term, but mostly deals with conserving and restoring biodiversity. There is a lot of interest in using our tools for working with threatened and endangered species. Aquatics managers are now looking into genetic considerations when it comes to matters such as stocking fish for example among many other applications. Our lab also acquired a super high throughput next generation sequencer (NGS). This will allow us to sequence genes at a speed and depth unattainable before. Our lab is also expanding by hiring new personnel. More staff means more flexibility and efficiency in the projects we take on.
2015 is shaping up to be a great year for the lab. We will process a record number of invasive carp samples and get to explore many new ideas and projects not possible before.