Magic...

. . . not really, but that's what DNA extraction seemed to me before learning the science behind it. In fact, it's quite simple! The process varies from kit to kit and lab to lab, but over all follows the same major steps. The DNA I am trying to extract came from all the mouths and cloaca of the frogs that were swabbed in the field. I want to get this DNA to come off the cotton swab and sit suspended in a solution which I will be able to pipette into the PCR machine (we'll talk about the PCR later).

My lab station set up with samples ready to go!

Maintaining constant pH throughout the process is very important -- this can be done with the aid of a buffer, such as phosphate buffered saline, added to the vial containing the swab before starting the extraction process. Next, a detergent and lysis agent are added to the sample. The detergent (not to be confused with laundry soap . . . although somewhat similar) breaks apart the cell membrane and nuclear envelope. This is important because it exposes the DNA which is wound around a protein called a histone. The lysis agent, usually a proteinase K, cleaves the histone releasing the DNA into the solution. Adding alcohol to the sample causes the DNA pieces to come out of the solution and clump together. And finally, filtering out the ethanol and adding a final buffer to the sample ensures that DNA is evenly distributed within the liquid in the vial and ready to be tested or frozen for later use. Tada! DNA has been successfully extracted from a cotton-tipped swab!

Pipetting... measuring is done in MICROliters... tiny amounts!

Samples floating in a warm water bath to speed up the detergent and lysis solution processes

Samples ready to be spun in the centrifuge

Lessons in catching frogs

1. Choosing a pond . . .
      Knowing what type of environment the frog of interest likes to live in greatly helps you choose where you will start searching. The northern cricket frog lives near slow moving water and enjoys being in the sun, so a good pond is key. Shallow clear water surrounded by an easily accessible bank will help you get close and see the frogs if they submerge. Cricket frogs are found throughout Illinois, but for this project, we decided to focus on areas along the Mississippi River.

Sites at which I was able to find cricket frogs

2. Catching frogs . . .
     Cricket frogs do a great job camouflaging with their environment with a variety of patterns of grey, brown and green. The easiest way to find these little critters is to walk slowly around the pond and after seeing one jump away, sneak up and slowly try to lead it into the baggie.

Northern cricket frog blending in... can you spot it?

     Since I am looking for an highly contagious ranavirus, I had to make sure to use a sterile technique as to not contaminate materials that might transfer the virus from a sick to a healthy frog. Being in the field makes sterility more difficult to achieve than in a hospital or lab setting, but luckily, with cricket frogs being so little, getting them to jump into a plastic baggie was relatively easy and we did not have to use nets. 

Success! Cricket frog in bag. 

     Choosing a sample population in a field setting is a different story than in a lab. Ideally I wanted at least 15 frogs per site . . .  however, with nature being somewhat unpredictable, I was lucky to catch one or two in some areas. The amount of ponds which were ideal for cricket frogs, but had none or very few, astounded me . Are frogs in these ponds at a different stage of development than others? Are predators more abundant in these locations? Or is a disease such as frog virus 3 wiping them out? Even though 15 frogs would be a better sample statistically, one FV3 positive individual would be an important red flag for wildlife veterinarians and scientists. The prospect of this deadly disease existing in Illinois makes it imperative to test even the sites where only one individual was found.

3. Keeping clean . . .
     Although it is possible to keep materials and frogs from contamination, keeping yourself mud-free is not. You just have to deal with this.

Thank you rubber boots!

4. Collecting the sample . . .
     Since I released the frogs back into their own pond, I had to make sure to bring all my supplies with me to the site. This included plastic ziplock bags, nitrile gloves (previously rinsed with water to remove toxins that might be dangerous to frogs' permeable skin), cotton tipped swabs, polypropylene tubes that snap closed, a permanent marker, and a notebook.

Supplies laid out in the field.

Frogs waiting to be sampled and released.

         A great way to test for FV3 without performing a necropsy , is to swab the oral cavity and cloaca for any  shedding virus. I placed each swab into its own polypropylene tube as to not contaminate them. Later DNA will be extracted from these swabs and tested to see if any were positive for FV3.  While collecting the sample , I performed a quick physical exam for any symptoms of ranavirus. Signs of the disease include papules or plaque in the mouth , ocular and nasal discharge , reddening of the skin (especially the      legs) , loss of pigment , and edema (swelling).

5. Saying goodbye . . .
     I'm very grateful for being able to catch as many frogs as I did , but letting them go back to their home environment felt great. Hopefully the results of this research will help not only them , but all amphibians and animals in the western Illinois area.

Bullfrog 

* Frog fun fact: The northern cricket frog averages an inch in length and can jump up to 6 feet (72 times its length)!!!

Let's talk frog...

You may be wondering why I want to spend hours of my life trudging through mud and sitting in a lab just to find out if a certain tiny non-living entity (FV3) can be found in frogs in Illinois... Well, besides considering this to be fun, there exist a countless amount of reasons why protecting wild populations of frogs, toads, and other amphibians is extremely important. These reasons may not affect your daily life in the near future, but if you have ever marveled at the beauty of the world and the perfection that exists in natural, unspoiled wild areas, then you'll understand.

                                 Red-eyed tree frog - Photo by Christian Ziegler

Frogs are designed perfectly to suit their wet and dry environment. Unlike other amphibians, such as salamanders, they lack a tail and have strong hind legs for jumping while on land and swimming in the water. Their highly permeable skin with superficial blood vessels allows them to absorb oxygen without having to breathe underwater. While on land, they inhale air through their nostrils into their lungs similar to other terrestrial animals. Bodies of water, however, are crucial for all frog species. This is where most lay eggs and spend the first part of their lives as tadpoles before metamorphosing into adults.

                               Wallace's flying frog - Photo by Tim Laman

But why are frogs so important to the health of the environment? Being a predator and prey, they are an indispensable part of the food web which could crumble without them, having dire effects on animals which eat them. This trend could continue throughout the ecosystem and affect species not even directly related to the frogs... and potentially humans. Without frogs, insect populations could grow uncontrollably as well as the diseases they spread such as West Nile virus and malaria.

                                 Frogs are important in the food chain.
                                       Photo by Jeremy Woodhouse

                                Frogs are important in the food chain.
                                         Photo by Tyrone Hayes

                                 Frogs are important in the food chain. 

                                         Photo by Tyrone Hayes

Having permeable skin and a life cycle which requires 2 types of environments make frogs amazingly unique but also highly susceptible to small changes in the environment. With an ever-changing world quickly becoming more urban, frog habitats are becoming smaller and more polluted. Due to the nature of their skin, frogs are very vulnerable to toxins and diseases and are considered a bioindicator. The overwhelming decrease in amphibian populations in the last several decades shows that the environment is in a crisis and it is up to us  to help these critters out.

'Pinocchio frog' discovered in 2010

Photo by Tim Laman

**Frog fun fact: Unlike mammals, frogs have a 3-chambered heart. You would think that the oxygenated and deoxygenated blood gets mixed in the single ventricle, but due to the perfectly timed contractions of the atria, most of the blood stays separate making this quite an efficient system. 

The journey begins...

Hello everyone! Welcome to my summer journey as I search the wetlands of Illinois for a deadly virus, Frog Virus 3 to be specific. FV3 is the most prevalent ranavirus in the United States and responsible for large frog and toad mortality events. What, did you think I was dealing with a virus that could kill me? No way! Anyway, I'm a veterinary student with an interest in wildlife and ecosystem health… and what better way to learn about it than getting out, catching frogs, and gettin’ my hands dirty!

I have already traveled throughout western Illinois to collect samples (you will hear more about this in future posts), and am about to start lab work consisting of extracting DNA and running PCR . All this is to determine if any of the frogs in these wild populations were positive for the disease . Sounds pretty fancy, doesn’t it? Well, follow me this summer and find out just how fun conducting research is and why we want to save the frogs!


I would love to thank the Merial Veterinary Scholars Program for my stipend and the Summer Research Training Program at the University of Illinois. Together, they are making this research possible!

 You can find more information on both right here...                                                    
- http://vetmed.illinois.edu/research/summertraining/
- http://www.merialscholars.com

                                  The journey begins...