Looking under the hood: how warm temperatures affect gene processing in turtle eggs

Featured Scientist: Rosario Marroquín-Flores (she/her/hers), PhD (Anticipated May 2022), Illinois State University, Biological Sciences.

Rosario standing in the water next to a boat, wearing waders. She holds a red-eared slider turtle in her hands and smiles at the camera.
Trapping turtles at our field site, Banner Marsh State Fish and Wildlife Area

Birthplace: Albuquerque, New Mexico

My Research: I study temperature-dependent sex determination (TSD) in turtles. Turtles that have TSD will become male or female based on the temperature that they experience as an egg. 

Research Goals: While I really enjoy fieldwork and my current research, I am interested in studying something different when I graduate. I am interested in doing research in college classrooms. I want to see if writing science communication articles can help students to understand primary literature. I also want to see if reading science communication articles can help students get excited about research.

Career Goals: I am interested in teaching and doing research at the university level.

Hobbies: I enjoy camping, backpacking, and spending time with my partner and my pets. 

Favorite Thing About Science: I enjoy the autonomy of science. I like that I get to decide what I want to study. I get to ask my own questions and find my own answers. I also like how dynamic it is. Every day I get to do something different. I’m very busy, but I’m never bored! 

My Team: I am the first author on the publication, but this has been a collaborative project. My advisors, Dr. Rachel Bowden and Dr. Ryan Paitz, helped me come up with the project, assisted with data analysis, and helped in writing the publication. My wonderful lab mate, Anthony Breitenbach, helped me organize my eggs into treatments, helped me with dissections, and provided lots of emotional support and very loud jokes.

Organism of Study: I study the red-eared slider turtle (Trachemys scripta)

A recently hatched turtle. The turtle is still half inside the egg and is roughly the size of a golf ball.
A hatchling red-eared slider turtle emerging from its shell

Field of Study: Eco-physiology

What is Eco-Physiology? Eco-physiology is the study of the relationship between the body and the environment. In my research, I study how temperature affects the way turtles develop in the egg.

Check Out My Original Paper: “Brief exposure to warm temperatures reduces intron retention in Kdm6b in a species with TSD”

A QR code that links to the original publication.
QR code to the original publication

Citation: Marroquín-Flores RA, Bowden RM, Paitz RT. 2021 Brief exposure to warm temperatures reduces intron retention in Kdm6b in a species with TSD. Biology Letters. 17: 20210167.20210167.

Research at a Glance: Temperature can impact how animals develop, and this is particularly true for animals with TSD. The red-eared slider turtle has TSD, which means that the temperature that the turtle experiences as an egg will determine whether it develops into a male or a female. In this species, warm temperatures result in female hatchlings and cool temperatures result in male hatchlings. Researchers have been studying TSD for over 50 years, but we still don’t understand how the turtle detects the temperature and how that temperature leads to male or female development. But we do know some things. We know many genes in the body of a turtle that turn on in response to temperature. We also know that these genes can lead to male or female development. Genes are made up of DNA and carry instructions that the body uses to make proteins, which go on to play important roles in the body. In this case, genes that turn on in response to temperature will make proteins that lead to male or female development. However, before genes can make a protein, they need to be processed. Usually, the body will process them by removing portions of the DNA, called introns. Once the introns are removed, the body will read the instructions in the gene and make a protein. This is where my research comes in. One really important gene in turtles is called histone H3 lysine 27 (H3K27) demethylase, or Kdm6b. Kdm6b is one of the earliest genes to respond to temperature. It turns on in response to cool temperatures and leads to male development. Kdm6b is special because it is not processed like other genes. Normally, introns are removed from genes when the DNA is processed. In Kdm6b, however, one intron stays in the DNA at cool temperatures, but is removed at warm temperatures.

Researchers who study TSD usually incubate eggs under a constant cool temperature or constant warm temperature. Then, they will look at how turtle genes respond to these temperatures. In my research, we use temperatures that mimic those that a turtle would experience in the wild. We use temperatures that go up and down, or fluctuate, to mimic a daytime and nighttime environment. Everything we know about how Kdm6b is processed is based on research that incubated eggs under constant temperatures. In my research, we wanted to understand how Kdm6b would be processed when we incubated eggs under fluctuating temperatures. To do this, we put eggs into incubators that fluctuated between 22°C and 28°C (25.0 ± 3°C) to mimic a cool, male-producing environment. Then, we moved some of the eggs to a warm, female-producing environment that fluctuated between 26.5°C and 32.5°C (29.5 ± 3°C). We then measured how often the intron stayed in Kdm6b at both temperatures. We learned that Kdm6b is processed very quickly in response to temperature. Within two days of exposure to warm temperatures, the intron was removed from Kdm6b. This shows us that even short bursts of warm temperature can impact how genes are processed. We learned about how Kdm6b is processed, but we still need more information to understand how the intron impacts male development. We are also still trying to figure out if the intron that stays in Kdm6b leads to a different protein.

Highlights: In this research, we found that Kdm6b is processed differently at male and female temperatures. We also found that it happens very quickly. When we isolate the intron containing version of Kdm6b, we see that it goes down when the egg is exposed to warm, female-producing temperatures (Figure 1). Figure 1 is a boxplot that shows how the intron containing version of Kdm6b changes in response to temperature. The y-axis shows gene expression, or how much Kdm6b is present in the body of the turtle. The x-axis shows the day that the eggs were sampled on and the temperature that the eggs were incubated under.

The letters in Figure 1 show the results of the data analysis. When the letters are different, that means that the expression of the intron containing version of Kdm6b changed. We see that the expression did not change between incubation day 26 and day 28 in eggs incubated under cool temperatures because both blue boxplots have the letter “a” above them. On the other hand, we see that expression was much lower on day 26 and day 28 in eggs incubated under warm temperatures because both pink boxplots have the letter “b” above them. Figure 1 shows that the expression of the intron containing version of Kdm6b went down in response to warm temperatures.

A data figure with boxplots that shows how much of the intron containing version of Kdm6b is expressed in the body of a growing turtle. The blue boxes show data from eggs reared at cool (25.0 ± 3°C), male-producing temperatures. These blue boxes are larger and have more spread than the pink boxes, which show data from eggs reared at warm (29.0 ± 3°C), female-producing temperatures. The blue boxes have an average near 0.001 and the pink boxes have an average near 0.  In other words, the intron-containing version Kdm6b is expressed more under male-producing temperatures than female-producing temperatures.
Figure 1. The expression of the intron containing version of Kdm6b at cool, male-producing temperatures and at warm, female-producing temperatures. The blue boxes show the expression of Kdm6b when eggs are incubated under male temperatures. The small pink boxes show the expression of Kdm6b when eggs are incubated under female temperatures. Figure adapted from Marroquín-Flores et al. 2021.

What My Science Looks Like: One important part of our research was the temperatures that we used for our eggs. We used temperatures that mimic those that a turtle would naturally experience in the wild. In our Illinois population, turtles nest from May to late June. When we measure soil temperatures during that time, we find that eggs are often exposed to cool, male-producing temperatures. This leads us to ask the question, how do female turtles hatch in this cool environment? In recent research from our lab, we found that short bursts of exposure to warm temperatures can lead to female hatchlings. Even just 5 days of warm temperature is enough to make some turtles hatch as female. We used this information when we designed our temperature treatments for this experiment.

We programmed incubators to fluctuate within the range of male-producing and female-producing temperatures. Turtles start to respond to incubation temperature during the thermal sensitive period (TSP) of development. The TSP takes place during the middle third of development and exposure to warm temperatures during the TSP can make turtles hatch as female. Longer exposure to warm temperatures during the TSP will result in more female hatchlings. All of our eggs were incubated for the first 24 days at a cool temperature. At these temperatures, incubation day 24 is when the TSP starts. After incubation day 24, eggs either stayed in the cool temperature incubator or were moved to a warm temperature incubator. Our goal was to expose eggs to a short burst of warm temperature, similar to what we see in the wild. Then, we sampled our eggs on incubation days 24, 26, and 28 to see how the intron in Kdm6b responded to the change in temperature over time. The image below shows the incubations that we used.

Eggs were incubated under a male-producing temperature of 25.0 ± 3°C for the entire experiment or were moved to a female-producing temperature of 29.5 ± 3°C after incubation day 24.
Eggs were incubated under a male-producing temperature of 25.0 ± 3°C for the entire experiment or were moved to a female-producing temperature of 29.5 ± 3°C after incubation day 24.

Finally, the image below summarizes the results of our research. When eggs are exposed to warm temperatures, the intron is removed from Kdm6b and these warm temperatures lead to female development. This shows us that temperature can impact how genes are processed, but we are still working to figure out how the intron impacts male development.

A picture of a red thermometer that points to a turtle egg. The image zooms into the inside of the egg to show the intron in the Kdm6b gene.  It shows that the intron is cut out of the gene under warm temperatures.
How Kdm6b responds to warm incubation temperature in a turtle egg. The green boxes represent parts of the Kdm6b gene. The thin green line represents the intron. At warm temperatures, the intron is removed from the Kdm6b gene. Image adapted from Marroquín-Flores et al. 2021.   

The Big Picture: One of the effects of climate change is that we now have warmer temperatures and more days at those warm temperatures. These changes in temperature can affect how animals grow and develop. There are many reptiles that have TSD and all of them are likely to be impacted by warm temperatures. My research helps us to understand how the body will respond to warm temperatures in species that have TSD. This research can also give us insight into how genes are processed more generally. Many of the genes that lead to male or female development are shared between species, including humans. All living things have evolved from a common ancestor and share certain traits. This is why scientists use animals in research to study human diseases. Kdm6b is an important gene in turtles because it turns on another gene called Doublesex and mab-3 related transcription factor 1, or Dmrt1. Dmrt1 is important for male development in humans, mice, chickens, fruit flies, and many other animals. In humans, males are born with a Y-chromosome and that chromosome carries genes that the child needs to develop as male. In humans, Dmrt1 is turned on by a gene on the Y-chromosome. In turtles, Dmrt1 is turned on by Kdm6b and Kdm6b is turned on by temperature. We are starting to learn that many genes can be activated by temperature, even by regular changes in body temperature. This type of research can help us understand how temperature impacts gene processing in many animals. In future work, we will use the same type of incubation conditions to see how temperature effects other important genes linked to male and female development.

Decoding the Language:

Autonomy: Autonomy is the ability to act on your own values and interests. It is having the power to make your own decisions independently and do the things that are important to you.

Boxplot: A boxplot is a graph that shows you information about the spread of your data. The top line of the box shows the 75th percentile. This is where 75% of the data fall below the line, and 25% of the data fall above the line. The line in the middle of the box shows the 50th percentile, or the middle of your data. The bottom line shows the 25th percentile. This is where 25% of the data fall below the line and 75% of the data fall above the line. The lines that extend from the box in either direction are called whiskers. They show the range of the highest values and the lowest values of the data set. Outliers fall outside of the rest of the data and are shown using a dot. As you can see in Figure 1, there was on outlier an incubation day 26 at warm temperatures. It shows up as a dot above the box. That particular sample had higher normalized expression than the other samples gathered on that day and at that temperature.

Chromosome: A chromosome is a long DNA molecule that carries genes. It is found inside your cells. A Y-chromosome is a specific type of chromosome that you inherit from your father and leads to male development. In humans, males have one Y chromosome (inherited from dad) and one X chromosome (inherited from mom). In humans, females have two X chromosomes, with one inherited from each parent.

Climate change: Climate change refers to increasing global temperatures and the resulting shifts in global weather patterns. Human use of fossil fuels releases greenhouse gasses into the atmosphere, which contributes to climate change.

Data analysis: Data analysis in biology involves comparing data (in this case, the amount of the intron-containing version of Kdm6b in turtles) across groups (known as “treatments”) in an experiment. Data analysis is essential to draw accurate and meaningful conclusions about a topic (like the role of Kdm6b in turtle sex determination) after an experiment.

Doublesex and mab-3 related transcription factor 1 (Dmrt1): Dmrt1 is a gene that is important for male development in many species. It helps male reproductive organs to grow. In turtles, Dmrt1 is turned on by Kdm6b. If Dmrt1 is intentionally turned off by researchers, then the turtle will develop as female.

Deoxyribonucleic acid (DNA): DNA is like a blueprint of the body. It is a molecule that contains all the instructions that the body needs to grow, develop, and function. 

Enzyme: An enzyme is a type of protein that can make a chemical reaction go faster.

Fieldwork: Fieldwork is a type of research that takes place outside. People who do fieldwork usually work with plants and animals. For my research, fieldwork includes trapping turtles and digging up turtle eggs at the Banner Marsh Fish and Wildlife Area.

Gene: A gene contains the instructions that your body needs to make a protein. Genes are heritable, which means that the genes you have in your body came from your parents. 

Hatchling: A hatchling is any animal that has recently come out of an egg.

Intron: An intron is a part of the gene that is usually removed during DNA processing. During processing, DNA is converted to RNA. RNA is a messenger that carries the information contained in a gene to the place in the body that is responsible for making proteins. Introns are removed at the RNA stage because introns usually don’t contain information that the body needs to make the protein. Once the introns are removed, the RNA can be re-organized to make different proteins. 

Histone H3 lysine 27 (H3K27) demethylase (Kdm6b): Kdm6b is a gene that is important for male development in TSD species. When turtles are incubated under cool, male-producing temperatures, the expression of Kdm6b increases. If turtles are incubated under male-producing temperatures, but Kdm6b is intentionally turned off by researchers, then the turtle will develop as female. Kdm6b is important because it responds directly to temperature and it turns on another gene that is critical for male development.

Primary literature: In science, primary literature consists of the published results of an original research project. Published research is evaluated by several other researchers to make sure that it is valid and high quality. The research paper referenced under the “citation” section of this article is an example of primary literature. It contains the published results of my original research.

Protein: A protein is a naturally-occurring compound made up of several amino acids. It is present in all living organisms and it performs very important functions in the body. Enzymes and antibodies are examples of proteins.

Temperature-dependent sex determination (TSD): TSD is a special form of sex determination, where temperature will determine whether an individual will develop as a male or female. There are several types of TSD. For some animals, like the red-eared slider turtle, warm temperatures create females and cool temperatures create males. For other animals, cool temperatures make females and warm temperatures make males. In other animals, like the American alligator, males are produced at high and low temperatures, but females are produced at intermediate temperatures.

Thermal sensitive period (TSP): The TSP is the window of development when the body of the growing turtle will respond to temperature and become male or female. If temperature is changed before or after the TSP, it will have no effect on the sex of the turtle.

Learn More:

National Aeronautics and Space Administration (NASA), USA site on Global Climate Change

University of Michigan Animal Diversity Web, information on the red-eared slider turtle

The Embryo Project Encyclopedia, information on temperature-dependent sex determination in reptiles

XBio educational video on how the body makes proteins

Recent research papers that provide more information about Kdm6b:

Deveson, I. W., Holleley, C. E., Blackburn, J., Graves, J. A. M., Mattick, J. S., Waters, P. D., & Georges, A. (2017). Differential intron retention in Jumonji chromatin modifier genes is implicated in reptile temperature-dependent sex determination. Science advances3(6), e1700731. *note: this paper calls Kdm6b “JMJD3”

Ge, C., Ye, J., Weber, C., Sun, W., Zhang, H., Zhou, Y., … & Capel, B. (2018). The histone demethylase KDM6B regulates temperature-dependent sex determination in a turtle species. Science360(6389), 645-648.

Weber, C., Zhou, Y., Lee, J. G., Looger, L. L., Qian, G., Ge, C., & Capel, B. (2020). Temperature-dependent sex determination is mediated by pSTAT3 repression of Kdm6b. Science368(6488), 303-306.

Synopsis edited by Jaclyn Everly, PhD (Anticipated: 2024), Illinois State University, School of Biological Sciences, and Naiomy Deliz Rios Arce, PhD, Lawrence Livermore National Laboratory, CA.

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