CHS Blog

Investigating the Enduring Mystery of Temperature-dependent Sex Determination

May 01, 2021
Jessica Leivesley, PhD candidate, Njal Rollinson Lab, University of Toronto

Temperature-dependent sex determination

In my opinion, temperature-dependent sex determination (TSD) is one of the weirdest but most interesting things about reptiles. In animals, sex determination is typically thought of in terms of sex chromosomes. For example, in species with an XY sex-chromosome system (such as humans), embryos possessing two X chromosomes will be genetically female, while those with both an X and a Y chromosome will be genetically male (there is also a ZW chromosome system in which ZZ is male and ZW female). But TSD is unusual and distinct in that there are no chromosomes involved. Instead, the incubation temperature that an egg experiences during development determines whether the embryo develops as a male or a female. Although not all reptiles display TSD, it’s almost universal in crocodilians, widespread in turtles, and found in some lizards as well as the Tuatara, sole surviving member of the ancient reptile order Rynchocephalia. Generally speaking, warmer temperatures are associated with the production of females, though in Tuatara, males result from warmer temperatures. Otherwise, the two main patterns observed are MF TSD (males produced at low temperatures, females at high temperatures), and FMF TSD (females produced at both low and high temperatures, males at intermediate temperatures).

TSD occasionally appears in the news because widespread feminization of populations is expected to occur within some TSD species due to current global warming trends. This phenomenon has been well studied in sea turtles: as temperature increases in certain locales, increased numbers of MF TSD hatchlings are predicted to be female, leading to populations that are nearly 90 per cent female [1]. The acceleration of climate change along with the novelty and interest in TSD has created a rush to understand why this trait is so important to these species, and if they can survive the rapid climate changes Earth is currently experiencing.

The Charnov-Bull Hypothesis

Although TSD was first discovered in 1966 by Madeleine Charnier [2], we still don’t fully understand the benefits the system provides over sex chromosomes. One idea, termed the Charnov-Bull Hypothesis [3], suggests that each sex is produced at the temperature that benefits it most. For example, in MF TSD species this would mean males are produced at cooler temperatures and females at warmer temperatures because each performs better at these respective regimes. Despite attempts to test this hypothesis, there’s still no accepted widespread mechanism that supports this framework. This is mostly because to date, traits found to follow the Charnov-Bull Hypothesis have been species specific; for example, in Painted Turtles, hatchling size follows the hypothesis, while in the Common Keelback Snake it’s swimming speed. (Note that the Common Keelback Snake is a species with sex chromosomes and not TSD, but tests of the Charnov-Bull in non-TSD species are still informative.)

Our experiment

The question of why TSD exists and how it is beneficial drew my attention at the start of my PhD, so I set out to design an experiment to test the Charnov-Bull Hypothesis in a way that had yet to be tried. First, we used a rarely measured trait—immune system strength—which is central to survival. All organisms must devote some of their energy reserves to having a strong immune system. This choice was inspired by my previous work with Soay sheep, in which we found evidence that investing in reproduction came at the cost of a weakened maternal immune system [4]. The immune system is also strongly linked to survival in early life, as young individuals with stronger immune systems are more likely to survive to adulthood.

We also wanted to test the Charnov-Bull Hypothesis under more natural conditions. Generally, tests have involved eggs incubated under two or more constant temperatures and a single trait then measured and compared across incubation regimes and both sexes. In the example of body size, for instance, males would be expected to be larger under male-producing temperatures and females larger under female-producing temperatures.

My PhD work is centered on a Common Snapping Turtle population in Algonquin Provincial Park. Common Snapping Turtles have FMF TSD, however, for the purpose of this experiment I ignored the cooler female-to-male transitional temperature range and focused on the upper male-to-female transition. In this system, 24°C represents a male-producing temperature and 28°C represents a female-producing temperature. Because constant temperature is an unnatural incubation condition and results from constant-temperature experiments generally don‘t hold outside of the laboratory, we incubated eggs under two constant (24°C, 28°C) and two variable (24 ± 4°C, 28 ± 4°C) temperature regimes. Finally, an exciting aspect of the experiment was to “decouple” sex and temperature by applying a hormone treatment (estrogen blocker) to create males at female-producing temperatures. In this way we could determine if males produced at female-determining temperatures had a weaker immune response than males produced at male-determining temperatures.

Sterile experiment setup

The immune system is a complex organization of cells, antibodies, and numerous types of responses that keep organisms safe from invading foreign material. There are many ways to measure the strength of the immune system, but each method only measures one part of the complex response. We chose to carry out a bacteria-killing assay, in which we added a novel strain of E. coli to hatchling blood to see how well the blood killed the bacteria. This was accomplished by measuring how much light passed through the mixture (spectrophotometry). The more light that passed through a sample, the fewer bacteria remained. This suggested how well hatchlings might respond to an infection with their first line of defense—the innate immune system comprised of bacteria-killing cells—before they had fully developed their adaptive immune system (antibodies, etc.).

Bacteria-killing assay

Our results

Contrary to expectations, males did not have a stronger immune response at 24°C than at 28°C. Nor did females have a stronger immune response at 28°C compared to 24°C. Thus, we found no support for the Charnov-Bull Hypothesis; perhaps the immune system isn’t the key to unlocking the enigma of TSD. We did, however, find that hatchlings incubated at constant temperatures had weaker immune response compared to those incubated under variable temperatures. Finally, and interestingly, we found that hatchlings from larger eggs had a better immune response than those from smaller eggs.

Overall, the evolutionary and adaptive significance of TSD remains unresolved, but we now know that using constant incubation temperatures might mask effects that we’re interested in and provide results that differ from those that could accrue under more natural temperature variation. Here in the Rollinson Lab, we have more experiments in the pipeline aimed at disentangling why TSD might be important—so watch this space!

You can find the full article on the Internet at this DOI: 10.1242/jeb.237016.


  1. Jensen, M. P., Allen, C. D., Eguchi, T., Bell, I. P., LaCasella, E. L., Hilton, W. A., Hof, C. A. M. and Dutton, P. H. (2018). Environmental Warming and Feminization of One of the Largest Sea Turtle Populations in the World. Curr. Biol. 28, 154–159.
  2. Charnier, M. (1966). Action of temperature on the sex ratio of the Agama agama (Agamidae, Lacertilia) embryo. C. R. Seances Soc. Biol. Fil. 160, 620–622.
  3. Charnov, E. L. and Bull, J. (1977). When is sex environmentally determined? Nature 266, 828–830.
  4. Leivesley, J. A., Bussière, L. F., Pemberton, J. M., Pilkington, J. G., Wilson, K. and Hayward, A. D. (2019). Survival costs of reproduction are mediated by parasite infection in wild Soay sheep. Ecol. Lett. 22, 1203–1213.