Anole Annals

Doctoral candidate Ariel Kahrl presents her research on sperm evolution at SICB 2017.

Every year since 2013, the Division of Ecology and Evolution (DEE) hosts the Huey Award Symposium at the annual SICB meeting. The Huey award is given for the best student presentation in DEE, in honor of Ray Huey, professor emeritus at the University of Washington. Ray’s career featured a lot of key research on anoles, and so there is often good representation by anole biologists at the Huey award. At this year’s symposium, Ariel Kahrl, a graduate student in Bob Cox’s lab at the University of Virginia, presented her research on pre- and postcopulatory selection in Anolis lizards.

We know that male competition for mates occurs both before copulation (mating success) and after copulation (sperm competition). Her research focuses on investigating the evolutionary connection between these two phases of competition. She found that larger males have smaller relative testis size, indicating a tradeoff between pre- and postcopulatory success, as larger males will have better success gaining access to females, but less sperm available for mating.

When she looked at testis and sperm morphology in greater detail, a few interesting patterns emerged. First, she found that testis size evolves faster than body size, consistent with other studies showing that reproductive organs evolve faster than other body traits. She also found that the midpiece section evolved faster than the head and the tail of the sperm. Importantly, the midpiece section of the sperm was strongly associated with male condition and sperm swimming speed, whereas the head and tail of the sperm were not associated with male  condition or sperm swimming performance. She further hypothesized that sperm count may be a more important target of selection than sperm morphology.

The effects of climate change and urbanization on reptiles and amphibians has been a major topic at this year’s SICB. Both are expected to cause drastic changes in the climate, which will likely be severely detrimental to many species. We hope that many species will be able to tolerate these changes by adapting or acclimating, either by thermoregulating or changing their physiology. Adults of many species are able to acclimate in this way, but Josh Hall of the Warner lab wanted to test if eggs (which are unable to move to thermoregulate) are able to acclimate their physiology in response to higher average temperatures and to spikes in temperature that you might find in urban environments. Josh collected two populations of wild A. cristatellus from Miami, an urban population and a forest population, brought them back to the lab, and collected their eggs.

Josh Hall’s design, with 5 temperature regimes. City=light blue, Forest=green.

He then put the eggs into five different thermal conditions 1) higher “urban” temperatures, 2) cooler “forest” temperatures, 3) “urban” temperature with a large temperature spike on day 8, 4) “forest” temperature with a large spike on day 10, and 5) “forest” temperature with a small spike on day 10. He predicted that eggs that had a higher baseline temp (i.e. the urban eggs), would be able to tolerate spikes in temperature better than eggs at lower temperatures and would have higher survival and less of a physiological stress response due to the temperature spike. Contrary to his hypothesis, he found that high temperatures, and spikes were both detrimental to the survival of eggs and hatchlings, and affected baseline and max heart rate in embryos. This is somewhat concerning because it suggests that even a single short burst of heat can kill embryos, and have lasting affects on juveniles. The bursts, which you might expect in urban areas, have a more profound affect when the background temperature is higher, which will likely happen due to climate change.

Effects of incubation treatments on embryonic heartbeat, egg and juvenile survival.

Image from Miguel Webber.

Frequent readers of Anole Annals are likely to recall the amazing convergent evolution of morphology related to habitat use in Caribbean anoles that coincides with similarly striking convergent evolution of social behavior. Most of what we know about behavior of Caribbean anoles is how males behave: there are major differences among ecomorphs in how often males use their colorful dewlaps and how often they mate with females. Such male-typical behavior seems intuitively linked to species differences in testosterone signaling. Previous work has shown, though, that these differences do not seem to be related to levels of testosterone in the blood, so Miguel Webber of Michele Johnson’s lab at Trinity University examined whether the receptors for testosterone varies in a manner consistent with the behavior for six Dominican Republic species of anoles and one U.S. species.

Hormones can only cause effects on tissues that have receptors for them, so Miguel looked at receptors for testosterone (androgen receptors) in the muscles responsible for moving those fabulous dewlaps (the ceratohyoid muscle), expecting to find a correlation across species between the number of androgen receptors in the muscle and the rate of dewlap extensions. Although the data are still preliminary, there was a trend for males with higher dewlap extension rates to have more androgen receptors in the ceratohyoid muscle. His next steps are to look for an association between rates of copulation and androgen receptors in the muscle used by males to copulate (retractor penis magnus muscle – yes, it does what you would guess based on the name…). He also wants to see if there is a correlation among species in the behavioral traits and androgen receptors in regions of the brain that are important for social behavior regulation.

Above: Dani Douglas presenting on her research on measuring heart rates of brown anole eggs at the annual Society for Integrative and Comparative Biology Meeting in New Orleans, Louisiana.

Heart rate can tell us a lot about how an animal’s physiology is influenced by environmental conditions. Even embryos can provide valuable heart rate information. Scientists have used the Buddy® system, a digital egg heart rate monitor, to measure heart rate in large eggs, such as those from chickens or iguanas. But can the Buddy® system detect heart rate measurements from much smaller eggs?

Cassie Guiffre, Austin Hulbert, and Dani Douglas, students at Auburn University working with PI Dr. Dan Warner, took heart rate measurements from brown anole (Anolis sagrei) eggs using the Buddy® system.

Compared to a chicken egg, a brown anole egg is tiny – smaller than a quarter. Guiffre, Hulbert, and Douglas kept the anole eggs in an incubator that varied temperature over the course of each day. At different points during the day, they removed eggs from the incubator to measure heart rate with the Buddy®.

The students were elated to find that the Buddy® system could reliably measure heart rate in those small anole eggs. This finding is especially exciting because the Buddy® system is non-invasive, so scientists can measure heart rate over the development of the eggs.

They also found that anole egg heart rates were positively correlated with temperature fluctuation. Anole egg heart rate was not related to the age of the egg.

All research comes with its own set of challenges. When each anole egg is removed from the incubator, its temperature begins to go down immediately. The students needed to measure heart rate quickly to avoid confounding effects of cooling, which can be tricky.

Challenges aside, it is promising thatthe students in the Warner lab ave confirmed that heart rate can be measured in tiny anole eggs.

The Johnson lab has another strong showing here at SICB 2017 with lots of presentations and posters! I stopped by two of their (many) posters on the evolution of reproductive behaviors and sexually-selected traits in anoles.

Adam Zeb, Amy Payne, and Hannah Hall presenting their posters at SICB 2017.

Adam Zeb and Amy Payne presented their poster that compared reproductive behaviors in anoles to the size of the neuromuscular junctions (NMJs) in the muscles responsible for dewlap extension (ceratohyoid) and hemipenes retraction (retractor penis). They predicted that species with higher dewlap extension rates and copulation rates would have larger NMJs because the NMJ is where the neuron communicates with the muscle fiber, initiating contraction. To ask this question they observed and collected 15 species of anoles from the Dominican Republic, Puerto Rico and the Bahamas. For each species they measured dewlap display rate, copulation rate, and also collected the ceratohyoid muscle and the retractor penis muscle. These tissues were sectioned and stained with acetylchloine iodide and lithium iodide to find the and measure the NMJs. This is still a work in progress, but preliminary evidence doesn’t suggest that NMJ area is correlated with retractor penis muscle size or with ceratohyoid size. However, there was a strong difference in NMJ size between those two muscle types, where the ceratohyoid has over 3x larger NMJs than the retractor penis muscle. This somewhat supports their original hypothesis that NMJ size would be correlated with use, as the dewlap is used much more frequently than the retractor penis muscle. Hopefully next SICB we’ll hear more!

Another Johnson lab member, Hannah Hall, has been working on a project to look at the relationship between pre- and postcopulatory traits in Anolis and to characterize the architecture of the Anolis testis. We know that Anolis have highly variable sperm morphology, but we do not know if a portion of that variation may be due to variation in the structure of the testis. To test this, Hannah collected testis sections from eight species of anoles, and measured the cross-sectional area of the seminiferous tubule, the lumen and  the epithelial height. She also collected measurements of sexual size dimorphism (as a proxy for the strength of precopulatory selection) and gonadosomatic index (GSI), which is the ratio of testis mass and body mass (as a proxy of the strength of postcopulatory selection). She found a negative correlation between SSD and GSI, suggesting a trade-off between pre- and postcopulatory selection. She also found significant positive correlations between cross-sectional area of the testis and sperm head size, and between lumen size and sperm tail size. This suggests that larger structures in the testis are needed to produce sperm with larger morphology. Hannah is still working on characterizing the testis structure of many anole species, so stay tuned for more developments!

Caty Tylan measuring anole footpad thickness as a indicator of immune function.

A major challenge for organisms is to protect themselves from pathogens, things in the environment, including other organisms or toxins, that can cause disease and harm them. Animals, including anoles, have several different types of immune responses by which they can respond to pathogens. Ecologists are particularly interested in how these immune responses work in natural settings, how effective they are at protecting organisms, and how other aspects of an organism’s environment, including diet, stress, and reproduction, may positively or negatively impact immunity. However, immune systems are very complex, and measuring immune function, especially in the field, can be quite challenging!

Ecoimmunologists have developed various procedures to test different aspects of immune function, but ideally these procedures should be validated, or proved to be meaningful, in each organism they are used in. The phytohemagglutinin (PHA) skin test is one such assessment of immune function that is commonly used. This procedure involves injecting a small amount of phytohemagglutinin, a plant protein that provokes an immune response in animals, into the tissue of an organism of interest, waiting a given amount of time, and measuring the resulting swelling which is then used as an index of immune response. Researchers like this test because it is minimally invasive, works in almost any species, and is simple and easy to do in the field. Though this test has been used in multiple reptile species, it has never been validated in a reptile.

Enter Caty Tylan, a Ph.D. student and diploma-carry veterinarian from Penn State University, who addressed this issue in her talk “Local and systemic immune response to phytohemagglutinin: Validation of the PHA skin test in the green anole, Anolis carolinensis.” Caty chose to validate this test in green anoles (Anolis carolinensis), a model anole species, using two different types of PHA, PHA-L and PHA-P, which are available to researchers. She injected PHA into anole footpads and compared swelling in those feet over 48 hours post-injection. By comparing swelling in these feet to those which were only injected with sterile saline, Caty showed that PHA does induce a swelling response over 48 hrs, and that this response is the same for both types of PHA. She also examined the white blood cell counts in these anoles and found that PHA-L, a more purified and specific PHA, induced the stronger lymphocyte response, an immune measure that many ecoimmunologists look to quantify. In the future, Caty will examine histological sections of injected anole feet to examine the local immune response to PHA injections and fully validate this assay. This work should allow effective use of the PHA assay in future anole research and support investigations into how various environmental variables affect cell-mediated immune function in reptiles.