Anole Annals

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.

Emily Watts with her poster in NOLA at SICB 2017.

Great new aggression work from the McGlothlin lab! Emily Watts, an undergraduate student, presented a poster on Saturday about differences in aggression between island populations of A. sagrei.

Previously, the McGlothlin lab quantified aggression of brown anoles from Eleuthera, Exuma, North Andros, and San Salvador in the Bahamas. They found that individuals from Exuma and North Andros were more aggressive than from Eleuthra and San Salvador. Emily wanted to know if these population-level differences were a result of genetics or of the environment.

To test this, she bred lizards from Eleuthra and Exuma in the lab using a full cross design and raised the offspring in a common garden environment. When the males were one year old, Emily used enclosure trials to quantify aggression for each individual. The results were not what she expected. She found that aggression was independent from where a lizard’s parent came from, but was instead influenced by the environment.

Next, Emily will be testing offspring from the North Andros – San Salvador crosses they also performed. Additionally, because aggression was not repeatable for individuals, Emily will be testing each male again against a mirror to quantify individual aggression independent of partner aggression.

Thanks for sharing, Emily! We look forward to next year’s presentation.

Sukalia Miller with her poster at SICB.

This post was written by Miguel Angel Webber, an undergraduate in Michele Johnson’s lab at Trinity University.

An animal’s position in a social hierarchy can influence many aspects of its behavior. In the green anole (Anolis carolinensis), a growing body of literature indicates that dominant males behave differently than subordinate males, and that these behavioral differences may be present even prior to the establishment of a dominance hierarchy. Sukalia Miller, a recent undergraduate in Walt Wilczynski’s lab at Georgia State University, designed an experiment to determine whether subordinate and dominant males differed in their rates of courtship, aggression, and exploratory behavior.

After taking baseline measures of behavior, size-matched green anoles were paired and allowed to establish dominance relationships over the course of six days. Following this pairing, each lizard was then tested again to determine post-pairing measures of behavior. Miller found that subordinate males did not differ from dominant males in their rates of aggression (as simulated in a mirror test) or courtship (tested by placing a female in the habitat) prior to pairing, but that bouts of aggression and courtship practically disappeared in the subordinate group after pairing. Additionally, Miller found that there was a trend in the dominant lizards, such that individuals with higher rates of pre-pairing aggression had higher rates of aggression post-pairing. However, no such relationship was found between pre- and post-pairing rates of courtship in either the dominant or subordinate group, and no Miller observed no differences in exploratory behavior between either group of lizards.

These results suggest that the influence of social status on behavior may be limited to social behaviors in green anoles, and furthermore, that these behavioral differences may not be detectable prior to the establishment of a dominant-subordinate hierarchy.

Maria Jaramillo with her poster, doing her best anole impersonation.

Behavior can be extremely variable, even within a species. To control for this inherent variability while assessing individual responses to a visual stimulus, some researchers have begun using videos in their experiments. Maria Jaramillo, an undergraduate student in Michele Johnson’s lab, is curious about how anoles process visual information and if videos and live encounters are processed similarly in the brain. On Saturday, Maria provided us with an update of her work.

For this experiment, Maria used 40 adult male A. carolinensis which were exposed to one of four treatments for 15 minutes: 1) another live anole, free to display; 2) a looped video of an anole, which displayed for about 30 seconds, waited 15 seconds, then displayed again; 3) a scrambled version of the previous video; 4) or a control video of a lizard perch. She recorded the behavior of these lizards, then sacrificed them in order to collect their brains.

Maria found that the lizards paid close attention to the live anole, the video of the anole, and the scrambled anole video, but significantly less attention to the control video. Interestingly, though, males displayed significantly less to the scrambled and control videos than to the live anole and the normal video.

Next, Maria will use immunocytochemistry to quantify c-fos+ neurons in five brain regions associated with visual processing and the social behavior network. c-fos is an immediate early gene that is transcribed when neurons are activated, and c-fos+ neurons were likely stimulated in response to the visual stimuli Maria presented. She will then use the ratio of ­c-fos+ neurons to total number of neurons in that brain region to quantify neural activity in the five brain regions and see how these differ between stimulus treatments.

This is really exciting work and we can’t wait to see the end result!