Anole Annals

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!

Urban environments are spreading and can influence the biotic and abiotic components of an ecosystem. Brown anoles (Anolis sagrei) that live in urban environments are larger in body size than their counterparts that reside in more natural areas. Body size is important for brown anoles because larger individuals can run faster, eat larger prey, and are more competitive. But what’s driving the variation in body size across urban to natural environments? Zac Chejanovski, a PhD student at the University of Rhode Island sought to answer this question.

Chejanovski did his research in south Florida at 38 sites along an urban to natural gradient. He measured food availability, abundance of conspecifics, and abundance of a known predator of anoles, the curly-tailed lizard. He also captured 15 male and 15 female anoles and measured body size, body temperature, and the thermal environment.

Curly-tailed lizard abundance had a strong positive relationship with body size for both male and female anoles. There was a weak positive relationship between the thermal environment and body size for males, but not females. Neither food availability nor abundance of conspecifics were related to body size. The findings of this research suggest that predation pressure from curly-tailed lizards might be one factor driving selection on body size variation in brown anoles.

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

Samantha Adams presenting at SICB.

The parietal eye, a photosensitive organ located on top of lizards’ heads, has long been thought to play an important role in regulating lizards’ circadian rhythm and body temperature. The eye detects UVB rays, mediating the release of melatonin from the pineal gland and evoking a behavioral response. Samantha Adams, an undergraduate at Marosh Furimsky’s lab at Westminster College, PA, conducted a study on bearded dragons (Pogona vitticeps) and green anoles (Anolis carolinensis) to examine the effects of masking their parietal eye on their thermoregulatory behavior. Adams took individuals from each species and set up a testing arena, placing a UV-B light-only source and an infrared heat-only source on opposite sides. She then tracked the amount of time each lizard spent under either lamp, doing so before masking the parietal eye, while the eye was painted with black non-toxic paint, and again after uncovering it.

Adams found that bearded dragons rely heavily on the eye for thermoregulation – while lizards ordinarily spend less than 20% of their time basking underneath the heat source, lizards with their parietal eye masked spent the vast majority of their time under the heat lamp. Additionally, all of her bearded dragons experienced erratic locomotion in the day post-masking, running so frequently from side to side of the cage that she had trouble characterizing the lizard’s lamp preference. Once the black paint was removed, lizards took one to two weeks to resume ordinary behavioral patterns. However, she found that the green anoles in her study seemed unaffected by the experimental manipulations; lamp preference was unchanged by covering the parietal eye, and anoles spent roughly 25% of their time under the heat lamp in both the control and experimental treatments. The anoles displayed none of the erratic locomotor patterns Adams found in the bearded dragons, and other than a qualitative account of more time spent being brown, the lizards seemed unperturbed by the black paint on their parietal eye.

Adams’ results shine a new light on the parietal eye, long thought to be a structure essential to all lizards that possess it, as a potentially vestigial structure in green anoles. Future work is necessary to understand any other roles the eye could serve in anoles, but the stark difference with bearded dragons in this study helps illuminate the wild evolutionary path of our favorite lizards.

Hello from unexpectedly cold New Orleans! Our coverage of SICB 2017 continues and we hope you will enjoy the increased posting over the next few days.

On Friday, David Delaney presented more data from his Master’s thesis from Dan Warner’s lab further exploring how adult and juvenile A. sagrei interact. Previously David showed that the presence of adults did not affect microhabitat use, but that high densities of adult males decreased survival in juveniles, especially in smaller lizards.

This year David presented intriguing data suggesting that the presence of adult males influenced how juveniles sat on their perches. He found that the presence of adult males alter both horizontal and vertical orientation of juveniles. When one male is present, juveniles increase horizontal orientation over time, but if three males are present, juveniles face upwards more than when no or only one male is present. Additionally, they also face the trunk of the tree more than away from the tree when they are horizontal and adults are present. Because of the chance of juvenile saurophagy, David suggests this helps juveniles to monitor where the adult males are to prevent being eaten.

Check back often to read about more great research being presented at SICB 2017!