Anole Annals

Neuropeptide Y (NPY) is a hypothalamic appetite-stimulating regulator of food intake that has been suggested to interact with components of stress response, including the release of the stress hormone corticosterone (CORT). Recent work suggests that NPY can interact directly with the adrenal gland to promote CORT secretion, raising the question of whether NPY can stimulate a stress response and whether NPY requires an active stress response to regulate food intake. This interaction has been examined in mammals but the role of NPY has not been explored in reptiles. To answer questions about the relationship between NPY, stress, and food intake in reptiles, Micaela Castro, a student with H. Bobby Fokidis at Rollins College, performed two manipulative experiments, one in the field, and one in the laboratory, on the brown anole (Anolis sagrei). These experiments utilized injections of NPY and dexamethasone (DEX), an agonist that inhibits CORT secretion, to test the hypotheses that NPY promotes CORT secretion and food intake and that CORT secretion is required for NPY to exert its appetite-stimulating effect.

In the field, adult male brown anoles were captured and injected with varying levels of either NPY, DEX, or saline as a control. An hour after injection, blood was collected and CORT levels were measured. From this study, it was found that NPY injections promoted CORT secretion while DEX injections decreased CORT secretion relative to the saline control. In the laboratory, adult male brown anoles were fasted for either 24 hours or 48 hours, injected with either NPY, DEX, DEX followed by NPY after an hour, or saline as a control, and were observed for differences in food intake. From this study, it was found that DEX injections decreased food intake relative to controls while NPY injections increased food intake relative to controls, but only when anoles were fasted for 48 hours. DEX injections followed by NPY injections resulted in similar food intake to control animals. All together, these results suggest that NPY and CORT are codependent, with NPY capable of stimulating CORT secretion and CORT being required by NPY for it to exert its appetite-stimulating effects.

Sperm storage is widespread in all major reptilian taxa and in combination with multiple mating it could have indirect benefits in polyandrous systems for example by increasing genetic diversity among offspring. Hannah Marshall, a junior majoring in Biomedical Sciences at Auburn University in Tonia Schwartz’s lab, set out to test the utility of microsatellite markers in paternity analysis in a population of brown anoles, Anolis sagrei, in Florida and to assess the extent and pattern of sperm storage from field matings.

Brown anoles from the field were housed in pairs (control) and in groups of four (2M:2F) and six (3M:3F) in 23 experimental laboratory enclosures. Eggs were collected over one breading season and hatchlings and their candidate parents were genotyped at seven microsatellite loci. The software CERVUS was used to determine the most probable parental pair for each hatchling and to disentangle paternity from experimental males to sperm storage.

Results show that these markers are sufficiently polymorphic to allow paternity assignments with high confidence. With regards to the use of stored sperm, 58% of the eggs produced in the lab were from field matings, which is consistent with previous findings in Anolis sagrei. However, Hannah’s data suggest that these lizards continue to use their stored sperm up to 4 months, longer than previously documented.

These findings are preliminary and Hannah is currently collecting and analyzing more data from these experimental enclosures. Understanding the dynamics of reproductive output in this focal population is valuable for planning further experiments to measure fitness.

Urbanization changes many factors, such as temperature and food availability, that influence body size in animals. Last year at SICB, Zach Chejanovski presented on this topic in brown anoles from Miami (Anolis sagrei). He found that predator (curly-tailed lizards) abundance was highly associated with body size in anoles. As predator abundance increases, anole body size increases. Chejanovski, a PhD student at the University of Rhode Island, then formed a new question based on his previous findings: Are larger anoles actually predated on less often than smaller anoles?

Male brown anole showing his dewlap. Photo by Renata Brandt

To answer this question, Chejanovski performed a tethered intruder experiment with male brown anoles of variable sizes. For each trial, he tied an anole at the end of a pole and presented the anole to a curly-tailed lizard. He then recorded the amount of time for the predator to get within 20 cm of the anole. Results from a survival analysis show that smaller lizards were attacked more often and more quickly than larger anoles. According to this experiment, larger body size in brown anoles results in less predation from curly-tailed lizards. However, is body size genetically determined?

Curly-tailed lizard

Chejanovski then set up  a common garden experiment with female anoles from urban sites with and without curly-tailed lizards. Eggs were collected from these anoles, incubated, and allowed to hatch. Hatchlings were raised in identical lab conditions and measured for body size to calculate growth rate. Male anoles from predator sites grew faster than males from non-predator sites. These results suggest that body size has some genetic control in males. However, female growth rates did not differ between sites. The discrepancy between sexes may be due to different selective pressures, such as sexual selection. This work highlights the importance of body size  in urban environments with predators.

Urbanization is a global issue that alters the way many natural populations survive and reproduce. The construction of new developments, housing, and other man-made structures alters the environment available to many species of lizard, and anoles perhaps most famously. Urban anoles in Florida and other parts of the southern United States are a common feature in many cities, why, everybody that attended SICB 2015 in West Palm Beach, Florida remarked that there were anoles on almost every tree! Particularly, the addition of artificial and smooth substrates poses a concern to many species of arboreal lizard that need rough and heterogeneous surfaces in order to climb and run effectively. The differences in structural habitat available to these anoles can in turn affect their morphology, leading to evolutionary changes in body shape and form over time to better adapt to urban lifestyles. Andrew Battles, a PhD student with Jason Kolbe at the University of Rhode Island, recognized this problem and designed a clever experiment to understand just how smoother surfaces impact the running ability of anoles.

Andrew sampled crested anoles from two sites: an urban and a natural site, and used a series of running experiments to understand how the addition of smooth, urban substrates affects the ability of anoles to move. Using two different inclinations (37 and 90 degrees), 2 substrates (smooth and rough), and the running power of 13 crested anoles, they found that anoles exhibit a decrease in speed due to increasing incline, and exhibited slower speeds on a smoother track relative to a rougher one. They also found that stride length decreased on smooth and vertical tracks, and that urban and natural anoles responded similarly to these changes in substrate and incline. They also found that anoles will try to change their gait and increase their stride width due to incline, but not so much on different substrates. Their major take-away was that smoother substrates do decrease lizard sprint performance, which is a fundamental trait for a lizard to survive and reproduce. And while there are no differences between habitat types, the build-up of urbanization over time might lead to evolutionary shifts for crested anoles in urban environments so that they might better adapt and live in cities. Keep up the stellar experiments, Andrew!

As many of us I’m sure know, many lizard species have the ability to lose their tail in order to escape predators or competitors. However, the tail doesn’t grow back as originally lost! The bone, muscle, and fat is replaced by a cartilaginous rod. Lizard species that use their tail in social communication might suffer a severe cost associated with losing their tail because they might lose their ability to communicate information to predators or members of the same species. Amy Payne, a student with Michele Johnson at Trinity University, recognized this issue and used a cross-species comparison to determine just what factors influence tail breakage.

Amy set out to test the hypotheses that species that use their tail in a social context will have lower rates of tail loss, species that use their tail in a predatory context will have higher rates of tail loss, and that species that primarily store fat in their tail will have lower rates of tail loss. They used curly-tail lizards, earless lizards, house geckos, and crested and green anoles in their work and found that curly tail lizards exhibit the greatest tail loss despite using their tails constantly. Earless lizards exhibited the lowest rates of tail loss and they used their tails quite frequently, and anoles had intermediate rates of tail loss based on social use. Tail use in a predatory context was rare for anoles, but quite common for curly tails (that exhibited a higher rate of tail loss) and earless lizards (that exhibited a lower rate of tail loss). They also found no relationship between tail autotomy and energetic storage in the tail. Their major takeaways are that species that use their tail very frequently in communication can exhibit either large or low rates of tail loss, and that there are a lot of intermediate rates of tail loss perhaps due to the multi-faceted role tails play in the evolution, ecology, and behavior of lizards. They are going to follow up this work using bomb calorimetry to more precisely measure species differences in the energy stored in the tail, and increasing the species sampling to incorporate more species (especially more anoles!)

Anolis RPM muscle cross-section. The darkened area in the middle of the muscle shows white blood cell infiltration, which indicates damaged tissue.

Martin measured the muscle damage of the RPM for 5-10 males in 27 different species of anoles. This estimate was made by calculating the cross sectional area (CSA) of the RPM and the CSA area of muscle damage of each muscle. He then calculated a ratio of muscle damage (damaged CSA / total CSA) for each RPM and then averaged to give each animal a single value. These species were also observed in the field to measure an observed copulation rate (totaling ~1000 hours of observation). He and his collaborators used phylogenetical generalized least squares regressions to test for a correlation between observed copulation rates and the average ratio of muscle damage across these species.

Positive correlation between observed copulation rates and the average ratio of damage of the RPM for 28 species of anole.

They found a significant and strong positive correlation between these estimates, suggesting that examining muscle damage may be an efficient way to estimate behavioral rates. Martin drove home the point that measuring the damage in the RPM of these species took him 2 orders of magnitude less time than estimating copulation rates in the field. This suggests that researchers may be able to more easily estimate behavioral rates of different species, as well as examine individual variation within species. In the future, this group hopes to explore the relationship between muscle damage, copulation rate, and recovery so they can more accurately describe the window of behavior they observe through muscle damage.

The color change from a bright, vibrant green to a dull, muddy brown is one of the most characteristic qualities of the green anole (Anolis carolinensis) (I am aware that some dewlap enthusiasts may take issue with this statement). Given the dramatic differences between the colors, Daisy Horr, a Junior in the Johnson Lab at Trinity University in San Antonio, TX, wondered how social behavior and body temperature may influence body color, and whether these relationships differ between males and females.

First, Daisy observed green anole social behavior in Palmetto State Park, TX, and found that males exhibited green body color more frequently than females, and males that performed more pushup and head bob displays also changed colors more frequently (between green and brown). She also found that females were more often green during social interactions.

Because green anoles are ectothermic, more commonly known as cold-blooded, ambient temperature plays an important role in nearly every aspect of their lives. Therefore, there might be a relationship between body color and thermoregulation. Daisy spent a lot of time searching the Trinity University campus for green anoles, and measured body temperatures, the temperatures of the perch on which she found them, and the distance to the nearest alternative perch (a measure of exposure). While she did not find any association between body color and temperature, she did show that males used warmer substrates than females, and that males were typically more exposed (greater distance to the nearest perch). Additionally, she found that males are generally greener than females throughout the day.

Daisy plans to pursue graduate school after she finishes at Trinity University, and we all hope she continues to contribute to anole research! Graduate advisors, you don’t want to miss out on a fabulous researcher!

(This post’s author’s diploma from Trinity University does not affect his assertion that Trinity graduates are among the best minds in biological research.)

Effects of urbanization pose major challenges to biological systems globally. One example that impacts the thermal environments of urban areas is the urban heat island effect, where urbanization creates an environment that is hotter than nearby natural areas. In Shane Campbell-Staton’s talk “Temperature-mediated shifts in performance and gene expression between populations of the Puerto Rican crested anole in natural and urban habitats” he sought to investigate divergence in thermal physiology and gene expression between urban and natural populations of anoles in Puerto Rico.

In situ, he investigated whether there were differences in urban and natural microhabitats, lizard thermal tolerance between urban and natural populations, and if there were differences in thermal physiology if this was a plastic or genetic response. He found that urban microhabitats were warmer, and that lizards from urban environments maintained function at higher temperatures when compared to their natural environment counterparts. This increase in thermal tolerance is a plastic response in the urban lizards. He then investigated the transcriptomics to investigate if there is evidence for temperature-mediated selection in urban heat islands, and found that selection on ancestral plasticity may play a role in acclimation to urban heat stressors. Future work includes identify genes involved in this accommodation. Amazing things to come!

Leah Elkhoury, an undergraduate student in the Fokidis Lab at Rollins College, investigated whether access to food would increase agonistic behaviors in the notoriously aggressive brown anole. First, to see if fasting would affect brown anole aggression, she tested wild caught brown anoles by feeding control lizards a regular diet and fasting treatment lizards for either 24, 48, or 72 hours. The treated anoles were then size-matched with a control lizard and placed in an enclosure, where their agonistic displays were recorded and analyzed.  The results of that portion of the experiment indicated that fasting does not increase agonistic behavior.

Next, lizards were separated into two groups: one with predictable feeding schedules, and one with a randomized feeding schedule for one month. This lizards then were tested for aggression in a similar way to the first experiment. The anoles were then sacrificed, and their blood was tested for stress steroid, fat mobilization, and fat deposition. Their brains were then collected and regionally divided to in order to measure stress steroid. Interestingly, lizards in the unpredictable feeding group exhibited more dewlap flashes, one of the brown anole’s agonistic behaviors. They also showed an increase in fat deposition and plasma corticosterone (stress steroid).  Though there were no differences in corticosterone between brain regions.

These exciting results show the “choices” brown anoles make about energy allocation under stress, and Leah plans to continue to ask questions about the link between feeding and aggression. We can’t wait to hear about her work in the future!