Anole Annals

The insulin signaling network has an essential role in growth, reproduction, and aging. Insulin-like growth factors, or IGFs, are important protein hormones within this network and are typically conserved across vertebrates. However, some proteins in the insulin signaling network have experienced selection in reptiles. Also, not a whole lot is known about the specific functions of components of this network within reptiles.

Amanda Clark, a PhD student in Dr. Tonia Schwartz‘s lab at Auburn University,  investigated the the function of purified IGFs on cell function for brown anoles (Anolis sagrei) and crested anoles (Anolis cristatellus). She had five different treatments for cell plates from both species: brown anole (BA) IGF-1, BA IGF-2, green anole IGF-1, a positive control, and a negative control. Cell proliferation was not different among all of the treatments, possibly due to incorrect protein folding or low concentrations of IGF. As expected, cell viability was also not affect by the IGFs. In the future, this experiment will be conducted again with increased sample size and an improved positive control.

Across species, bigger brains usually mean better cognition. But, this relationship rarely holds when considering individual differences within a species. Within species, the number of neurons in the brain may be a better proxy for cognitive ability than brain size. Further, the number of neurons may be independent of brain size.  But how to measure neuron number?

Levi Storks, a graduate student in Manuel Leal’s lab at the University of Missouri, set out to do just that. He adapted a protocol that has previously been used in mammals, birds, and crocodiles, but never before in lizards. In brief, he dissected the telencephalon, cerebellum, and other regions of the brain of an Anolis cristatellus and used the isotropic fractionator method to determine neuron number in each of the three. After homogenizing each tissue, he used a double-labeling technique with DAPI to stain each nucleus and neuronal nuclei antibody to stain each neuron, and used a hemocytometer to count the cells under magnification. Now that this protocol is working, look out for Levi’s future results on anole brain structure and cognition!

Muscle growth and development occur via different physiological mechanisms across the animal kingdom. Variation in behavioral uses of muscle may lead to the evolution of different muscle sizes across animal species. Different-sized muscles may vary in their capacity for strength or frequency of use and larger muscles may develop as the result of possessing higher numbers of muscle fibers, larger muscle fibers, or a combination of the two. Jesus Vega, an undergraduate student with Michele Johnson at Trinity University, was interested in learning how muscle size evolves across anole species by studying the retractor penis magnus (RPM), used to retract the hemipenes back into the tail.

Testing a hypothesis that larger RPM muscles will have more or larger muscle fibers, due to an expected evolutionary trade-off between fiber number and size, Jesus examined copulation behavior data and RPM muscle traits of 24 species of anoles. Behaviorally, there was no correlation found between copulation rate and RPM muscle fiber size or number. Physically however, species that have larger RPM muscles have more RPM fibers, species with larger muscle fibers have RPMs with more fibers, and species with larger bodies have more RPM muscle fibers and larger RPM muscles. These results show that larger muscles evolve due to increased muscle fiber size and number and also suggest that copulation behavior is not associated with muscle size evolution in anoles.

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!