The Effect of Androgen Exposure on Cerebral Lateralization in the American Alligator (Alligator mississippiensis)

Abstract

The division of the brain manifests in lateralized physical behaviors, where specific tasks originate from one side of the body. Previous studies have shown that birds and reptiles mediate aggression in their right hemisphere and focus on opponents with their left eye. Degree of lateralization varies between sexes, likely due to androgen inhibition of lateralization in mammals, birds, and fish, but remains untested in herpetofauna. In this experiment, we investigated the effect of androgen exposure on cerebral lateralization in the American Alligator, Alligator mississippiensis. Alligator eggs were collected and incubated at female producing temperature with a subset dosed with methyltestosterone in ovo. Dosed hatchlings were randomly paired with control individuals and their interactions were recorded. The number of bites initiated by focus from each eye and the number of times an animal was bitten on each side of the body was recorded for each individual to elucidate cerebral lateralization in aggression. Control alligators had a significant bias towards left-eye bite initiation whereas androgen exposed alligators used both eyes indiscriminately. No significance was found in injury patterns. This study suggests that androgen exposure inhibits cerebral lateralization in alligator brains and corroborates right-hemisphere mediation of aggression, something previously unstudied in crocodilians.

Introduction

Cerebral lateralization is the separation of the brain into two hemispheres, each of which is specialized to perform specific functions (Rogers, 1990). The right hemisphere is used in processing strong emotions and spatial awareness while the left hemisphere is required for visual discrimination tasks and learning (Rogers, 1990). This neural organization often manifests in lateralized behaviors, with certain tasks originating from one side of the body. These behaviors include viewing objects such as food, potential mates and threats, turning behavior, and limb preference (Deckel, 1995; Hews & Worthington, 2001; Rogers, 1990; Vallortigara et al., 2002). Initially thought to be a uniquely human trait, researchers have discovered evidence of lateralized behaviors in more ancient lineages. Asymmetrical predation scars have been found on fossils of trilobites, indicating lateralized predator evasion (Babcock, 1993). In modern taxa, evidence of behavioral lateralization has been recorded in several species of mammals, birds, amphibians, and fish (Rogers and Andrew 2002), but has remained largely unstudied in reptiles. In more recent years, evidence of lateralization has been seen in the predatory responses of the ornate dragon lizard, Ctenophorus ornatus (Robins et al. 2005), escape responses of common wall lizards, Podacris muralis (Bonati et al. 2010), and several other reptiles, advancing the study of reptilian lateralization.

In avian and reptilian brains, retinal input is processed by contralateral superficial input layers in the brain (Güntürkün et al., 2020), indicating objects viewed with the left eye are processed with the right hemisphere. Studies conducted on birds and herpetofauna show consistent results: aggressive behaviors are elicited more readily when viewing stimulus with the left eye and discrimination of small objects uses viewing from the right eye (Rogers et al., 2013). These analogous results suggest all tested taxa mediate aggression in their right hemisphere and food drive with their left. By recording side bias during certain actions, it becomes increasingly clear where certain functions are processed in the typical brain.

The hemisphere in which tasks are processed is conserved within species, but the strength of lateralization varies depending on certain factors; one of which is sex (Pfannkuche et al., 2009). Not only do male humans show increased left-handedness, but studies have shown different patterns in lateralization between males and females among humans, birds, and fish (Schaafsma & Groothuis, 2011, 2011; Schwarz & Rogers, 1992; Zappia & Rogers, 1987). After finding sex-dependent asymmetry in chicks, follow up studies revealed these differences are likely due to sex steroid hormones (Adret & Rogers, 1989). Exposure to both estradiol and testosterone prevents asymmetry in the development thalamofugal projections (Rogers 2020; Rogers & Rajendra 1993). Testosterone exposure has also been shown to inhibit and sometimes reverse asymmetry in the lateralized behaviors of some non-human taxa (Schwarz & Rogers, 1992; Zappia & Rogers 1987), but the biological mechanisms are still largely unknown. A popular hypothesis postulates elevated levels of testosterone inhibit the growth of the dominant left hemisphere (Geschwind & Galaburda, 1985). By inhibiting the faster development of the left hemisphere, both sections grow more evenly, reducing lateralization and left-hemisphere dominance.

The effects of testosterone on lateralization and behavior have been studied in several species of mammals, birds, and fish, but have not yet been investigated in herpetofauna (Pfannkuche et al., 2009).. To better understand the homologous and analogous structures and functions in the avian and mammalian brain, archosaur brains need to be better understood. Crocodilians are the closest extant relative to Aves (Green et al., 2014) having diverged around 254 million years ago (Janke & Arnason, 1997), yet there have been no studies conducted on crocodilian cerebral lateralization. Understanding crocodilian neurobiology may be the first step in revealing cerebral evolution in archosaurs.

In this experiment, we investigated the effect of androgen exposure on cerebral lateralization in the American Alligator, Alligator mississippiensis. We hypothesized unexposed alligators will exhibit a left eye preference during aggressive encounters. Although unstudied in Reptilia, we predict American Alligators will respond similarly to other studied taxa, and that individuals exposed to testosterone will exhibit decreased lateralization. We also hypothesize that individual aggression behaviors should mimic eye use, with unexposed individuals biting predominantly on one side of their opponent relative to exposed individuals.

Methods

One hundred sixty-seven American Alligator (Alligator mississippiensis) eggs from seven clutches were collected from private property near Rockefeller Wildlife Refuge, LA and transported to Southeastern Louisiana University in June of 2020 for a separate experiment. Eggs were randomized and housed individually in two incubators at a female-producing temperature (28° C) to ensure a female sexual differentiation. Incubators were held at 95% humidity and temperature was maintained using a Penn A421 rheostat. Eggs were randomly assigned to a treatment group; control or MT (Methyl-Testosterone)-dosed, using a random number generator. MT-dosed eggs received a 5μL topical bolus of 100 μg/ml of 17α-MT in 95% ETOH solution at stage 18 (Ferguson, 1985) prior to the temperature sensitive period during which sex is determined. Control individuals received a 5μL topical bolus of 95% ethanol vehicle. This dosage for treatment groups is known to masculinize experimental alligator embryos in ovo (Murray et al., 2016). Hatchling alligators were individually marked via caudal scute removal and housed in individual opaque 36”×18” Rubbermaid bins with ~8 cm of water and a basking area on a 12:12 hour light: dark cycle. Feeding and water changes were performed biweekly.

Behavioral trials were conducted in pairs for a different experiment. Individuals were naïve to other hatchlings until randomly paired with the opposite treatment group (i.e. MT-dosed with control) in a 100-gallon opaque tub with 10 cm of water and a 12” × 12” basking rock under a heat lamp. Pairs were not allowed acclimation time because exploratory behavior that resulted in interaction between the pair was the focus of the trial. Trials were done in a locked room with no windows set to 77° F. Pairs were left together for one hour and recorded using a Panasonic digital camcorder on a tripod. One animal in each trial was marked on the back with a dot of pink non-toxic nail polish that was gently removed at the end of each trial. Researchers analyzing the recordings and quantifying behavior were naïve to the random marking.

Video footage was reviewed for aggressive interactions such as bites and attempted bites. Any biting attempt by an individual whether or not contact was made was counted. For each event, two values were recorded: the eye the biter used to view the opponent directly before the bite and the side of the opponent that the bite landed on. For statistical analysis, the number of bites originating from each eye and the number of times each side of an animal was bitten was collected for each individual. Two sample T-tests were conducted comparing biting events originating from the left eye to those originating from the right. Variances were assumed equal. The same test was conducted for bite side in all groups. Biting attempts, eye side preference, and bite were assumed to be independent of one another. Retaliatory bites, bites that occurred immediately being bitten, were not counted.

Results

After reviewing footage of 36 individual trials, 107 individual biting events were recorded and analyzed (Table 1). Overall, alligators exhibited left viewing bias when biting opponents (df = 68, t = 2.456, p = 0.017). In total, the left eye was used during biting 63.6% of the time (n=68) compared to 36.4% of bites originating from right eye focus (n=39). As a result of biting events, 55 injuries accumulated on the left side of the body while 52 injuries occurred on the right. No bias was noted in the distribution of injuries (df = 68 t = 0.249, p = 0.804).

Table 1.

Table showing the sum and percent values for each group and test conducted. “Eye Focus” is the eye side used to focus on the opponent while “Bite Side” values are the side of the opponent the bite landed on. The bolded control group in the eye focus test is the only group in which there was a significant difference between the left and right side.

Test	Group	Left Side	Right Side	% Left	% Right
Eye Used	Control	68	45	60.2	39.8
(Initiated) Testosterone Treated		23	16	59	41
Side Bitten	Control	38	30	55.9	44.1
(Received) Testosterone Treated		17	22	43.6	56.4

For control alligators, there was a significant bias towards left eye use during biting events (df = 52, t = 2.711, p = 0.009) (Fig 1.A). Out of the 68 events, the left eye was used to focus 66.2% of the time (n=45). Control organisms focused with the right eye during biting encounters only 23 times. Compared to control alligators, specimens exposed to androgens had no eye preference (df = 14, t = 0.884, p = 0.392) (Fig 1. B). Androgen exposed individuals were just as likely to bite while focusing with the right eye (n=23) as the left (n=16). Control specimens did not exhibit bias in the side of injury when bitten (df = 68, t = 0.249, p = 0.804). Further, control specimens had the same likelihood of biting a conspecific on their left side (n=38) as on their right (n=30). There was also no bias found in the injured sides of those bitten by testosterone exposed alligators. As with control trials, the likelihood of a specimen with increased androgen biting a conspecific on the left (n=17) is the same as on the right (n=22). Although not statistically significant, it is interesting to note that unlike any other category, the right side had a higher total number of injuries for individuals facing androgen exposed partners.

Figure 1.

Graphs comparing eye side use side bitten between control and androgen exposed alligators. A. In control alligators, individuals were more likely to use their left eye when focusing on an opponent before attacking. Only 23 of the 68 events began with right eye focus. B. Alligators exposed to testosterone did not have any preference when biting opponents, attacking with an eye focus split of 59% and 41% split. C. Control alligators bit opponents evenly on the left and right side, causing a total of 38 bites on the left and 30 injuries on the right. D. Alligators exposed to testosterone also bit opponents evenly, causing 17 injuries on the left and 22 injuries on the right.

Discussion

Here, we tested the effect of in ovo androgen exposure on cerebral lateralization in the aggressive behavior of crocodillians, a previously untested vertebrate clade. Control individuals (female) viewed opponents preferentially using of their left eye before aggressive encounters, whereas female individuals exposed to androgens exhibited a lack of lateralization in their viewing behavior. Although no studies have been conducted on other crocodilians, previous studies on species of Squamata show corroborative results. During aggressive encounters, members of Anolis preferentially look out of their left eye (Deckel, 1995), and male tree lizards (Urosaurus) using their left eye when viewing intruding males responded more aggressively (Hews & Worthington, 2001). These finding support the notion that, like many other taxa, aggression is mediated in the right hemisphere of the alligator brain and is androgen dependent as opposed to sex.

For androgen-exposed individuals, there was no difference between left or right eye use when exhibiting aggressive behavior, indicating that androgen exposure impacted lateralization. In domestic chicks (Gallus gallus), administration of testosterone to the egg early in development inhibited and slightly reversed asymmetry in formation of thalamofugal visual projections (Schwarz & Rogers, 1992). These results suggested that testosterone inhibited, instead of reversed, lateralization, and the effects of androgen exposure on the alligator brain mimic the effect of darkness on the chick brain. Rogers (1990) speculates that during chick development in the egg, the right eye is facing out and exposed to light. Light stimulates the growth of neural projections in the left and forebrain, creating asymmetry (Rogers, 1990). Chicks hatched in completely dark environments don’t have this asymmetry and are subsequently less lateralized. In a study comparing highly and poorly lateralized chicks, chicks with high lateralization responded faster when a simulated predator appeared on their left side than their left. Those that were poorly lateralized responded uniformly when approached from either side (Vallortigara et al., 2002). The lack of discrimination in eye use of MT alligators during aggressive encounters indicates that androgens inhibit lateralization in the crocodilian brain.

Both the control and androgen-exposed specimens were indiscriminately bit on both sides of the body. Although control individuals were viewing out of their left eyes preferentially, bites did not accumulate on the right side of the body. During biting events, opponents were not always oriented parallel to one another, so bites were biased based on orientation. During non-aggressive behaviors, lizards use both eyes indiscriminately (Deckel, 1995). If opponents are not displaying aggressive behaviors when bitten, the likelihood of them viewing conspecifics with either eye, and exposing either side, is equal.

Organisms with decreased lateralization have difficulty completing certain tasks compared to those with normal asymmetry. Lateralization is correlated with multitasking ability, as poorly lateralized chicks are unable to efficiently forage while simultaneously scanning for predators (Dharmaretnam & Rogers, 2005). When poorly lateralized chicks are able to detect predators, it is often much later than their lateralized individuals (Rogers, 2008). These characteristics accompanying decreased lateralization negatively impact survivability (Rogers, 2008). Inability to discriminate between food and nonfood items increases the likelihood of ingesting harmful material, decreased predator detection increases susceptibility, and this inability of multitasking can result in unnecessary energy expenditure.

Here, we use a synthetic androgen to organizationally masculinize female alligators in ovo to isolate the effects of androgens on lateralization without the bias of sex. However, these findings also have applied science implications to crocodilians exposed to endocrine disrupting contaminants (EDCs). 17α-methyltestosterone (MT) is an environmental androgen known to bias sexual differentiation in wild American crocodile populations in Guanacaste, Costa Rica (Murray et al. 2015, 2016, 2017). Results from the present study may indicate that exposure to MT during development will inhibit lateralization in wild crocodilian populations, adding to the already-present conservation concern.

In summary, we tested the effects of in ovo androgen exposure on cerebral lateralization in a crocodilian model organism. Increased exposure to androgen potentially inhibits cerebral lateralization, impacting crocodilian behavior. To predict the extant of which this finding may impact natural populations, we recommend behavioral studies comparing highly and poorly lateralized individuals. Previous research exploring androgen effects on lateralization focused on the relationship between cerebral development and sex. This study provides framework to investigate the organization effects of hormones on cerebral lateralization in organisms without the confounding factor of sex. This study also supports right-hemisphere mediation of aggression, something previously unstudied in crocodilians.

• Alligators exhibit a left-eye bias during aggressive behavior encounters

• In ovo exposure to androgens inhibits behavioral lateralization

• Crocodilians mediate aggression with their right cerebral hemisphere

• Androgens likely inhibit reptile cerebral lateralization