A comparison of non-surgical methods for sexing young gopher tortoises (Gopherus polyphemus).

Many turtle species have temperature-dependent sex determination (TSD), raising the prospect that climate change could impact population dynamics by altering sex ratios. Understanding how climate change will affect populations of animals with TSD requires a reliable and minimally invasive method of identifying the sexes of young individuals. This determination is challenging in many turtles, which often lack conspicuous external sexual dimorphism until years after hatching. Here, we explore four alternatives for sexing three age classes of captive-reared young gopher tortoises (Gopherus polyphemus), a terrestrial turtle of conservation concern native to the southeastern United States: (1) naive testosterone levels, (2) testosterone levels following a follicle stimulating hormone (FSH) challenge, (3) linear morphological measurements, and (4) geometric morphometrics. Unlike some other turtle species, male and female neonatal gopher tortoises have overlapping naive testosterone concentration distributions, justifying more complicated methods. We found that sex of neonates (<7 days old) is best predicted by a "random forest" machine learning model with naive testosterone levels and morphological measurements (8% out-of-bag error). Sex of hatchlings (4-8 months old) was predicted with 11% error using a simple threshold on naive testosterone levels, or with 4% error using a simple threshold on post-FSH testosterone levels. Sex of juveniles (approximately 3.5 years old) was perfectly predicted using a simple threshold on naive testosterone levels. Sexing hatchlings at >4 months of age is the easiest and most reliable non-surgical method for sex identification. Given access to a rearing facility and equipment to perform hormone assays, these methods have the potential to supplant laparoscopic surgery as the method of choice for sexing young gopher tortoises.

Endocrine indicators of a stress response in nesting diamondback terrapins to shoreline barriers in Barnegat Bay, NJ.

Anthropogenic stressors such as habitat loss are a global problem for wildlife. Coastal development in the United States has replaced estuary shorelines with hard erosion barriers. In Barnegat Bay, New Jersey, the diamondback terrapin (Malaclemys terrapin) encounters these barriers when approaching upland beaches for nesting. To determine the effects of shoreline barriers on this threatened species' nesting abilities, we measured adrenocortical response (i.e., stress response) by comparing natural corticosterone and testosterone levels of 91 terrapins following in situ exposure to either an experimentally blocked, or open nesting beach. In addition, we exposed 15 individuals, from various nesting beaches, to handling stress to identify acute corticosterone secretion, finding a significant increase over 60min to 8ng/ml. Corticosterone did not reach this level in terrapins exposed to barriers. Corticosterone and testosterone levels were not significantly higher among terrapins exposed to barriers compared to those at open reference beaches. This lack of a stress response suggests that terrapins do not physiologically respond to barriers when they approach nesting beaches and thus are not stressed. This may be due to an adaptive trait to help female turtles complete the nesting process despite the natural stresses inherent to coming on land. Our study suggests that this lack of stress response is also applied to non-natural, human made nesting barriers. If terrapins are not physiologically capable of adapting to shoreline barriers, future erosion control structures could support terrapin nesting with periodic upland access points. This endocrinological study provides a more quantitative approach to guiding management of anthropogenic stressors upon wildlife.

Strong male-biased operational sex ratio in a breeding population of loggerhead turtles (Caretta caretta) inferred by paternal genotype reconstruction analysis.

Characterization of a species mating systems is fundamental for understanding the natural history and evolution of that species. Polyandry can result in the multiple paternity of progeny arrays. The only previous study of the loggerhead turtle (Caretta caretta) in the USA showed that within the large peninsular Florida subpopulation, multiple paternity occurs in approximately 30% of clutches. Our study tested clutches from the smaller northern subpopulation for the presence of multiple paternal contributions. We examined mothers and up to 20 offspring from 19.5% of clutches laid across three nesting seasons (2008-2010) on the small nesting beach on Wassaw Island, Georgia, USA. We found that 75% of clutches sampled had multiple fathers with an average of 2.65 fathers per nest (1-7 fathers found). The average number of fathers per clutch varied among years and increased with female size. There was no relationship between number of fathers and hatching success. Finally, we found 195 individual paternal genotypes and determined that each male contributed to no more than a single clutch over the 3-year sampling period. Together these results suggest that the operational sex ratio is male-biased at this site.

Effects of training and testosterone on muscle fiber types and locomotor performance in male six-lined racerunners (Aspidoscelis sexlineata).

Testosterone (T) is thought to affect a variety of traits important for fitness, including coloration, the size of sexual ornaments, aggression, and locomotor performance. Here, we investigated the effects of experimentally elevated T and locomotor training on muscle physiology and running performance in a nonterritorial male lizard species (Aspidoscelis sexlineata). Additionally, several morphological attributes were quantified to examine other characters that are likely affected by T and/or a training regimen. Neither training alone nor training with T supplementation resulted in increased locomotor performance. Instead, we found that T and training resulted in a decrease in each of three locomotor performance variables as well as in hematocrit, ventral coloration, and testis size. Strikingly, neither the size nor the fiber composition of the iliofibularis or gastrocnemius muscles was different among the two treatments or a group of untrained control animals. Hence, the relationships among T, training, and associated characters are not clear. Our results offer important insights for those hoping to conduct laboratory manipulations on nonmodel organisms and highlight the challenges of studying both training effects and the effects of steroid hormones on locomotor performance.

Seasonal variation in testosterone and performance in males of a non-territorial lizard species.

The ability to perform key behaviors, particularly those associated with reproduction, has been linked to reproductive success. However, it remains unclear as to which underlying mechanisms govern variation in the performance capacities of these behaviors. Due to its ability to mediate physiological and morphological changes, the steroid hormone testosterone has been hypothesized to influence performance. This hypothesis, however, has not been tested in a broad range of taxa or mating systems. In this study, seasonal patterns of bite force and locomotor performance were quantified concurrently with circulating testosterone levels in males of a non-territorial polygynous lizard species (Aspidoscelis sexlineata). Bite force and one measure of locomotor performance were significantly greater in the breeding season than in the post-breeding season, and this seasonal variation in performance was correlated with seasonal changes in testosterone levels. These results suggest that the performance capacities of behaviors important for acquiring mating opportunities in this species are maximized during the breeding season by elevated testosterone levels.

Ultrasonography of reproductive structures and hormonal correlates of follicular development in female American alligators, Alligator mississippiensis, in southwest Louisiana.

Ultrasonography has been used effectively to study reproduction in a variety of reptile species, but its application to crocodilians has been relatively limited. We present results from a study testing the efficacy of using ultrasonography to monitor reproduction in the American alligator, Alligator mississippiensis. Ultrasound results were then compared with plasma hormone levels. A total of 124 females were examined during March, April, May, and early June (2001-2003). Ultrasound results were validated on a series of reproductive females (n=14) necropsied for other studies. Previtellogenic follicles, vitellogenic follicles, recently shelled eggs, fully developed well-calcified eggs, and atretic follicles were readily discernible with ultrasound in mature females. Reproductive structures were observed in 57 females of which 43 were actively reproductive, while 14 were non-reproductive, but contained large atretic follicles from prior years. Oviducts were discernible in females with eggs. Ovarian state was also correlated with hormone levels. These results are in agreement with previous studies that showed that 50% or less of the adult female alligator population is reproductively active in a given year. Ultrasonography can be used to make an accurate assessment of reproductive condition in wild alligator populations.

Variation in plasma lipids during the reproductive cycle of male and female desert tortoises, Gopherus agassizii.

Plasma triacylglycerol, phospholipid, cholesterol, cholesterol esters, fatty acids, and total lipids were measured in 30 female and 20 male desert tortoises (Gopherus agassizii) during the annual reproductive cycle in the eastern Mojave desert, Nevada. Blood samples were collected at monthly intervals from April to October. All lipid fractions, with the exception of free fatty acids, were significantly higher in female plasma than in male plasma in all months of the year. In contrast, free fatty acids were higher in male plasma than in female plasma in all months. The seasonal pattern in estradiol secretion mirrored that of triacylglycerol, phospholipid, cholesterol, and total lipid, all of which showed a significant correlation with the hormone. Estradiol and the vitellogenesis-associated lipids were all significantly higher in August, September, October, and April than in June. The seasonal variation in cholesterol ester levels in females did not correlate with any of the reproductive events and did not appear to be involved in yolk precursor formation. Total lipid in males showed a negative correlation with testosterone and spermatogenesis. Individual fatty acids in the June and August samples (at the highest and lowest estradiol levels) were compared in male and female plasma. The percent of C18:3n3, C18:2n6, C18:1n9, C20:5n3, and C22:5 were significantly higher in the June female plasma sample than in the August sample. Docosahexanoic (C22:6n3) acid was barely detectable in female plasma in either month.

Puberty in male Japanese macaques (Macaca fuscata): Social and sexual behavior in a confined troop.

The social and sexual behaviors of four groups of males (4, 5, 9, and 10 years old) were compared throughout one complete mating season in a confined troop of Japanese macaques (Macaca fuscata). The aim of this research was to document social behavior changes that occurred during adolescence and to relate them to changes in sexual behavior that occurred at puberty. The 4-year-old males all mounted females and ejaculated but they had significantly fewer ejaculations and female partners than did the older males. The 4-year-old males also showed a pattern of delayed development in tail carriage and courtship, and they played more frequently than the older males. There were no significant differences in the frequency of aggression among the groups, but both 4-year-old and 5-year-old males were displaced by adult males and females more frequently than were the older males. We concluded that the development of adult patterns of social behavior in this genus is not climactic, but occurs slowly one to two years after physiological puberty has been reached.