Allan R. Woodward

Serum Concentrations of Various Environmental Contaminants and Their Relationship to Sex Steroid Concentrations and Phallus Size in Juvenile American Alligators

Abstract. Recent studies have reported a number of abnormalities in the hatchling and juvenile alligators of Lake Apopka, FL (USA). These abnormalities include modifications of plasma concentrations of sex steroids in males and females as well as abnormalities in gonadal morphology, gonadal enzyme activity, and steroidogenesis. Embryonic exposure to environmental contaminants in the eggs has been hypothesized to be the causal agent for these changes. However, posthatchling exposure can also contribute to changes in reproductive and endocrine functioning. We have detected serum concentrations of 16 of 18 organochlorine pesticides or metabolites (OCs) and 23 of 28 congener-specific polychlorinated biphenyls (PCBs) examined in juvenile alligators from Lake Apopka, Orange Lake, and Lake Woodruff National Wildlife Refuge. Lake Apopka juveniles had significantly elevated serum concentrations of p,p′-DDE, dieldrin, endrin, mirex, oxychlordane, ΣDDTs, and ΣPCBs compared to juveniles from the other lakes. Further, we observed no correlations between serum contaminant concentrations and sex steroid concentrations (estradiol-17β and testosterone). However, serum testosterone was significantly lower in males from Lake Apopka and Orange Lake compared to Lake Woodruff NWR. We did not observe relationships between phallus size or other body parameters and serum contaminant levels. Phallus size was smaller in males from Lake Apopka even after adjustment for body size. We suggest that the observations previously reported for juvenile alligators—and observed again in this study—are apparently not associated with the current serum levels of the environmental contaminants we measured, but could be due to exposures during embryonic development to these or other pollutants. Future studies must determine if a causal relationship exists between the contaminants found in alligator eggs and abnormalities observed in the hatchlings and persisting in juveniles.

Effect of Acute Stress on Plasma Concentrations of Sex and Stress Hormones in Juvenile Alligators Living in Control and Contaminated Lakes

Environmental contaminants can act as stressors, inducing elevated circulating concentrations of stress hormones such as corticosterone and corticod sol. Development in contaminated eggs has been reported to modify circulating sex steroid hormone concentrations in alligators (Alligator mississippiensis). We ex- amined plasma concentrations of testosterone (T), estradiol-17p (E2), and corticosterone (B) immediately upon and two hours after capture in male and female juvenile alligators living in either contaminated or relatively pristine lake systems. We observed that plasma T concentration was significantly depressed in males from the contaminated lake, whereas plasma E2 showed significant variation between sexes but not between lakes. Initial plasma B concentrations were similar between alligators from both lakes or either sex. Two hours of capture and restraint did not effect plasma T or E2 concentrations but was associated with a dramatic rise in plasma B concentrations. These data suggest that juvenile alligators exposed to contaminants in ovo are apparently unaffected in their rapid glucocorticoid response to acute stress.

CLINICAL AND NECROPSY FINDINGS ASSOCIATED WITH INCREASED MORTALITY AMONG AMERICAN ALLIGATORS OF LAKE GRIFFIN, FLORIDA

From December, 1997, through November, 2000, 306 deaths were documented among adult and subadult American alligators (Alligator mississippiensis) of Lake Griffin, Florida (USA). Some live alligators were lethargic and unresponsive to approach. To determine the cause, we examined ten alligators captured from Lake Griffin between December 1997 and June 1999. Initially, four alligators, three of which were clinically unresponsive, were sacrificed for routine diagnostic necropsy. The other six Lake Griffin alligators, and five control alligators captured from Lake Woodruff National Wildlife Refuge, Florida, where mortality was negligible, were studied extensively by clinical neurologic examination, electromyography, hematology, serum chemical analyses, and blood culture, then sacrificed and necropsied. Samples of brain, spinal cord, peripheral nerves, skeletal muscle, and major internal organs were examined by light microscopy for abnormalities. Samples of nervous tissue also were examined by electron microscopy, and samples of various tissues were collected for toxicologic analyses. Clinical signs included swimming in circles, inability to submerge, lethargy, weakness, unresponsiveness, slow reflexes, dragging the dorsal surfaces of the hind feet, head tilt, and anisocoria. Lake Griffin alligators had significantly lower distal sciatic nerve conduction velocities than Lake Woodruff alligators, and the most severely affected alligators had the lowest velocities; but morphologic abnormalities in peripheral nerves were not evident in most cases. Three severely affected alligators had acute focal necrosis of the torus semicircularis in the midbrain, two had skeletal myofiber atrophy, another had diffuse nonsuppurative encephalomyelitis, and one mildly affected alligator had skeletal myodegeneration. The cause or causes have not yet been identified.

Formation and regression of the corpus luteum of the American alligator (Alligator mississippiensis)

Luteal morphology of the American alligator is unique when compared to other reptiles but is similar to that of its phylogenetic relatives, the birds. The theca is extensively hypertrophied, but the granulosa never fills the cavity formed following the ovulation of the ovum. The formation of the corpus luteum (CL) is correlated with elevated plasma progesterone concentrations, which decline dramatically after oviposition with the onset of luteolysis. Unlike those of most other reptiles, the central luteal cell mass is composed of two cell types; one presumably is derived from the granulosa, whereas the other is from the theca interna. Both cell types are present throughout gravidity but only one cell type is seen during mid to late luteolysis. A significant decline in luteal volume occurs following oviposition and continues throughout the post‐oviposition period. The fastest decline in luteal volume occurs in the month immediately after oviposition; this rate then slows. Luteolysis appears to continue for a year or more following oviposition, as distinct structures of luteal origin can still be identified in animals 9 months after oviposition. The size of persistent CL can be used to determine whether a given female oviposited during the previous nesting season. Females with CL having volumes greater than 0.2 cm2 or CL diameters greater than 0.4 cm were active the previous season.

PATHOLOGY, PHYSIOLOGIC PARAMETERS, TISSUE CONTAMINANTS, AND TISSUE THIAMINE IN MORBID AND HEALTHY CENTRAL FLORIDA ADULT AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS)

An investigation of adult alligator (Alligator mississippiensis) mortalities in Lake Griffin, central Florida, was conducted from 1998–2004. Alligator mortality was highest in the months of April and May and annual death count peaked in 2000. Bacterial pathogens, heavy metals, and pesticides were not linked with the mortalities. Blood chemistry did not point to any clinical diagnosis, although differences between impaired and normal animals were noted. Captured alligators with signs of neurologic impairment displayed unresponsive and uncoordinated behavior. Three of 21 impaired Lake Griffin alligators were found to have neural lesions characteristic of thiamine deficiency in the telencephalon, particularly the dorsal ventricular ridge. In some cases, lesions were found in the thalamus, and parts of the midbrain. Liver and muscle tissue concentrations of thiamine (vitamin B1) were lowest in impaired Lake Griffin alligators when compared to unimpaired alligators or to alligators from Lake Woodruff. The consumption of thiaminase-positive gizzard shad (Dorosoma cepedianum) is thought to have been the cause of the low tissue thiamine and resulting mortalities.

Spacing Patterns in Alligator Nests

The pattern of alligator nest location is measured for four consecutive years across a set of quadrats on Orange Lake (Alachua County) Florida. The nests are clumped, and this clumping is evident even given controls for habitat heterogeneity. The recent recovery of Alligator mis- sissippiensis from endangered status has generated a significant renaissance of research into this reptiles biology. It is particularly important for biologists and management specialists alike to learn what factors might limit the density of alligator populations. For over 20 years workers in Louisiana have known that

Alligator diet in relation to alligator mortality on Lake Griffin, FL

Abstract Alligator mississippiensis (American Alligators) demonstrated low hatch-rate success and increased adult mortality on Lake Griffin, FL, between 1998 and 2003. Dying Lake Griffin alligators with symptoms of poor motor coordination were reported to show specific neurological impairment and brain lesions. Similar lesions were documented in salmonines that consumed clupeids with high thiaminase levels. Therefore, we investigated the diet of Lake Griffin alligators and compared it with alligator diets from two lakes that exhibited relatively low levels of unexplained alligator mortality to see if consumption of Dorosoma cepedianum (gizzard shad) could be correlated with patterns of mortality. Shad in both lakes Griffin and Apopka had high levels of thiaminase and Lake Apopka alligators were consuming greater amounts of shad relative to Lake Griffin without showing mortality rates similar to Lake Griffin alligators. Therefore, a relationship between shad consumption alone and alligator mortality is not supported.

Morphological Variation in Hatchling American Alligators (Alligator mississippiensis) from Three Florida Lakes

Morphological variation of 508 hatchling alligators from three lakes in north central Florida (Lakes Woodruff, Apopka, and Orange) was analyzed using multivariate statistics. Morphological variation was found among clutches as well as among lakes. Principal components analysis was used to determine the proportion of the variation attributable to shape as opposed to size. Shape accounted for 42% and 36% of the variation among individuals and clutches, respectively. Linear discriminant analysis with cross-vali- dation was performed to determine whether the clutch from which an individual came or the lake from which a clutch came from could be predicted. Success of predicting clutch membership varied from 67% for Lake Apopka to 74% for Orange Lake. The lake from which a clutch was obtained could be predicted with a success rate of 49%. The optimal subset of measurement variables, obtained from stepwise discrim- inant analysis, was used to illustrate the morphological variation among clutches and lakes in a scatterplot of the first two canonical variables. Assuming factors such as genetics, nutrition, age, size, and stress affect hatchling morphology, one would suspect hatchlings from the same clutch to be more similar than hatch- lings from different clutches. Likewise, we suggest that a lake effect could be the result of intrapopulational similarity of environmental factors, such as food and resource availability, nutrient levels, contaminant levels, and parental lineage (genetics). We hypothesize that demographic and environmental influence on the ma- ternal contribution to the embryonic environment, rather than genetic influence, is responsible for the ob- served pattern of morphological variation.

Effects of egg and hatchling harvest on american alligators in Florida

Harvest of crocodilian eggs and young for captive rearing (ranching) has been used worldwide as an option for producing crocodilian skins and meat from wild stock. The long-term effects of harvesting a certain proportion of early age class, wild American alligators (Alligator mississippiensis) without repatriation is unknown. We removed an estimated 50% of annual production of alligators on Lakes Griffin and Jesup in central Florida over an 11-year period and monitored population levels via night-light counts. Densities of the total alligator population increased (P 0.117), and subadult (122-182 cm TL) alligators increased (P < 0.011) on harvest areas. The density of juveniles on the control area increased (P = 0.006), and the density of subadults showed some evidence of increasing (P = 0.088). No changes were detected in size distributions on the treatment areas. Nest production, as observed from aerial helicopter surveys, increased (P < 0.039) on Lake Woodruff NWR and Lake Jesup and showed some evidence of an increase on Lake Griffin (P = 0.098) during 1983-91. A 50% harvest rate of eggs or hatchlings did not adversely affect recruitment into the subadult or adult size classes.

Possible Generational Effects of Habitat Degradation on Alligator Reproduction

Abstract Population decline of the American alligator (Alligator mississippiensis) was observed in Lake Apopka in central Florida, USA, in the early 1980s. This decline was thought to result from adult mortality and nest failure caused by anthropogenic increases in sediment loads, nutrients, and contaminants. Reproductive impairment also was reported. Extensive restoration of marshes associated with Lake Apopka has been conducted, as well as some limited restoration measures on the lake. Monitoring by the Florida Fish and Wildlife Conservation Commission (FFWCC) has indicated that the adult alligator population began increasing in the early 1990s. We expected that the previously reported high proportion of complete nest failure (θ0) during the 1980s may have decreased. We collected clutches from alligator nests in Lake Apopka from 1983 to 2003 and from 5 reference areas from 1988 to 1991, and we artificially incubated them. We used a Bayesian framework with Gibbs sampler of Markov chain Monte Carlo simulation to analyze θ0. Estimated θ0 was consistently higher in Lake Apopka compared with reference areas, and the difference in θ0 ranged from 0.19 to 0.56. We conducted change point analysis to identify and test the significance of the change point in θ0 in Lake Apopka between 1983 and 2003, indicating the point of reproductive recovery. The estimated Bayes factor strongly supported the single change point hypothesis against the no change point hypothesis. The major downward shift in θ0 probably occurred in the mid-1990s, approximately a generation after the major population decline in the 1980s. Furthermore, estimated θ0 values after the change point (0.21) were comparable with those of reference areas (0.07–0.31). These results combined with the monitoring by FFWCC seem to suggest that anthropogenic habitat degradation caused reproductive impairment of adult females and decreases in θ0 occurred with the sexual maturity of a new generation of breeding females. Long-term monitoring is essential to understand population changes due to habitat restoration. Such information can be used as an input in planning and evaluating restoration activities.