Plasma steroid concentrations in relation to size and age in juvenile alligators from two Florida lakes

Previous studies have reported a number of physiological differences among juvenile alligators from two well-studied populations (Lake Apopka and Lake Woodruff) in north central Florida. These studies obtained alligators of similar size from each lake under the assumption that the animals were of similar age. Lake Apopka is a hypertrophic lake with a 50-year history of contamination from agricultural and municipal operations, whereas Lake Woodruff is a eutrophic lake and part of a National Wildlife Refuge that receives little point source pollution. If growth rates differ among these areas, it could be argued that differences in endocrine parameters reported previously (e.g. steroid or thyroid hormone concentrations) could be the result of differences in the animals' ages. Using growth annuli in cross-sections of femurs, we estimated the ages of juvenile alligators and compared the relationship of estradiol-17beta (E(2)) and testosterone (T) to size and age within each lake and sex. No differences were detected in the relationship between size and age between the two areas indicating similar growth rates between lakes. Plasma E(2) was positively related to size in females from Lake Apopka, and age in Woodruff females. Males from Lake Apopka had elevated plasma E(2) compared with Lake Woodruff males and did not differ from Woodruff females. No significant relationships were detected for T from either lake, and no differences in plasma T were detected among lakes or sexes. Our data indicate that both size and age can have a significant relationship with steroid concentrations. However, the relationship between steroid concentrations and size or age differed between lakes. We suggest both factors should be considered when conducting physiological studies where there is evidence to suggest growth rates may differ among populations.     

Altered histology of the thymus and spleen in contaminant-exposed juvenile American alligators

Morphological differences in spleen and thymus are closely related to functional immune differences. Hormonal regulation of the immune system has been demonstrated in reptilian splenic and thymic tissue. Spleens and thymus were obtained from juvenile alligators at two reference sites in Florida, USA: Orange Lake and a National Wildlife Refuge, Lake Woodruff, as well as from a contaminated lake, Lake Apopka. Lake Apopka has been extensively polluted with agricultural pesticides. Tissues were prepared for histological analysis to determine if previously detected endocrine abnormalities associated with contaminant exposure might also be reflected in morphological differences in splenic and thymic structures important for immunological response. Similar tissues were taken from captive-raised juvenile female alligators (3 years old) that were hatched from eggs collected on Lake Woodruff and Lake Apopka. Differences in thymic ratios (medulla/cortex) were found among alligators collected from the two lakes (P = 0.0051). Alligators from Lake Apopka had smaller thymic ratios than animals from either reference lake. Males from Lake Woodruff had significantly smaller lymphocyte sheaths in the spleen than females (P = 0.0009), indicative of a normal sexual dimorphism. Lymphocyte sheath width differed among females obtained from the three lakes, with females from Lake Apopka having the smallest sheath width and those from Orange Lake having the largest. Malpighian body area was largest in alligators from Orange Lake, intermediate in Lake Woodruff, and smallest in Lake Apopka. In contrast to that observed for wild-caught animals, no difference was found in the thymic medulla/cortex ratio of captive-raised female alligators (P = 0.378). Captive-raised female alligators from Lake Apopka and Lake Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals; Lake Apopka alligators had smaller lymphocyte sheath width compared to Woodruff alligators (P = 0.0396). In contrast to wild-caught animals, area of the Malpighian bodies did not differ by lake in the captive-raised female alligators (P = 0.066). The enlarged thymic cortex suggests a change in T-lymphocyte maturation within the thymus of alligators from a contaminated environment, Lake Apopka. The results point to alterations in the histology of the thymus and spleen. Further studies are required to examine the functional significance of these observations.     

Altered neonatal development and endocrine function in Alligator mississippiensis associated with a contaminated environment

Reduced reproductive success, altered reproductive tract development, and differences in circulating hormones have been documented in American alligators (Alligator mississippiensis) from Lake Apopka, FL, compared to less contaminated sites, such as the Lake Woodruff National Wildlife Refuge, FL. Comparative studies among alligators of varying size and age suggest that in ovo contaminant-induced alterations of endocrine function are further modified during postembryonic development and/or through environmental exposure. In the present study, we examined developmental and endocrine-related indices in neonatal (age, <1 mo) alligators from Lake Apopka in comparison to those of a reference population (Lake Woodruff), thereby limiting contaminant exposure to that derived via maternal contribution. We compared several reproductive and developmental parameters, including hatching success, primary sex determination, and somatic indices. Furthermore, we examined circulating testosterone concentrations and aromatase activity in an effort to establish relative gonadal endocrine function shortly after hatching. Finally, we compared phallus size among males and oviduct epithelial cell height (ECH) among females (androgen- and estrogen-dependent tissues, respectively). Significant differences between populations were noted for body size and spleen somatic index. Neonatal alligators from Lake Apopka exhibited higher plasma testosterone, but no differences were detected in gonadal aromatase activity compared to Lake Woodruff. Phallus tip length and cuff diameter were smaller in males from Lake Apopka, whereas no differences were noted in oviduct ECH. Our data establish basic indices of development and endocrine function in neonatal alligators before environmental exposure to contaminants. These results should begin to help separate developmental abnormalities resulting from in ovo exposure, presumably of maternal origin, from physiological alterations induced through environmental exposure to contaminants.     

Developmental effects of embryonic exposure to toxaphene in the American alligator (Alligator mississippiensis)

A variety of organochlorine pesticides have been shown to adversely affect embryonic development. A number of abnormalities have been documented in alligators (Alligator mississippiensis) from highly-contaminated Lake Apopka, FL, USA that are similar to the results of experimental studies exposing embryos to pesticides. In the current study, we exposed developing alligator embryos to varying concentrations of toxaphene, a broad-spectrum pesticide found in relatively high concentration in Lake Apopka alligator egg yolk. The toxaphene, dissolved in 50 microl of ethanol, was applied topically to the eggshell just prior to the sex-determining period of development. Shortly after hatching, we examined a number of morphological and physiological endpoints to determine the consequences of sub-lethal embryonic exposure to toxaphene. Our results indicate that toxaphene had little or no effect on the morphological endpoints examined including body mass (BM) and size, liver, thyroid, and gonad development. In addition, toxaphene failed to affect sexual differentiation, or in vitro thyroxin, testosterone (T), and estradiol production. However, male plasma T concentration was higher in animals treated with 10 and 0.01 microg toxaphene/kg (based on mean egg mass) than control males. Because in vitro T production was not different among control groups, we suggest the difference in plasma T could be due to differences in hypothalamic-pituitary stimulation of the gonad or hepatic steroid degradation. This study indicates that technical grade toxaphene, at the applied doses, does not induce the same developmental abnormalities associated with alligators living in Lake Apopka. Future studies should consider the effects of embryonic exposure to a mixture of chemicals, including toxaphene metabolites, on development in alligators to better evaluate the consequences of environmental contamination.     

Seasonal variation in plasma thyroxine concentrations in juvenile alligators (Alligators mississippiensis) from three Florida Lakes

Circulating concentrations of thyroxine (T(4)) vary seasonally in many vertebrates. This study examined the seasonal variation in plasma concentrations of T(4) in juvenile American alligators (Alligator mississippiensis) from three populations in central Florida, USA. One site, Lake Woodruff National Wildlife Refuge, is considered a reference site whereas the other two lakes, Lake Apopka and Orange Lake, are significantly impacted by human activity. Juvenile American alligators ranging from 75-150 cm in total length were hand-captured at night from November 2000-April 2002. Plasma thyroxine concentrations were analyzed using a radioimmunoassay (RIA) previously validated for alligator plasma. Juvenile American alligators display seasonal variation in circulating T(4) concentrations. Plasma T(4) concentrations decrease from August/September to November and then begin a slow rise until April, at which point they plateau. Sex of juveniles influenced plasma concentrations of T(4) in some months but did not appear to alter the pattern in seasonal variation. The pattern we observed in plasma T(4) concentrations is not directly related to an environmental factor such as ambient temperature but is similar to that seen in plasma sex steroid concentrations during the reproductive cycle of adult alligators. Although the pattern and plasma concentration of T(4) exhibits significant variation among the three lakes studied, the pattern in seasonal variation appears similar. Comparing the seasonal pattern in plasma T(4) with plasma concentrations of sex steroids (testosterone and estradiol-17beta) or corticosterone could provide important information on the peripubescent life stage of the American alligator.     

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 B(1)) 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.     

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.     

Organochlorine pesticides and thiamine in eggs of largemouth bass and American alligators and their relationship with early life-stage mortality

Thiamine deficiency has been linked to early mortality syndrome in salmonids in the Great Lakes. This study was conducted to compare thiamine concentrations in American alligators (Alligator mississippiensis) and Florida largemouth bass (Micropterus salmoides floridanus) eggs from sites with high embryo mortality and high exposure to organochlorine pesticides (OCPs) (Lakes Apopka and Griffin, and Emeralda Marsh, Florida, USA) to those from sites that have historically exhibited low embryo mortality and low OCPs (Lakes Woodruff and Orange, Florida). During June-July 2000, 20 alligator clutches were collected from these sites, artificially incubated, and monitored for embryo mortality. Thiamine and OCPs were measured in one egg/clutch. During February 2002, 10 adult female bass were collected from Emeralda Marsh and Lake Woodruff and mature ovaries analyzed for thiamine and OCP concentrations. Although ovaries from the Emeralda Marsh bass contained almost 1,000-fold more OCPs compared with the reference site, Lake Woodruff, there were no differences in thiamine concentrations between sites (11,710 vs. 11,857 pmol/g). In contrast, alligator eggs from the reference site had five times the amount of thiamine compared with the contaminated sites (3,123 vs. 617 pmol/g). Similarly, clutches with >55% hatch rates had significantly higher concentrations of thiamine compared with clutches with <54% hatch rates (1,119 vs. 201 pmol/g). These results suggest that thiamine deficiency might be playing an important role in alligator embryo survival but not in reproductive failure and recruitment of largemouth bass. The cause(s) of this thiamine deficiency are unknown but might be related to differences in the nutritional value of prey items across the sites studied and/or to the presence of high concentration of contaminants in eggs.     

Detection by microsatellite analysis of early embryonic mortality in an alligator population in Florida

In the 1980s, alligators (Alligator mississippiensis) of Lake Apopka (Florida, USA) underwent a population decline associated with decreased egg viability, effects that have been associated with endocrine-disrupting, persistent organochlorine pesticides. It is currently unknown whether the decreased egg viability is due to fertilization failure or early embryonic death. Therefore, we conducted a preliminary study to evaluate the use of microsatellite DNA loci to determine the fertilization status of nonviable eggs. Using microsatellite analysis, we compared genotypes from blasto-disks and embryos with the genotypes from females trapped at the nest. Four of five nonviable egg samples tested yielded evidence of fertilization. No evidence of unfertilized eggs was obtained, but amplifiable DNA could not be obtained from one entirely nonviable clutch. Thus, we demonstrate that early embryonic mortality in alligators can be detected by microsatellite analysis, but also suggest substantial effort is needed to improve the recovery of DNA and amplification of alligator microsatellite loci.     

Microsatellite DNA analyses support an east-west phylogeographic split of American alligator populations

We examined the population genetic structure of American alligators (Alligator mississippiensis) sampled from 12 localities across the southeastern United States. The primary goal of this study was to determine the extent of population differentiation among alligators from four Florida lakes using eight microsatellite loci and compare the results to additional sites located at varying distances from them. Analyses of population structure revealed little differentiation (F(ST)=0.039; Rho=0.012) among the four Florida lakes, Apopka, Griffin, Orange and Woodruff, which are all located in the St. John&'s River watershed in north-central Florida. Further, there was little differentiation among these samples and samples collected from the Everglades National Park (F(ST)=0.044; Rho=0.009) and south Georgia (F(ST)=0.045; Rho=0.032). Therefore, these six samples were pooled together as a "FL/sGA group." Similarly, samples collected in the western extent of the range, Anahuac National Wildlife Refuge in Texas and Salvador Wildlife Management Area, Marsh Island Wildlife Refuge and Rockefeller Wildlife Refuge in Louisiana, also lacked population structure (F(ST)=0.024; R(ST)=0.040). These four populations were pooled into the "TX/LA group." Comparisons of these two groups with samples taken from the Santee Coastal Reserve in South Carolina and Mobile, Alabama yielded three to four times more differentiation among groups (F(ST)=0.131; Rho=0.187). These and other analyses support the hypothesis of an east-west phylogeographic split in American alligator populations and are consistent with studies of many freshwater fish and aquatic and terrestrial turtles distributed throughout this same geographic region.     

Evaluation of recent limnological changes at Lake Apopka

Recent changes in submersed macrophytes and water quality variables have been offered as the strongest evidence that the current restoration program at Lake Apopka will be effective (Lowe et al., 2000); however, the new beds of submersed plants in Lake Apopka are found only on hard substrates on the fringes of the lake within 40 m of shore and are protected from waves by cattails (Typha spp.). They occupy only 0.02% of the lake area, and there is no indication that they can colonize the flocculent sediments that make up 90% of the lake area. There is no correlation between annual inputs of phosphorus and total phosphorus concentrations in the lake, and patterns of change in chlorophyll and other water quality variables do not follow changes in phosphorus loads. Rather than reflecting decreases in phosphorus loading, the recent changes could be related to the harvest of benthivorous fish or are just the normal fluctuations found in lakes that have not been perturbed. Regardless of the reason the macrophytes were lost in the 1940s, the new analyses confirm our previous findings that the high turbidities in Lake Apopka are due to the resuspension of sediments, and that the fluid mud cannot support the colonization of submersed aquatic macrophytes. Even without the fluid mud, the target phosphorus concentration of 55 mg m^–3 is too high to bring about the restoration of the former macrophyte beds in the lake.     

Cyanobacterial dynamics in shallow Lake Apopka (Florida,U.S.A.) before and after the shift from a macrophyte‐dominated to a phytoplankton‐dominated state

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Chronic incidental lead ingestion in a group of captive-reared alligators (Alligator mississippiensis): possible contribution to reproductive failure

An American alligator (Alligator mississippiensis) breeding facility using male and female alligators raised from artificially incubated eggs was established in 1975. These alligators first reproduced at 6 years of age as compared to 10-12 years in wild alligators, but the eggs produced showed a lower hatching rate than those collected from the wild. By age 21 reproduction had failed almost completely. The alligators were sacrificed and tissues collected at necropsy from 44 captive and 15 wild animals and assayed for metals. Results showed that captive alligators had significantly higher tissue levels of lead than wild alligators. Cadmium did not differ between wild and captive and selenium was 50% higher in wild than captive alligator kidneys. Bone lead in captive alligators was 252,443 +/- 20,462 ng/g. High yolk lead was suggested as a probable cause for early embryonic death in alligator eggs. The high tissue lead levels in captive alligators was attributed to long-term consumption of nutria (Myocastor coypus) meat contaminated with lead shot. Liver, ovary, and testis were assayed for lipid peroxidation using the thiobarbituric acid (TBA) test. Captive alligators had 3.6 fold increased TBA-reactive materials in the liver tissue compared to wild. Lipid peroxidation was strongly suspected as having been enhanced by consumption of rancid nutria meat containing lead.     

Inter lake comparison of liver morphology and in vitro hepatic monodeiodination of L-thyroxine in sexually mature coho salmon, Oncorhynchus kisutch Walbaum, from Lakes Erie, Ontario, Michigan and Superior

The hepatosomatic index (HS1) of female sexually mature coho salmon, Oncorhynchus kisutch , from Lake Superior was significantly higher (P<0.01) than in males, whereas that of females from Lake Erie was significantly lower (P<0.05) than in males; there was no significant sex differences in coho salmon from Lakes Ontario or Michigan. Hepatic nuclear areas (HNA) were significantly higher ( P <0.01) in males compared with females in fish collected from Lakes Ontario and Erie, but there was no sex differences in HNA of coho salmon from Lakes Superior or Michigan. In all four lakes, hepatocyte cytoplasmic volume was significantly higher ( P <0.01) in males than in females from the same lake, and the cytoplasmic volume: nuclear volume ratio of males was corresponding significantly higher ( P < 0.05) than in females from the same lake.
Two hepatocytic cell types were evident, termed' light and dark cells, based on the electron density of their cytoplasmic matrix. There were considerable intersex and interlake differences in the relative populations of light 'and dark' cells in coho salmon from Lakes Ontario, Michigan, Erie and Superior, and also in the hepatocytic content of lipid, glycogen, mitochondria and ribosomal and smooth endoplasmic reticulum.
There were no sex differences with regard to in vitro hepatic monodeiodination of L-thyroxine (T4) to triiodo-L-thyronine (T3) in salmon from any of the lakes. However, T4 to T3 conversion was significantly lower (P < 0.05) in liver homogenates prepared from Lake Ontario coho salmon than in comparable preparations of liver from Lake Superior, Lake Erie, or Lake Michigan salmon.