Increased posthatching mortality and loss of sexually dimorphic gene expression in alligators (Alligator mississippiensis) from a contaminated environment

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.     

Form and function of the feeding apparatus of Alligator mississippiensis

The architecture of the jaw muscles and their tendons of Alligator mississippiensis is described and their function examined by electromyography. Alligator grabs its prey with forward lunges or rapid lateral movements of the head. It does not engage in regular masticatory cycles. Prey is manipulated by inertial movements and the tongue does not appear to play any role in transport. The Mm. adductor mandibulae externus, adductor mandibulae posterior, and pterygoideus activate bilaterally and simultaneously during rapid closing or crushing. The M. pterygoideus does not act during prey holding whereas the Mm. adductor mandibulae externus, adductor mandibulae posterior continue to be active. The Mm. depressor mandibulae and intramandibularis are variably active during both jaw opening and closing.     

Gonadal development and differentiation in Alligator mississippiensis at male and female producing incubation temperatures

The development and differentiation of the gonads of embryonic alligators incubated at 30 °C (100% female producing) and 33 °C (100% male producing) was investigated histologically. The stage of development of the gonad and differentiation into an ovary or a testis occurred at essentially the same time at both temperatures. This contrasts with the overall development of the embryos which was slower at the lower temperature. A few days prior to differentiation, gonads grew more quickly at 33 °C than they did at 30 °C. However, once differentiated into a presumptive testis, gonads reduced in volume so that at hatching presumptive testes were smaller than presumptive ovaries. It is hypothesized that synchrony/asynchrony of development of the gonad and the rest of the embryo may account for temperature-dependent sex determination.     

Morphometric analysis of embryonic development in Alligator mississippiensis, Crocodylus johnstoni and Crocodylus porosus

The size of embryos at various stages of development was determined in three species of crocodilian ( Alligator mississippiensis, Crocodylus johnstoni and C. porosus). Various morphometric measurements were taken of embryos throughout development and were described for each stage of development. Increase in size from stage to stage was faster in A. mississippiensis than in C. porosus and C. johnstoni. Hatchlings of A. mississippiensis were large in length but light in mass compared with the hatchlings of C. johnstoni and C. porosus which were heavier per unit length. These morphometric parameters can be used to determine the stage of embryonic development by size. The use of principle component analysis improves this technique further by dampening any anomalous data points.
The rate of embryonic growth in A. mississippiensis appeared to be under greater genetic control than in the two species of Crocodylus. The evolutionary advantages of this phenomenon probably relate to the biology of A. mississippiensis. Due to the northerly range of this species it is advantageous for alligators to hatch as soon as possible, as large as possible, to maximize the period prior to winter hibernation and reduce predation. Tropical crocodiles have fewer selection pressures for rapid development and have slower rates of embryonic growth. Genetic aspects of crocodilian embryonic development have been largely ignored but may help explain some aspects of crocodilian growth under farming conditions.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Differences in renal-cloacal function between Crocodylus porosus and Alligator mississippiensis have implications for crocodilian evolution

Major electrolytes and nitrogenous excretory products were analysed in the blood plasma, ureteral urine and cloacal urine of juvenile Alligator mississippiensis and Crocodylus porosus in fresh and hypoosmotic salt water (206 mosmol · l⁻¹). Both species coped well with saline water, showing little (Alligator) or no (Crocodylus) change in plasma composition. Comparisons of renal-cloacal function point to major differences in their osmoregulatory physiology. The cloaca of C. porosus is a very active osmoregulatory organ in salt and fresh water, contributing to water conservation and NaCl excretion through the lingual salt glands. In contrast, the cloaca of Alligator has little impact on the composition of excreted urine. It seems likely that A.␣mississippiensis is largely constrained to a renal response to osmotic and ionic stress while C. porosus is able to call on a more complex mix of renal response, post-renal modification of urine in the cloaca, and excretion of excess NaCl through the salt glands. The results support the idea that there are deep-seated differences in the osmoregulatory physiology of alligatorids and crocodylids (Eusuchia), an understanding of which should provide valuable insights into their evolution and zoogeography. Accepted: 7 September 1996     

Dorsal galnds of Alligator mississippiensis: a histological and histochemical study

Several investigators have described in some crocodilians a row of round or oval organs, called dorsal glands, lying under scutes on each side of the dorsal midline. The function of these glands is unknown, but they are hypothesized to produce skin-conditioning secretions. We investigated the anatomy and histochemistry of the dorsal glands of adult American alligators ( Alligator mississippiensis ). Twenty to 22 pairs of glands containing a viscous, often black, material were observed lying from the mid-cervical to the anterior caudal regions in the axial musculature or on the inner surface of the dermis. The capsule of each gland consists of dense collagenous fibres and numerous short elastic fibres, and is surrounded by skeletal muscle. The single lumen is lined by one to several layers of cuboidal to columnar epithelium in varying stages of degeneration, indicating a holocrine secretory mode. The epithelial cell membranes often interdigitate and tight junctions and desmosomes occasionally are observed between them. The epithelial cells and secretory product contain slight to considerable amounts of lipid; glycoproteins may be present. Crystals exhibiting a dense core and/or layering occur in the epithelial cells and secretory product. Energy-dispersive X-ray analysis demonstrates calcium, copper, iron, lead, potassium, and zinc in the crystals. Mitochondria, vacuoles, and short segments of rough endoplasmic reticulum also occur in the cells.     

Multiyear multiple paternity and mate fidelity in the American alligator, Alligator mississippiensis

We examined multiple paternity during eight breeding events within a 10-year period (1995–2005) for a total of 114 wild American alligator nests in Rockefeller Wildlife Refuge in south-west Louisiana. Our goals included examining (i) within population variation in multiple paternity among years, (ii) variation in multiple paternity in individual females and (iii) the potential for mate fidelity. To accomplish this, in the current study, eggs were sampled from 92 nests over 6 years and analysed along with 22 nests from a previous 2-year study. Genotypes at five microsatellite loci were generated for 1802 alligator hatchlings. Multiple paternity was found in 51% of clutches and paternal contributions to these clutches were highly skewed. Rates of multiple paternity varied widely among years and were consistently higher in the current study than previously reported for the same population. Larger females have larger clutches, but are not more likely to have multiply sired nests. However, small females are unlikely to have clutches with more than two sires. For 10 females, nests from multiple years were examined. Seven (70%) of these females exhibited long-term mate fidelity, with one female mating with the same male in 1997, 2002 and 2005. Five females exhibiting partial mate fidelity (71%) had at least one multiple paternity nest and thus mated with the same male, but not exclusively. These patterns of mate fidelity suggest a potential role for mate choice in alligators.     

Development of the dentition in Alligator mississippiensis: upper jaw dental and craniofacial development in embryos, hatchlings, and young juveniles, with a comparison to lower jaw development

Development of the upper dentition in Alligator mississippiensis was investigated using a close series of accurately staged and aged embryos, hatchlings, and young juveniles up to 11 days posthatching, as well as some young and old adult specimens. Studies from scanning electron microscopy, light microscopy, acetate and computer reconstructions, radiography and macroscopy were combined to elucidate the details of embryonic dental development, tooth initiation pattern, dentitional growth, and erupted functional dentition. The results were compared with those from the lower jaw and related to the development of other craniofacial structures. Approximately 17 early teeth in each jaw half develop as surface teeth, of which 13 project for 1 to 12 days before sinking into the mesenchyme. The first three teeth initiate directly from the oral epithelium at Ferguson stages 14-15 (days 15-19 after egg laying), before there is any local trace of dental lamina formation. All other teeth develop from a dental prolamina or lamina; and with progressive lamina development, submerged teeth initiate from the aboral end leading to the formation of replacement teeth. All teeth form dentin matrix, but 12 early teeth do not form enamel. Approximately 20 embryonic teeth are resorbed, 6 are transitional, and 42 function for longer periods after hatching. The embryonic tooth initiation pattern (illustrated by defining a tooth position formula) does not support the previous models of Odontostichi, Zahnreihen, and Tooth Families, each of which postulates perfect regularity. Up to three interstitial tooth positions develop between sites of primary tooth initiation, and families with up to five generations at hatching are at first arbitrarily defined.     

Scale morphogenesis and ultrastructure of dermis during embryonic development in the alligator (Alligator mississippiensis, Crocodilia, Reptilia)

Formation of scales in different body regions of embryonic alligators is described using light and electron microscopy. Transformation of the skin surface to produce scales takes place between stages 19 and 23, after which the shape of scales is complete over most of the embryonic surface. Scalation is not synchronous; different regions develop scales at different rates. Initially scales are formed on the back and dorsal side of the proximal tail and appear as undulations of the epidermis which form symmetrical (bumps) or asymmetrical (serrated) scale anlagen. No dermal condensations are apparent beneath the epidermis, although in some areas of the skin (belly, limbs) mesenchymal cells are more numerous within the bumps than in other areas. At stage 21, scalation has spread to the neck and belly but is absent or poorly developed over most areas of the flank, gular, jaw, limb and head regions. Grooves form between the outer edges of adjacent scales or interbump regions. A superficial denser dermis and a reticulated deep subdermis are visible in many scales from stage 21. The dermis forms a superficial loose and a deep dense layer from stage 22. Both loose and deep dermis, and sometimes the deep reticulate subdermis, move towards the surface to form the dermal core of scales, although the mechanism of this movement is not known. Bundles of collagen fibrils, with almost no elastic fibrils, are progressively deposited, especially in the denser dermis. At stage 22, the flank, gular and proximal areas of limbs form scales, but the head, jaw, distal limbs and digits still lack scales. The digits become scaled at stage 23 when scalation is well advanced in the other regions. By stage 24 most of the body is scaled and subsequent scale modifications occur only by growth. Five main types of scales are recognized by their shape: symmetrical scutes, asymmetrical scutes, overlapping scutes, tuberculate scales, and elevated asymmetrical scutes (tail verticils). Pigmentation, mainly due to epidermal melanocytes, is visible at embryonic stage 23 and progresses through stages 24 and 25.     

Bone morphometrics and tetracycline marking patterns in young growing American alligators (Alligator mississippiensis)

Nine young American alligators (Alligator mississippiensis) were injected at monthly intervals with tetracycline to determine the bone apposition rate and the resorption patterns over a 3-mo period. The periosteal apposition rate increased progressively over the 3-mo period from 2.99 microns/day to 5.94 microns/day. Endosteal apposition rate was much slower with incomplete tetracycline lines being observed on the endosteum. This suggests that most modeling-resorptive activities occur on the endosteal envelope.     

Seasonal androgen cycles in adult male American alligators (Alligator mississippiensis) from a barrier island population

The seasonal patterns of two primary plasma androgens, testosterone (T) and dehydroepiandrosterone (DHEA), were assessed in adult male alligators from the Merritt Island National Wildlife Refuge, a unique barrier island environment and home to the Kennedy Space Center in Florida. Samples were collected monthly from 2008 to 2009, with additional samples collected at more random intervals in 2007 and 2010. Plasma T concentrations peaked in April, coincident with breeding and courtship, and declined rapidly throughout the summer. Seasonal plasma T patterns in smaller though reproductively active adult males differed from those in their larger counterparts during the breeding season. Both size classes showed significant increases in plasma T concentration from February to March, at the beginning of the breeding season. However, smaller adults did not experience the peak in plasma T concentrations in April that were observed in larger adults, and their concentrations were significantly lower than those of larger males for the remainder of the breeding season. Plasma DHEA concentrations peaked in May and were significantly reduced by June. This is the first study to demonstrate the presence of DHEA in a crocodilian, and the high plasma DHEA concentrations that paralleled the animals' reproductive activity suggest a reproductive and/or behavioral role in adult male alligators. Similar to androgen variations in some birds, plasma DHEA concentrations in the alligators were considerably higher than T concentrations during the nonbreeding season, suggesting a potential role in maintaining nonbreeding seasonal aggression.     

Animal-Borne Imaging Reveals Novel Insights into the Foraging Behaviors and Diel Activity of a Large-Bodied Apex Predator,the American Alligator (Alligator mississippiensis)

Large-bodied, top- and apex predators (e.g., crocodilians, sharks, wolves, killer whales) can exert strong top-down effects within ecological communities through their interactions with prey. Due to inherent difficulties while studying the behavior of these often dangerous predatory species, relatively little is known regarding their feeding behaviors and activity patterns, information that is essential to understanding their role in regulating food web dynamics and ecological processes. Here we use animal-borne imaging systems (Crittercam) to study the foraging behavior and activity patterns of a cryptic, large-bodied predator, the American alligator (Alligator mississippiensis) in two estuaries of coastal Florida, USA. Using retrieved video data we examine the variation in foraging behaviors and activity patterns due to abiotic factors. We found the frequency of prey-attacks (mean = 0.49 prey attacks/hour) as well as the probability of prey-capture success (mean = 0.52 per attack) were significantly affected by time of day. Alligators attempted to capture prey most frequently during the night. Probability of prey-capture success per attack was highest during morning hours and sequentially lower during day, night, and sunset, respectively. Position in the water column also significantly affected prey-capture success, as individuals’ experienced two-fold greater success when attacking prey while submerged. These estimates are the first for wild adult American alligators and one of the few examples for any crocodilian species worldwide. More broadly, these results reveal that our understandings of crocodilian foraging behaviors are biased due to previous studies containing limited observations of cryptic and nocturnal foraging interactions. Our results can be used to inform greater understanding regarding the top-down effects of American alligators in estuarine food webs. Additionally, our results highlight the importance and power of using animal-borne imaging when studying the behavior of elusive large-bodied, apex predators, as it provides critical insights into their trophic and behavioral interactions.     

Identification of new C27 and C24 bile acids in the bile of Alligator mississippiensis

Biliary bile acids of Alligator mississippiensis were analyzed by gas-liquid chromatography-mass spectrometry after fractionation by silica gel column chromatography. It was shown that the alligator bile contained 12 C27 bile acids and 8 C24 bile acids. In addition to the C27 bile acids, such as 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestanoic acid, 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid, 3 alpha,12 alpha-dihydroxy-5 beta-cholestanoic acid, 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholestanoic acid, and 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholestanoic acid, identified previously in the bile of A. mississippiensis, 3 alpha,7 beta-dihydroxy-5 beta-cholestanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholestanoic acid, 7 beta,12 alpha-dihydroxy-3-oxo-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestanoic acid, 3 alpha,7 alpha,12 alpha,26-tetrahydroxy-5 beta-cholestanoic acid, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholestanoic acid were newly identified. And in addition to the C24 bile acids, such as chenodeoxycholic acid, ursodeoxycholic acid, cholic acid, and allocholic acid, identified previously, deoxycholic acid, 3 alpha,7 alpha-dihydroxy-5 beta-chol-22-enoic acid, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-chol-22-enoic acid, and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid were newly identified.     

Altered mitochondrial function and metabolic inflexibility associated with loss of caveolin-1

Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility.     

Differences in tissue-specific lipid composition between embryos of wild and captive breeding alligators (Alligator mississippiensis)

Fertile eggs obtained from alligators reared in captivity typically exhibit high rates of embryonic mortality. Also, the fatty acid composition of the yolk lipid of the captive eggs is markedly different from that observed in eggs from wild alligators, possibly as a result of differences in maternal diet in the two situations. The fatty acid compositions of tissue lipids during the embryonic development of wild and captive alligators were compared. The lipids of liver, adipose tissue and heart of the two types of embryo displayed fatty acid profiles which generally reflected the acyl compositions of the respective yolks. Thus the lipids from these tissues of the captive embryos contained markedly higher proportionate levels of linoleic and linolenic acids, lower levels of palmitoleic acid, and, in general, lower levels of docosahexaenoic acid and other C20 and C22 polyunsaturates, in comparison to the values for the wild embryos. In contrast, the fatty acid composition of the brain phosphoglycerides was very similar in the two types of embryo. Thus, at least in those embryos which had survived during the developmental period studied, the brain was able to maintain a relatively constant fatty acid composition, in spite of major differences between the wild and captive eggs in the proportions of the various fatty acids supplied from the yolk. It is suggested that a major cause of embryonic mortality in the captive embryos could be a failure to maintain an adequate level of docosahexaenoic acid in the developing brain.