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To elucidate the molecular mechanism of the direct allosteric exchange of oxygen and hydrogen carbonate in the hemoglobins of crocodiles, the N-terminal sequence of the beta-chains of the crocodiles of the Nile (Crocodylus niloticus) and of the Mississippi (Alligator mississippiensis) was studied. The N-terminal end of the peptide is blocked. By mass spectrometry the N-terminal sequences of both species were found to be N-acetyl-alanyl-seryl-phenylalanine. These data explain the absence of hemoglobinphosphate interaction, the data are in good agreement with the stereochemistry postulated for allosteric exchange of oxygen and hydrogen carbonate in crocodilian hemoglobins.
Two molecular forms of prolactin (PRL), glycosylated and non-glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 M sucrose, 1 mM NH4HCO3, pH 6.3. Purification was performed by ion exchange chromatography on DE-52, gel filtration on Sephadex G-75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26,000 and 24,000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti-sea turtle PRL and anti-ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn-Ala-Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26,000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24,000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and Ile for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.
Several methods for external sampling of blood and administration of intravascular fluids are applicable to reptilian species. This paper reviewed some of the technics that have been previously reported and introduced several new procedures applicable to snakes, turtles, alligators, crocodiles, and lizards.
David Peakall and co-workers pioneered innovative approaches that utilized extra-embryonic membranes to assess accumulation of organochlorine pesticides in eggs. This technique provided the foundation for an entire line of research to improve non-lethal methods for assessing contaminant exposure in oviparous wildlife. Currently, analysis of chorioallantoic membranes (CAMs) provides predictable estimates of chlorinated contaminant presence in eggs and in maternal tissues. Field studies have been conducted with herons, stilts, alligators, crocodiles, and sea turtles. Controlled dose-response studies have been completed in chickens. The following manuscript presents the foundations for the CAM approach and a review of research findings involving this technique.
Developing organisms are sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in reproductive as well as nonreproductive organs, including persistent molecular alterations. Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans, some of which exhibit estrogenic or anti-androgenic activity by binding to estrogen or androgen receptors. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, should be identified to understand the molecular basis of estrogenic activity during embryonic development. To understand molecular mechanisms of estrogenic chemicals in developing organisms, we identified estrogen-responsive genes using cDNA microarrays and a quantitative reverse transcriptase-polymerase chain reaction, and genes related to estrogen-independent vaginal changes in mice induced by estrogens during a critical window. We also analyzed genes in Xenopus laevis embryos related to abnormalities induced by estrogenic chemicals using cDNA microarrays. Estrogen receptors and other steroid hormone receptors were cloned in various species, including the American alligator, crocodile, Florida red-bellied turtle, Silurana tropicalis, roach, Fundulus and mosquito fish. An environmental androgen, trenbolone, was found to induce persistent changes in the gonopodium and ovotestis in mosquito fish. In Daphnia magna, juvenile hormone agonists induce the production of male offspring. Tributyltin (TBT) induces imposex in rockshell through retinoid X receptor (RXR) activation. Microarray technology is a powerful tool for understanding the molecular mechanism of receptor-mediated toxicology in various animal species. Ecotoxicogenomics is a multidisciplinary field concerned with elucidating how the entire genome is involved in the biological response of wild animals exposed to environmental toxicants and stressors. Ecotoxicogenomics combines information from studies of genomic-scale mRNA profiling by microarray analysis (transcriptomics), protein profiling (proteomics), metabolite profiling (metabolonomics) and computational models to understand the roles of gene-environment interaction in the chemical toxicity. To understand the molecular mechanism of chemicals in developing wildlife, the establishment of ecotoxicogenomics is essential in the near future.
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Mercury methylation may be enhanced in wetlands and humic-rich, blackwater systems that crocodiles and alligators typically inhabit. Given their high trophic level and long life-spans, crocodilians could accumulate significant burdens of Hg. Our objectives were to survey Hg concentrations in alligators from several areas in the southeastern United States to test their utility as sentinels of Hg contamination, to examine relationships among Hg concentrations in various tissues and to look for any differences in tissue Hg concentrations among locations. We measured total Hg concentrations in alligators collected in the Florida Everglades (n = 18), the Okefenokee National Wildlife Refuge, Georgia (n = 9), the Savannah River Site (SRS), South Carolina (n = 49) and various locations in central Florida (n = 21), sampling tissues including blood, brain, liver, kidney, muscle, bone, fat, spleen, claws and dermal scutes. Alligators from the Everglades were mostly juvenile, but Hg concentrations in tissues were high (means: liver 41.0, kidney 36.4, muscle 5.6 mg Hg/kg dry wt.). Concentrations in alligators from other locations in Florida were lower (means: liver 14.6, kidney 12.6, muscle 1.8 mg Hg/kg dry wt.), although they tended to be larger adults. Alligators from the Okefenokee were smallest and had the lowest Hg concentrations (means: liver 4.3, kidney 4.8, muscle 0.8 mg Hg/kg dry wt.). SRS alligators had the greatest size range and intermediate Hg levels (means: liver 14.9, muscle 4.8 mg Hg/kg dry wt.). At some locations, alligator length was correlated with Hg concentrations in some internal organs. However, at three of the four locations, muscle Hg was not related to length. Tissue Hg concentrations were correlated at most locations however, claw or dermal scute Hg explained less than 74% of the variation of Hg in muscle or organs, suggesting readily-obtained tissues, such as scutes or claws, have limited value for non-destructive screening of Hg in alligator populations.
The concentrations of Na, K, Ca, Mg, Ba, Sr, Fe, Al, Mn, Zn, Pb, Cu, Ni, Cr, Co, Se, U and Ti were determined in the osteoderms and/or flesh of estuarine crocodiles (Crocodylus porosus) captured in three adjacent catchments within the Alligator Rivers Region (ARR) of northern Australia. Results from multivariate analysis of variance showed that when all metals were considered simultaneously, catchment effects were significant (P < or = 0.05). Despite considerable within-catchment variability, linear discriminant analysis (LDA) showed that differences in elemental signatures in the osteoderms and/or flesh of C. porosus amongst the catchments were sufficient to classify individuals accurately to their catchment of occurrence. Using cross-validation, the accuracy of classifying a crocodile to its catchment of occurrence was 76% for osteoderms and 60% for flesh. These data suggest that osteoderms provide better predictive accuracy than flesh for discriminating crocodiles amongst catchments. There was no advantage in combining the osteoderm and flesh results to increase the accuracy of classification (i.e. 67%). Based on the discriminant function coefficients for the osteoderm data, Ca, Co, Mg and U were the most important elements for discriminating amongst the three catchments. For flesh data, Ca, K, Mg, Na, Ni and Pb were the most important metals for discriminating amongst the catchments. Reasons for differences in the elemental signatures of crocodiles between catchments are generally not interpretable, due to limited data on surface water and sediment chemistry of
The concentrations of Na, K, Ca, Mg, Ba, Sr, Fe, Al, Mn, Zn, Pb, Cu, Ni, Cr, Co, Se, U, and Ti were determined in the flesh and osteoderms of estuarine crocodiles (Crocodylus porosus) captured in three adjacent catchments of Kakadu National Park, within the Alligator Rivers Region of northern Australia. This study provides, for the first-time, baseline concentrations of elements in both flesh and osteoderms of wild crocodiles. Multiple linear regression analyses were used to determine the effects of total crocodile length, estimated age, gender, inferred reproductive status, physical condition, and catchment of capture on element concentrations in both tissues. The Mg concentration (log10) in the flesh and osteoderms of C. porosus significantly (p < or = 0.001) decreased with increasing length (1.7-5.0 m) and estimated age (5-40 years). Similarly, the Ti concentration (log10) in flesh significantly (p < or = 0.01) decreased with increasing length. In contrast, Zn and Se concentration (log10) in flesh significantly (p < or = 0.001) increased with increasing length and/or age, suggesting that these relationships are mediated by biological rather than environmental chemical factors. In flesh, Fe and Na concentrations (log10) significantly (p < or = 0.05) increased as the physical condition of C. porosus deteriorated. No significant (p > 0.05) effects of gender or inferred reproductive status on element concentrations in the flesh and osteoderms were found. The mean concentrations (log10) of Al, Ba, Cr, Ni, and Pb in flesh and Co, Fe, Mg, Mn, and U in the osteoderms were significantly (p < or = 0.01) different between catchments. The significant (p < or = 0.05) effects of catchment on the concentrations of various elements indicate that C. porosus reflects the chemistry of its environmental milieu and therefore has a certain degree of catchment fidelity, even though the catchments are adjacent to one another. Such catchment-specific signals may be useful in the determination of the provenance of itinerant crocodiles. They also point to the utility of crocodiles as long-term biomonitors of their environment.
Based on published records and own data a summary is given of the geographical distribution of the currently known species of pentastomid parasites infecting crocodiles and alligators, as well as freshwater chelonians. A brief generic diagnosis is provided for each genus. Fourteen out of the currently 23 living crocodilian species have been recorded as being host to one or more pentastomes. Out of the 32 pentastome species six are considered species inquirendae. Presently, six genera of crocodilian pentastomes, Agema, Alofia, Leiperia, Sebekia, Selfia and Subtriquetra are recognized. African crocodiles harbour eight pentastome species, six of which have been recorded from the Nile crocodile, Crocodylus niloticus. Three species belong to the genus Sebekia, Alofia being represented by two and Leiperia by only one species. Two species, Alofia parva and Agema silvae-palustris, occur in the dwarf crocodile, Osteolaemus tetraspis, and the slender-snouted crocodile, Crocodylus cataphractus, exclusively, but a single Sebekia species is shared with the Nile crocodile. The genus Agema is endemic to the African region. Infective stages of the pentastome Subtriquetra rileyi, thought to utilize Nile crocodiles as final hosts, have been recovered only from fishes. The largest number of pentastome species is found in the Australasian region. Of these, the Indo-Pacific crocodile, Crocodylus porosus, harbours seven, representing the genera Alofia, Sebekia, Leiperia and Selfia. Selfia is exclusive to the latter host. The genus Subtriquetra has been reported from "Indian crocodiles", a term possibly referring to either Crocodylus palustris, Crocodylus porosus or Gavialis gangeticus. Ten species of pentastomes parasitizing the crocodilian genera Alligator, Caiman, Crocodylus and Melanosuchus have been recorded from the Neotropical region including the southern states of the North American continent. The two most wide-spread pentastome genera, Alofia and Sebekia, have been recorded together with representatives of the genus Subtriquetra and immature and larval forms of Leiperia. To date the two monospecific genera, Pelonia, from two terrapin species, Pelusios sinuatus and Pelomedusa subrufa, in South Africa, and Diesingia from Hydraspis geoffroyana and Hydromedusa tectifera in South America, are the only chelonian pentastomes recovered world-wide. A possible exception is the crocodilian pentastome Sebekia mississippiensis which can reach maturity in experimentally infected terrapins.
The date of a single divergence point--between living alligators and crocodiles--was estimated with quartet dating using calibrations of widely divergent ages. For five mitochondrial sequence datasets, there is a clear relationship between calibration age and quartet estimate--quartets based on two relatively recent calibrations support younger divergence estimates than do quartets based on two older calibrations. Some of the estimates supported by young quartets are impossibly young and exclude the first appearance of the group in the fossil record as too old. The older estimates--those based on two relatively old calibrations--may be overestimates, and those based on one old and one recent calibration support divergence estimates very close to fossil data. This suggests that quartet dating methods may be most effective when calibrations are applied from different parts of a clade's history.
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The complete mitochondrial genome of the alligator, Alligator mississippiensis, was sequenced. The size of the molecule is 16,642 nucleotides. Previously reported rearrangements of tRNAs in crocodile mitochondrial genomes were confirmed and, relative to mammals, no other deviations of gene order were observed. The analysis of protein-coding genes of the alligator showed an evolutionary rate that is roughly the same as in mammals. Thus, the evolutionary rate in the alligator is faster than that in birds as well as that in cold-blooded vertebrates. This contradicts hypotheses of constant body temperatures or high metabolic rate being correlated with elevated molecular evolutionary rates. It is commonly acknowledged that birds are the closest living relatives to crocodiles. Birds and crocodiles represent the only archosaurian survivors of the mass extinction at the Cretaceous/Tertiary boundary. On the basis of mitochondrial protein-coding genes, the Haemothermia hypothesis, which defines birds and mammals as sister groups and thus challenges the traditional view, could be rejected. Maximum-likelihood branch length data of amino acid sequences suggest that the divergence between the avian and crocodilian lineages took place at approximately equal to 254 MYA.
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