Genetic characterization of captive Cuban crocodiles (Crocodylus rhombifer) and evidence of hybridization with the American crocodile (Crocodylus acutus)

There is a surprising lack of genetic data for the Cuban crocodile (Crocodylus rhombifer), especially given its status as a critically endangered species. Samples from captive individuals were used to genetically characterize this species in comparison with other New World crocodilians. Partial mitochondrial sequence data were generated from cyt-b (843 bp) and the tRNA(Pro)- tRNA(Phe)-D-loop region (442 bp). Phylogenetic analyses were performed by generating maximum parsimony, maximum likelihood, and Bayesian-based topologies. In addition, in an effort to identify species-specific alleles, ten polymorphic microsatellite loci were genotyped. Distance and model-based clustering analyses were performed on microsatellite data, in addition to a model-based assignment of hybrid types. Both mitochondrial and nuclear markers identified two distinct C. rhombifer genetic sub-clades (alpha and beta); and microsatellite analyses revealed that most admixed individuals were F(2) hybrids between C. rhombifer-alpha and the American crocodile (C. acutus). All individuals in the C. rhombifer-beta group were morphologically identified as C. acutus and formed a distinct genetic assemblage. J. Exp. Zool. 309A:649-660, 2008. (c) 2008 Wiley-Liss, Inc.     

Alpha-1-antitrypsin (protease inhibitor) phenotypes and longevity

We have tested the hypothesis that the protease inhibitor phenotypes MZ and MS are disadvantageous and reduce survival by comparing the prevalence of these phenotypes in a group of 707 very old people (hospital patients) with the prevalences reported in younger populations of blood donors. The MS and MZ phenotypes appear to be no less common among those who have survived to old age, but a highly significant difference was found in the occurrence of the M subtypes. The M1 type was more common in the elderly, and the M heterozygotes were less common than would be predicted from the reported incidence in younger groups and from the Hardy-Weinberg equilibrium. This discrepancy appeared to be smaller in subjects of Mediterranean origin than in those of British or Irish genetic background.     

Evolutionary history of Cuban crocodiles Crocodylus rhombifer and Crocodylus acutus inferred from multilocus markers

Among crocodilians, Crocodylus rhombifer is one of the world's most endangered species with the smallest natural distribution. In Cuba, this endemic species coexists with the American crocodile (Crocodylus acutus). Hybridization between these two species is well known in captivity and might occur in the wild, but has never been demonstrated genetically. Here, we combined molecular data with environmental, geographic, and fossil data to infer the evolutionary history of Crocodylus in the Cuban Archipelago, and to evaluate genealogical support for species boundaries. We analyzed seven microsatellite loci plus DNA sequence data from nuclear (RAG-1) and mitochondrial (cytochrome b and cytochrome oxidase I) genes from 89 wild-caught individuals in Cuba, Grand Cayman Island, Jamaica, and Central America, and two samples from zoo collections. Microsatellites showed evidence of introgression, suggesting potential hybridization among Cuban groups. In Cuba, C. acutus contained one mitochondrial DNA (mtDNA) haplotype, whereas C. rhombifer contained two haplotypes. MtDNA data showed that C. acutus is paraphyletic with respect to C. rhombifer, revealing 1% sequence divergence between species within Cuba vs. 8% divergence between Cuban forms and mainland C. acutus. We suggest that hybridization has been a historical as well as a current phenomenon between C. acutus and C. rhombifer. These findings suggest that long-term conservation of crocodiles in Cuba will require identification of genetically pure and hybrid individuals, and a decrease in anthropogenic activities. We also recommend more extensive morphological and genetic analyses of Cuban population to establish clear boundaries of the hybrid zone between C. acutus and C. rhombifer.     

The reproductive cycle of the Nile crocodile (Crocodylus nilotkus)

The reproductive cycle of the Nile crocodile ( Crocodylus niloticus ) was studied in Zimbabwe. Females attained sexual maturity after they had reached a total length of about 262 to 287 cm and males, 270 to 295 cm. Some adult females did not reproduce every year. Follicle growth and vitellogenesis occurred from April to mid August during the dry winter. During this time reproductive females had elevated levels of plasma oestradiol-17β, testosterone, calcium and magnesium, but lowered levels of iron. The measuring of plasma calcium was an 'early pregnancy' test, reliable up to four months before nesting. Elevated levels of plasma testosterone in reproductive females corresponded to the time of courtship and mating. Ovulation occurred during the latter half of August. Males had viable sperm from mid May to mid September, during the winter. Courtship and mating occurred from late June to mid August, when crocodiles were confined to pools. Females nested on the higher sand ridges in the dry river bed from early September to early October, and eggs hatched during December.     

Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria

ABSTRACT: BACKGROUND: The Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains. METHODS: Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650M and the purified plasma was tested for antibacterial activity. RESULTS: Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates. CONCLUSIONS: The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.     

253 Novel polymorphic microsatellites for the saltwater crocodile (Crocodylus porosus)

Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253 novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an average polymorphic information content (PIC) of 0.425.     

Scanning electron microscopic study of intestinal mucosa of the Nile crocodile (Crocodylus niloticus)

As part of an ongoing study of the intestinal tract of the Nile crocodile Crocodylus niloticus, the appearance of the small intestinal mucosa was examined using a scanning electron microscope (SEM). The duodenum displayed ridge-like and complex primary folds, both of which were arranged in a longitudinally directed zigzag pattern. The two types of folds alternated with each other, with the second type following the zigzag contours of the first. The folds were covered with polygonal- and dome-shaped epithelial cells. The latter cell type was less common and was restricted in location to the proximal two thirds of the mucosal folds. Both types of epithelial cells were covered with stubby microvilli which displayed a distinct linear arrangement. Goblet cells were present between the absorptive cells. In the jejunum the basic pattern of the folds persisted except that both types of primary folds became tall and leaf-like. The angle of the zigzag pattern was less acute than in the duodenum. In the ileum the two types of alternating folds decreased in height and were arranged in a longitudinally directed wave-like fashion. The zigzag mucosal folds of the small intestine ended abruptly at the ileorectal junction. The rectum displayed low, irregular folds which formed occasional large, puckered, rosette-shaped structures. The particular arrangement of the intestinal folds fulfills the dual function of promoting the absorptive process and facilitating the smooth passage of the intestinal contents. © 1995 Wiley-Liss, Inc.     

Shifts in hatching date of American crocodile (Crocodylus acutus) in southern Florida

Globally temperature of marine environments is on the rise and temperature plays an important role in the life-history of reptiles. In this study, we examined the relationship between sea surface temperature and average date of hatching for American crocodiles (Crocodylus acutus) over a 37-year period at two nesting sites, Everglades National Park and Florida Power and Light Turkey Point Power Plant site in southern Florida. Our results indicate that hatch dates are shifting 1.5 days earlier every two years and at half that rate for the Turkey Point site, and with every 1 °C degree increase in temperature, hatching occurs about 10 days earlier in the Everglades and 6 days earlier at Turkey Point. Our results on shifting hatch dates for American crocodiles provide further details about the impacts of temperature change on crocodile life history and suggest that increased temperature may affect their phenology.     

Nest opening response of the Nile crocodile Crocodylus niloticus

Observations on captive Nile crocodiles, Crocodylus niloticus confirm earlier reports on parental care of nests and of young. Male and female appear to form a pair bond after courtship, the male defending nesting territory with the female rarely leaving the nest until the sounds of the young hatching stimulate her to open the nest to release them. The female transports unhatched eggs and live young in her buccal pouch to water and establishes a nursery where the young are defended for several weeks. Experiments involving playback of distress calls by tape recorder, offering of live young and eggs to the adults, the parent opening an artificial nest and vocalisations and resultant interactions between adults and young are described.     

Morphology and ultrastructure of possible integumentary sense organs in the estuarine crocodile (Crocodylus porosus)

The skins of crocodylids and gavialids can be distinguished from those of alligatorids by the presence of darkly pigmented pits, known as integumentary sense organs (ISOs), on the postcranial scales. The structure of ISOs, in Crocodylus porosus, was studied using light microscopy and scanning and transmission electron microscopy. The stratum corneum of the epidermis in the area of the ISO is thinner, while the stratum germinativum is thicker, relative to other regions of the integument. Beneath the epidermal layer the ISO region has a paucity of collagen fibers relative to the rest of the dermis. Widely dispersed fibrocytes, nerve terminals, and chromatophores occur throughout the ISO region of the dermis, but these elements are concentrated in the area immediately beneath the stratum germinativum in the ISO region. The morphology of the ISOs suggests that they are sensory organs. It has traditionally been assumed that sensory organs on the amniote integument have a mechanosensory function. However, alternate functional interpretations of this structure are possible, and a resolution awaits further work. © 1996 Wiley-Liss, Inc.     

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii)

The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5+/-0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species.     

Ultrastructure of the crocodile kidney (Crocodylus acutus) with special reference to electrolyte and fluid transport

The study of the ultrastructure of the kidney tubules of the crocodile was made to compare the cellular structure with the capacity for electrolyte resorption and the ability to create an osmotic gradient across the tubular wall. The crocodile tubular cells were found to differ from the mammalian tubular cells in that they do not have basal infoldings, but instead have open lateral spaces between the cells, similar in many aspects to those found in the mammalian gallbladder. The physiological role of these lateral spaces in solute and fluid transfer is discussed.     

Some properties of a ficin-papain inhibitor from avian egg white

A procedure has been established for the isolation, from sheep liver, of 6-phosphogluconate dehydrogenase which is homogeneous according to the criteria of the analytical ultracentrifuge, and isoelectric focusing. A systematic determination of the effects of pH, ionic strength, metal ions, and temperature, on the kinetic parameters of the isolated 6-phosphogluconate dehydrogenase has been carried out. Double-reciprocal plots of enzyme rate measurements as a function of substrate concentration indicate K_m values of 15 μm for 6-phosphogluconate, and 7 μm for NADP⁺, under optimum assay conditions, and show no effect of the concentration of one substrate on the K_m of the other substrate under the assay conditions employed. Ionic strength, monovalent and divalent metals, are apparently interchangeable in their ability to activate the enzyme, and act by decreasing the K_m values of the enzyme, not by increasing the reaction rate. Concentrations of metals above the optimum are strongly inhibitory. Plots of ?log K_m vs pH show inflection points at 8.3 for 6-phosphogluconate, and 6.5 for NADP⁺. At low substrate concentrations the pH optimum of the enzyme is at pH 7.7, but plots of V vs pH increase up to pH 9.1 (the enzyme is unstable at higher pH values). An Arrhenius plot shows a straight line between temperatures of 8.6 and 39.4 °C, and an energy of activation of 15,450 cal mole^?1.