High levels of population genetic differentiation in the American crocodile (Crocodylus acutus).

The American crocodile (Crocodylus acutus) is a widely distributed species across coastal and brackish areas of the Neotropical region of the Americas and the Greater Antilles. Available information on patterns of genetic differentiation in C. acutus shows a complex structuring influenced by interspecific interactions (mainly hybridization) and anthropogenic actions (mostly historical hunting, recent poaching, habitat loss and fragmentation, and unintentional translocation of individuals). In this study, we used data on mitochondrial DNA control region and 11 nuclear polymorphic microsatellite loci to assess the degree of population structure of C. acutus in South America, North America, Central America and the Greater Antilles. We used traditional genetic differentiation indices, Bayesian clustering and multivariate methods to create a more comprehensive picture of the genetic relationships within the species across its range. Analyses of mtDNA and microsatellite loci show evidence of a strong population genetic structure in the American crocodile, with unique populations in each sampling locality. Our results support previous findings showing large degrees of genetic differentiation between the continental and the Greater Antillean C. acutus. We report three new haplotypes unique to Venezuela, which are considerably less distant from the Central and North American haplotypes than to the Greater Antillean ones. Our findings reveal genetic population differentiation between Cuban and Jamaican C. acutus and offer the first evidence of strong genetic differentiation among the populations of Greater Antillean C. acutus.

Identification and characterization of dipeptidyl peptidase IV enzyme activity in the American crocodile (Crocodylus acutus).

Serum from the American crocodile was assayed for dipeptidyl peptidase IV (DPP4) activity. We measured the DPP4-mediated hydrolysis of Ala-Pro-AFC. The generation of AFC was dependent on the titer of serum, with significant DPP4 activity (0.20 + or - 0.03 nmol product formed) measured using only 2 microL of crocodile serum, with maximum activity measured using 500 microL of serum. The hydrolysis of substrate was inhibited in a concentration-dependent manner by diprotin A, a specific inhibitor of DPP4 activity, indicating that this activity was due to the presence of DPP4. The crocodile serum DPP4 exhibited classical Michaelis-Menten kinetics, with K(m) and V(max) extrapolated, by double-reciprocal plot, to be 14.7 + or - 1.3 microM and 75.5 + or - 4.3 nmol/min, respectively. Crocodile DPP4 catalyzed the hydrolysis of Ala-Pro-AFC rapidly, with substantial activity measured within 5 min of the addition of substrate. After an initial rapid increase in activity, near maximal activity (7.43 + or - 0.24 nmol product formed) measured at 180 min. Crocodile serum DPP4 activity was temperature-dependent, with steadily increased activity from 5 to 40 degrees C.

Identification and characterization of serum complement activity in the American crocodile (Crocodylus acutus).

Incubation of unsensitized sheep red blood cells with serum from the American crocodile (Crocodylus acutus) resulted in a concentration-dependent hemolysis. The hemolytic activity was heat-sensitive, and inhibited by EDTA in a concentration-dependent manner. The EDTA-inhibited SRBC hemolysis could be restored by the addition of excess Ca2+ or Mg2+, but not Ba2+ or Cu2+, revealing the specificity of this activity for these two divalent cations. The hemolytic activity of crocodile serum was titer-dependent, with 329 microL producing 50% of maximal SRBC hemolysis. The complement activity was also temperature-dependent, with decreased activity at lower temperatures (5-15 degrees C) and maximal activity occurred at 30-40 degrees C. The hemolysis occurred relatively slowly, with near zero activity after 10 min, 40% of activity observed within 15 min of exposure to SRBCs, and maximal activity at 30 min.