Plasma vitellogenin in Morelet's crocodiles from contaminated habitats in northern Belize.

Vitellogenin induction has been widely used as a biomarker of endocrine disruption in wildlife, but few studies have investigated its use in wild reptiles living in contaminated habitats. This study examined vitellogenin induction in Morelet's crocodiles (Crocodylus moreletii) from wetlands in northern Belize contaminated with organochlorine (OC) pesticides. Vitellogenin was measured in 381 crocodile plasma samples using a vitellogenin ELISA previously developed for this species. Vitellogenin was detected in nine samples, all from adult females sampled during the breeding season. Males and juvenile females did not contain detectable levels of vitellogenin; however, many of these animals contained OC pesticides in their caudal scutes, confirming contaminant exposure. The lack of a vitellogenic response in these animals may be attributable to several factors related to the timing and magnitude of exposure to endocrine-disrupting chemicals and should not be interpreted as an absence of other contaminant-induced biological responses.

Metals and organochlorine pesticides in caudal scutes of crocodiles from Belize and Costa Rica.

Despite high animal diversity in the Neotropics and the largely unregulated use and disposal of pesticides and industrial chemicals in Central America, few data exist regarding accumulation of environmental contaminants in Central American wildlife. In this study we examined accumulation of metals and organochlorine (OC) pesticides in caudal scutes of crocodiles from Belize and Costa Rica. Scutes from Morelet's crocodiles (Crocodylus moreletii) from two sites in northern Belize were analyzed for metals, and scutes from American crocodiles (C. acutus) from one site in Costa Rica were analyzed for metals and OC pesticides. All scutes (n=25; one scute from each of 25 individuals) contained multiple contaminants. Mercury was the predominant metal detected, occurring in all scutes examined from both species. Other metals detected include cadmium, copper, lead, and zinc. American crocodile scutes from Costa Rica contained multiple OC pesticides, including endrin, methoxychlor, p,p'-DDE, and p,p'-DDT, all of which occurred in 100% of scutes analyzed (n=6). Mean metal and OC concentrations varied in relation to those previously reported in crocodilian scutes from other localities in North, Central, and South America. OC concentrations in American crocodile scutes were generally higher than those previously reported for other Costa Rican wildlife. Currently, caudal scutes may serve as general, non-lethal indicators of contaminant accumulation in crocodilians and their areas of occurrence. However, a better understanding of the relationships between pollutant concentrations in scutes, internal tissues, and environmental matrices at sample collection sites are needed to improve the utility of scutes in future ecotoxicological investigations.

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.