Plasma vitellogenin in Morelet’s crocodiles from contaminated habitats in northern BelizeIntroductionMaterials and methodsStudy sitesSample collectionDetermination of plasma vitellogeninVitellogenin antibodyVitellogenin enzyme-linked immunosorbent assay (ELISA)Denaturing polyacrylamide gel electrophoresis (SDS-PAGE)ImmunoblottingStatistical analysisResultsDiscussionConclusionsAcknowledgmentsReferencesPlasma vitellogenin in Morelet’s crocodiles fromcontaminated habitats in northern BelizeThomas R. Rainwatera,*, Kyle W. Selcerb, Lisa M. Nespolib, Adam G. Fingera,David A. Rayc,1, Steven G. Plattd, Philip N. Smitha, Llewellyn D. Densmorec,Todd A. Andersona, Scott T. McMurryaaThe Institute of Environmental and Human Health and Department of Environmental Toxicology,Texas Tech University, Box 41163, Lubbock, TX 79409-1163, USAbDepartment of Biological Sciences, Bayer School of Natural and Environmental Sciences,Duquesne University, Pittsburgh, PA 15282, USAcDepartment of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USAdDepartment of Biology, P.O. Box C-64, Sul Ross State University, Alpine, TX 79832, USAReceived 15 May 2007; received in revised form 20 July 2007; accepted 25 July 2007Wild crocodiles living in habitats polluted with organochlorine pesticide sdid not exhibit contaminant-induced vitellogenin induction in blood plasma.AbstractVitellogenin induction has been widely used as a biomarker of endocrine disruption in wildlife, but few studies have investigated its use inwild reptiles living in contaminated habitats. This study examined vitellogenin induction in Morelet’s crocodiles (Crocodylus moreletii) fromwetlands in northern Belize contaminated with organochlorine (OC) pesticides. Vitellogenin was measured in 381 crocodile plasma samplesusing a vitellogenin ELISA previously developed for this species. Vitellogenin was detected in nine samples, all from adult females sampledduring the breeding season. Males and juvenile females did not contain detectable levels of vitellogenin; however, many of these animals con-tained OC pesticides in their caudal scutes, confirming contaminant exposure. The lack of a vitellogenic response in these animals may be at-tributable to several factors related to the timing and magnitude of exposure to endocrine-disrupting chemicals and should not be interpreted asan absence of other contaminant-induced biological responses.Ó 2007 Elsevier Ltd. All rights reserved.Keywords: Vitellogenin; Organochlorine pesticides; Endocrine disruption; Crocodiles; Belize1. IntroductionOver the last 15 years, increasing evidence of contaminant-induced endocrine disruption in wildlife has highlighted theneed for sensitive and reliable assays to screen populationsfor exposure to compounds that perturb the endocrine system(Colborn et al., 1993; Crain and Guillette, 1997; Hutchinsonand Pickford, 2002; Rotchell and Ostrander, 2003; Segneret al., 2003; Porte et al., 2006; Hutchinson et al., 2006). Induc-tion of the egg-yolk precursor protein, vitellogenin, has shownpromise as a sensitive, non-lethal, and non-destructive* Corresponding author: 155 Johnson Ferry Road, Marietta, GA 30068,USA. Tel.: þ1 806 239 5472; fax: þ1 806 885 2132.E-mail addresses: [email protected] (T.R. Rainwater),[email protected] (K.W. Selcer), [email protected] (L.M. Nespoli),[email protected] (A.G. Finger), [email protected] (D.A. Ray),[email protected] (S.G. Platt), [email protected] (P.N. Smith), [email protected] (L.D. Densmore), [email protected] (T.A.Anderson), [email protected] (S.T. McMurry).1Current address: Department of Biology, West Virginia University,Morgantown, WV 26506, USA.0269-7491/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.envpol.2007.07.018Available online at www.sciencedirect.comEnvironmental Pollution 153 (2008) 101e109www.elsevier.com/locate/envpolbiomarker of wildlife exposure to estrogenic xenobiotics, par-ticularly in aquatic systems (Sumpter and Jobling, 1995;Palmer and Selcer, 1996; Palmer et al., 1998; Folmar et al.,1996; Cheek et al., 2001; Tyler et al., 2002; Liao et al., 2006).A number of in vivo assays for endocrine disruption havebeen developed that are based on vitellogenin induction, thevast majority of which use fish as model organisms (e.g.,Sumpter and Jobling, 1995; Folmar et al., 1996; Fenskeet al., 2001; Parks et al., 1999; Hemmer et al., 2001; Royet al., 2004; Cheek et al., 2004; Nakari, 2004; Kirby et al.,2004; Meucci and Arukwe, 2005; Van Veld et al., 2005; Eidemet al., 2006). In contrast, vitellogenin assays have been devel-oped for only a few species of lizards (Brasfield et al., 2002),turtles (Palmer and Palmer, 1995; Irwin et al., 2001; Herbstet al., 2003; Kamata et al., 2005; Rie et al., 2005; Sifuentes-Romero et al., 2006) and crocodilians (Rey et al., 2006; Selceret al., 2006). In fact, reptiles are poorly represented overall instudies of endocrine disruption (Hopkins, 2000). There isa pressing need for more information on the effects of environ-mental contaminants on reptiles because they are experiencinga worldwide decline, and environmental toxicants are amongthe suspected causes for their reduction in numbers (Hopkins,2000; Gibbons et al., 2000).Environmental contaminants have been found in crocodiliantissues throughout tropical and subtropical areas worldwide(see Campbell, 2003; Rainwater et al., 2007). There is consider-able evidence that exposure of American alligators (Alligatormississippiensis) to organochlorine (OC) pesticides and otherpollutants has detrimental effects on reproduction and develop-ment (Matter et al., 1998a,b; Guillette et al., 2000; Milnes et al.,2005); however, little information is available on endpoints ofendocrine disruption in wild crocodilians outside of Florida,USA. Freshwater (Crocodylus johnstoni) and estuarine(Crocodylus porosus) crocodiles from Western Australia haverecently been shown to contain high levels of OC pesticidesin their tissues (Yoshikane et al., 2006), and OCs have alsobeen recently detected in American crocodiles (Crocodylusacutus) from Costa Rica (Rainwater et al., 2007). In addition,several previous studies have reported multiple OC pesticidespresent in the eggs and tail scutes of Morelet’s crocodiles (Croc-odylus moreletii) from northern Belize (Wu, 2000; Wu et al.,2000a,b, 2006; DeBusk, 2001; Pepper et al., 2004), creatingthe potential for endocrine disruption in these