Population-specific incidence of testicular ovarian follicles in Xenopus laevis from South Africa: A potential issue in endocrine testing
Introduction
Testicular ovarian follicles (TOFs, also reported in the literature as testicular oocytes; TOs) are a phenomenon that can be induced by exposure to estrogens such as 17β-estradiol and which have been suggested as a potential endpoint for characterizing exposures and responses to estrogenic compounds (Hecker et al., 2006, Jobling et al., 1998, Mackenzie et al., 2003). TOFs are female reproductive cells with an intact nucleus, nucleoli, and a surrounding squamous epithelial layer embedded in testicular tissue (Hecker et al., 2006) and can occur naturally but to varying degrees in the testes of some frog species (Reeder et al., 2005, Witschi, 1929, Witschi, 1930, Witschi, 1942). Results of studies on exposure to atrazine and its linkage to the incidence of TOFs fall into three categories. Some researchers report no incidence in unexposed animals but increased incidence of TOFs in developing larval frogs exposed to concentrations of ≥0.1 μg atrazine/L (Hayes et al., 2003). Others report the presence of testicular oocytes in unexposed as well as atrazine-exposed frogs in laboratory and field studies with no relationship to exposure concentration (Coady et al., 2005, Jooste et al., 2005, Murphy et al., 2006, Smith et al., 2005). Other studies report no incidence in frogs, whether they are exposed to atrazine or not (Kloas et al., 2009, Oka et al., 2008). In an ecoepidemiological study of the grey tree frog, Acris crepitrans, TOFs were observed in specimens collected both before and after the introduction of atrazine to the market (Reeder et al., 2005). The number of TOFs has been reported to decrease with age in Xenopus laevis (Everson, 2006, Jooste et al., 2005) and regressed TOFs are more frequently observed in older animals (Jooste et al., 2005). An EPA Science Advisory Panel recommended that, because of the uncertainty and differences in observations between laboratories and in the field, a definitive study of the phenomenon should be conducted (USEPA, 2003).
For many years, exports of X. laevis as laboratory animals took place from the W Cape area of South Africa (SA) (Tinsley and McCoid, 1996), but the origins of the many colonies of X. laevis in laboratories around the world are, for the most part, unknown. The taxonomic relationships among all species of Xenopus, including morphologically differentiated populations of X. laevis, have been characterized by both mitochondrial (mtDNA) and nuclear DNA (Evans et al., 1997, Evans et al., 2004, Evans et al., 2005, Evans et al., 2008, Grohovaz et al., 1996, Measey and Channing, 2003) and multiple putative subspecies of X. laevis have been identified (Tinsley et al., 1996). Based on mtDNA, there are at least three divergent lineages in X. laevis within South Africa (SA) (Grohovaz et al., 1996). The distribution of these mtDNA lineages is potentially consistent with a role played by the Cape Fold Mountains (Fig. 1), which separate the southern winter-rainfall and northern summer-rainfall areas of SA, in restricting gene flow within X. laevis. However, relationships between these mtDNA lineages and the incidence of TOFs or molecular variation in nuclear DNA have not been investigated. As has been pointed out, correct identification of test organisms provides a baseline for extrapolation as well as avoiding the propagation of conceptual and methodological errors (Bortolus, 2008).
In this study, we explored the morphology, atrazine exposure, molecular diversity, and incidence of TOFs in X. laevis individuals collected in habitats in SA with little or no atrazine exposure, ranging from downwind and near to the major atrazine use-area in maize crops to areas that are upwind, far from this region, and that have no atrazine exposure—current or historical (Sites A–I, Fig. 1, see SI for details on sites). DNA sequences were obtained from representative individuals from these localities and from additional samples from throughout sub-Saharan Africa. We included other African species (X. gilli, X. muelleri, and Scathophaga tropicalis) in our molecular analysis as outgroups. In addition to these wild-collected frogs, we analyzed DNA from X. laevis from Xenopus-1 Inc. and Xenopus Express who supply this species to the north-American market and maintain colonies developed from X. laevis originally from suppliers in the W Cape (Weldon et al., 2007).
Section snippets
Collection of test animals and residue analyses
Four to 10 baited bucket Xenopus traps were set in the water bodies at the selected collection sites (B–I; see Fig. 1). Up to a maximum of 50 male frogs were collected at each site. Actual number of males collected varied between 12 and >50 (see SI). Females were also collected for the purposes of morphological characterization. Sediment and water samples were taken in the shallow water in the each of four quadrants of the pond. Water samples were pooled into two 1 L sub-samples collected in 1 L
Pesticide exposures
Organochlorine, organophosphorus, and pyrethroid pesticides were not detected at any of these locations (see SI). Atrazine, its metabolites, and other triazines also were not detected, except at Site I, where low concentrations of atrazine, simazine, and terbuthylazine were observed (0.1, 0.4, and 0.3 μg/L, respectively). Because of the persistence of atrazine in surface waters (Giddings et al., 2005), these samples are representative of use within the previous 2 years in the watershed of lotic
Discussion
Phylogenetic analysis of mitochondrial and nuclear genes indicates that frogs from the SW Cape are evolutionarily divergent from those from NE South Africa and the rest of sub-Saharan Africa. Site E was either a point of secondary contact or a cline between a population in the W Cape (Sites A–D) and a population in the rest of SA (Sites F–I) plus Malawi. Six mtDNA haplotypes from Site E were closely related to those from the W Cape and four were closely related to those from Site F (Fig. 5). Of
Acknowledgements
The authors gratefully acknowledge Gideon Everson, North West University and Christa Maitland, University of Guelph, for technical assistance; Genome Canada (through the Ontario Genomics Institute) and Dr. Ben Evans, McMaster University, Hamilton ON for access to samples and for help with analysis of the DNA data. This study was funded by Syngenta Crop Protection Inc., the Nation Research Foundation of South Africa (FA2006040300033; L.D.P. and N.K.) through funding to the Canadian Barcode of
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