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Vitellogenin (VTG), a yolk-precursor protein in oviparous vertebrates, is a useful biomarker for reproductive physiology and environmental estrogenic pollution. To examine interspecific applicability of an enzyme-linked immunosorbent assay (ELISA) for quantifying Chinemys reevesii VTG, we observed cross-reactivity between a polyclonal antibody against Chinemys reevesii VTG and the VTGs from other turtle species: Chelydra serpentina (Chelydridae), Macrochelys temminckii (Chelydridae), and Pelodiscus sinensis (Trionychidae), which are phylogenetically distant from Chinemys reevesii (Geoemydidae). The VTGs of the three species were induced by injecting estradiol 17beta into the turtles and purified by using the EDTA-MgCl(2) precipitation method. The purified VTG appeared as a 200-kDa protein in sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating that the molecular mass of the VTGs of the three species was similar to that of Chinemys reevesii VTG. The purified VTGs were serially diluted (0.004-2 mug/ml) and applied to the ELISA. Although the VTGs of the two chelydrid turtles showed cross-reactivity in a concentration-dependent manner, the degree of cross-reactivity was only 22.8-41.2% (mean=30.0%) and 19.7-53.0% (mean=33.2%) for Chelydra serpentina VTG and Macrochelys temminckii VTG, respectively. The ELISA may therefore be theoretically applicable to measure relative levels of the VTGs of these two species, but the absolute concentration values may be inaccurate. Pelodiscus sinensis VTG showed almost no cross-reactivity (8.0-9.7%, mean=8.9%) at any concentration tested, thus indicating the inapplicability of the ELISA to quantify Pelodiscus sinensis VTG. There are thus limitations in extending the applicability of the ELISA across species, even within the order Testudines.

Vitellogenin (VTG), a biomarker for environmental estrogenic pollution, can be detected in the bloodstream of oviparous animals before morphological and functional abnormalities appear due to exposure to environmental estrogens. Reports observing VTG in turtles have been limited. We therefore cloned and sequenced a partial cDNA of VTG in Reeves' pond turtle, Chinemys reevesii. The cloned cDNA fragment possessed the start codon and 2,229 bp, encoding 743 amino acid residues. A sequence of deduced amino acid from the cDNA did not contain a high serine content, such as that which exists in phosvitin. Two N-glycosylation sites were found in the sequence. The sequence was compared to those of two birds (chicken and herring gull), one amphibian (Xenopus), and five fishes (carp, zebrafish, eel, haddock, and red seabream). The C. reevesii VTG was similar to that of herring gull (78%, value of positives), chicken (76%), Xenopus (69%), eel (63%), red seabream (62%), haddock (62%), carp (62%), and zebrafish (61%). The phylogenetic tree showed that C. reevesii VTG existed between the amphibian and birds, and it was present far from fish VTGs. A reverse transcription-polymerase chain reaction method was employed to detect the mRNA expression of the C. reevesii VTG through the use of primers designed from our sequence. The VTG mRNA expression (292 bp) was proven in the total RNA extraction from the liver of the juvenile turtles which were treated with estradiol-17beta. The information herein would be useful for ecotoxicological studies using freshwater turtles and these findings are expected to contribute positively towards wildlife conservation.

To ascertain whether wild male turtles were influenced by environmental estrogens, we examined serum vitellogenin (VTG) levels of male Reeves' pond turtles (Chinemys reevesii) collected from four study sites (A-D) in Kyoto, Japan. Sites A-C, which were impacted by domestic or industrial wastewater and effluents from sewage treatment plants, were chosen as contaminated sites, and site D was intended as a reference site. This contaminated/reference site characterization was confirmed by measuring estrogenic activities of the water samples collected at each site for over a year. Serum VTG levels in the turtles were quantified by an enzyme-linked immunosorbent assay established previously. Estrogenic activities of the water samples were measured using a previously validated yeast two-hybrid assay and expressed as the estradiol-17beta equivalent. Estrogenic activity was observed at high levels at sites A-C, but was almost undetected at site D throughout the sampling period: the mean and range were 0.74 (<0.07-2.1), 0.52 (0.17-1.6), 1.7 (<0.07-7.3), and 0.07 (<0.07-0.62) ng/l at sites A-D, respectively. Significant differences were found only in site D versus sites A, B, and C. Therefore, site D and sites A-C were confirmed to be a reference site and contaminated ones, respectively. Overall, 320 male turtles were captured and examined. The majority of the turtles showed normal VTG values (0.10-0.74 microg/ml). Although only five turtles from sites A-C showed unusually high VTG values (1.1-5.9 microg/ml, nearly one order of magnitude higher than normal values but much lower than values in adult females), there was no significant difference in the incidence of these high values between sites A-C and site D. Moreover, among the five turtles, one turtle was captured again 2 months later, but its VTG value dropped to the normal level. The unusually high VTG values may therefore be transient elevation caused by incidental and/or individually specific agents. Excluding the unusually high values, the mean serum VTG (accompanied with the range) was 0.22 (0.10-0.74), 0.27 (0.11-0.62), 0.27 (0.17-0.68), and 0.23 (0.10-0.57) microg/ml at sites A-D, respectively. There were no significant differences in the mean VTG values among sites A-D. Although it should be clarified how and why the unusually high VTG values occurred at sites A-C, our results suggested that wild male C. reevesii turtles would not be significantly affected by xenobiotic estrogens at environmentally relevant levels in terms of serum VTG elevation.

High abundance of invasive red-eared slider (RES) turtles (Trachemys scripta elegans) in Japan has been a serious issue in freshwater ecosystems. Information on reproductive parameters, such as sex ratios, of RES turtles is needed to effectively control the invasive species. We conducted a long-term field study on RES turtles at two study sites: the Taisho River (a shallow river) and Teradaike Pond (an irrigation reservoir that was irregularly drained to maintain the embankment and water quality) in western Japan. Using trapping and muddling methods, we captured RES turtles and checked for sex, carapace length, and body weight. We also examined whether the population of RES turtles could be reduced by continuously removing the turtles captured at each study site. The sex ratio (male/both sexes) of RES turtles that could be sexed by external morphology (presumably adult turtles) was 0.27 at the Taisho River and 0.26 at Teradaike Pond. At both sites, these sex ratios were almost the same as those of RES turtles that could be sexed only by necropsy after euthanasia (presumably juvenile turtles). These sex ratios were markedly biased toward females and consistent between the adult and the juvenile turtles, thus suggesting that more females had been produced than males. At the Taisho River, a total of 1,366 RES turtles were captured and eliminated over more than ten years. Nevertheless, the annual catch did not tend to decrease and only an effect of preventing population growth was observed. At Teradaike Pond, a total of 1,831 RES turtles were captured over nine years. Nearly half of them were caught for the initial two years, during which the pond was drained. Thereafter, the annual catch of RES turtles clearly decreased. These results suggest that it is difficult to control RES turtles established in rivers but that it is feasible to effectively control RES turtles in enclosed small water bodies, such as irrigation reservoirs, by intensively capturing and eliminating the turtles during the drainage period. [ABSTRACT FROM AUTHOR]

To demonstrate the annual ovarian cycle of a multiclutched turtle Chinemys reevesii, we quantified vitellogenin (VTG, a yolk-precursor protein) in the serum collected monthly from turtles kept in an outdoor enclosure. We also sacrificed wild adult females (one or two individuals per month) captured from a river site in Kyoto, Japan, and observed oviductal eggs and follicles assigned to five size classes: C1 to C5, in ascending order. The seasonal variation in the serum VTG level showed a sharp peak in late spring and a broad peak during autumn, indicating that vitellogenesis accelerated rapidly in spring, decreased in summer, increased slowly but steadily in autumn, and ceased in winter. From May to July, ovulations occurred in succession preceded by the C4-to-C5 growth of follicles, but without substantial growth of C1-C3 follicles. The vernal peak of vitellogenesis would therefore contribute largely to the sequential growth of the follicles prepared for the second and third clutches of the breeding season. In August, when the successive ovulations had been completed, no remarkable growth of C1-C3 follicles was observed anymore, reflecting the ovarian quiescence. Newly-formed C1 follicles appeared in September when C2 and C3 follicles markedly increased in number but C4 and C5 follicles were still absent. In October and November, C4 and C5 follicles were observed again, suggesting that the follicles for the first clutch of the next breeding season reached preovulatory size before hibernation. The production and remarkable growth of follicles occurring from September to November would account for the broad peak of vitellogenesis in autumn. Thus the observed seasonal variations in the serum VTG level and follicular growth were concordant with each other. [ABSTRACT FROM AUTHOR]