Examination of an amphibian metamorphosis assay under an individual-separated exposure system using Silurana tropicalis tadpoles
Highlights
► We developed an amphibian metamorphosis assay with a light burden on testers. ► The protocol is based on an individual-separated exposure system. ► This system served to reduce variability in tadpole growth and development. ► Using this system, thyroxine and propylthiouracil were successfully tested. ► Our results agree with historical data, showing the validity of our assay protocol.
Introduction
Amphibian metamorphosis, which is induced and regulated by thyroid hormones, accompanies drastic transformations in essentially every organ and tissue. Particularly in anurans, the process of morphological alteration caused by metamorphosis (tadpole-to-frog transformation) can be easily monitored by visual inspection. In addition, postembryonic development has been thoroughly studied as a biological model to elucidate thyroid functions (Shi, 2000). By exploiting these advantages, an in vivo assay to detect chemicals disrupting thyroid functions has been designed using Xenopus laevis, a worldwide experimental anuran species (Kloas, 2002, Kloas et al., 2003, OECD, 2004, Opitz et al., 2005). The experimental protocol of this assay has been standardized by OECD (2009). Meanwhile, similar metamorphosis assays have also been successfully developed using other anuran species, such as Silurana tropicalis (formerly called Xenopus tropicalis) (Mitsui et al., 2006) and Rana rugosa, an endemic species in Japan (Oka et al., 2009). In particular, S. tropicalis, which is phylogenetically related to X. laevis, has attracted attention as a new model species, due to its experimental superiority over X. laevis: a shorter life cycle and a diploid genome that increase the practicability of use for multi-generational tests and genetic analyses (Song et al., 2003, Fort et al., 2004a, Fort et al., 2004b, Kashiwagi et al., 2010).
However, traditional assays have practical difficulties when conducted by one or two individual researchers due to the large number of tadpoles required for the examination of morphological endpoints. As demonstrated by historical studies (Kloas et al., 2003, Opitz et al., 2005, Mitsui et al., 2006, Oka et al., 2009), tadpoles, even within a control, tend to develop enhancing individual differences in growth and development during exposure periods. This tendency would become more conspicuous under individual-grouped exposure systems where 20–30 tadpoles are held together in a single test tank, because the tadpoles can easily interfere with each other during feeding. An evaluation of results yielded using amphibian metamorphosis assays is based largely on statistical comparisons of morphological data concerning tadpole development. Accordingly, such large within-group variability necessitates the use of a large number of tadpoles for control and chemical treatment groups. In addition, when including replication, the number of tadpoles increases two- to four-fold. Therefore, an exposure system to exclude interactions among tadpoles can serve to minimize interindividual variability of morphological data and thereby allow the number of tadpoles to be reduced. The simplest design fit for this purpose is an individual-separated exposure system in which each tadpole is held in a single test vessel. This system also enables individual identification without marking of the tadpoles, which is technically difficult (Rice et al., 1998). However, in the individual-separated exposure system, which requires as many test vessels as the total number of test tadpoles, the test vessel must be reduced in size due to the limited volume of the temperature-constant chamber. This exposure system inevitably reduces the amount of swimming space available for each tadpole. For certain anuran species, it has been suggested that tadpole development can be accelerated by gradually reducing aqueous space that may stimulate the release of thyrotropin, a thyroid-stimulating hormone (Denver, 1998, Denver et al., 1998). The anuran metamorphosis assay under the individual-separated exposure system may therefore not truly detect thyroid-disrupting chemicals, unless sufficient volume of test solution is provided for each tadpole during the test.
The current work aimed to establish an amphibian metamorphosis assay practicable by one or two individual researchers, using S. tropicalis tadpoles as a test species. We first conducted a comparative rearing experiment between the individual-separated exposure system and the traditional individual-grouped exposure system in order to confirm that the former served to reduce the interindividual variability of the morphological without inducing developmental acceleration that would affect the test results. Subsequently, under the individual-separated exposure system, we tested thyroxine (T4, one of thyroid hormones) and propylthiouracil (PTU, an antithyroid chemical), both of which have been used as test chemicals in traditional metamorphosis assays. By comparing the results with the traditional data, we verified the validity of the amphibian metamorphosis assay under the individual-separated exposure system.
Section snippets
Animal husbandry
Adult pairs of S. tropicalis were obtained from the Institute of Amphibian Biology of Hiroshima University. The adult frogs were kept in polypropylene aquaria filled with dechlorinated tap water under the following conditions: water depth, 9 cm; water temperature, 25±1 °C; photoperiod, 12-h light/12-h dark; frog density, six frogs per 1800 cm2 of water surface area; feeding, thrice per week with a commercial diet for aquatic frogs (XL-2, Oriental Yeast, Tokyo, Japan).
After an acclimatization
Experiment 1
Throughout experiment 1, one dead tadpole was observed in the individual-grouped exposure system, however, no other external abnormalities were observed in either systems. As shown in Fig. 1, the tadpoles reared under the individual-separated exposure system developed with significantly small dispersions in developmental stage (median and range=stage 57 and stages 56–58), hind limb length (mean±SD=9.86±1.61 mm), and total body length (mean±SD=48.6±1.76 mm) compared with those reared under the
Discussion
The current study was conducted to establish an alternative testing protocol for an amphibian metamorphosis assay, which would be practicable to carry out by only one or two individual researchers. The subject of this study is therefore how to reduce practical burdens on testers. We addressed this issue by employing an individual-separated exposure system that would make tadpoles develop as uniformly as possible by excluding interference during feeding among the tadpoles. In other words, we
Conclusions
The current work examined an alternative testing protocol for amphibian (S. tropicalis) metamorphosis assays: an individual-separated exposure system that was expected to reduce interindividual variability in tadpole growth and development and thereby allow the number of test tadpoles to be reduced (n=30 for each group). Our expectation was verified by the comparative rearing experiment between the individual-separated exposure system and the traditional individual-grouped exposure system.
Acknowledgments
The frogs S. tropicalis were supplied from the Institute of Amphibian Biology of Hiroshima University as a part of the National BioResource Project by the Japan Ministry of Education, Culture, Sports, Science, and Technology. We thank A. Kashiwagi, M. Takase, and Y. Yaoita for technical advice on the keeping and breeding of S. tropicalis.
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