Corticosterone Level Changes throughout Larval Development in the Amphibians Rana sylvatica and Ambystoma jeffersonianum Reared under Laboratory, Mesocosm, or Free-living Conditions David L. Chambers 1,2 , Jeremy M. Wojdak 3 , Pang Du 4 , and Lisa K. Belden 1 Studies of a few ‘‘model’’ amphibians continue to advance our mechanistic understanding of the endocrine control of larval amphibian development and metamorphosis, but there are few studies examining steroid profiles across species during larval amphibian development. We used censored regression analysis to address our primary objective, which was to examine baseline corticosterone level changes and responses to a standardized stressor throughout larval development in two amphibian species: one anuran (Wood Frogs, Rana sylvatica) and one caudate (Jefferson Salamanders, Ambystoma jeffersonianum). In addition, we looked at two additional factors that could influence the study of corticosterone during larval development, namely the rearing location of the animals (free-living, mesocosm- held, or laboratory-held) and for A. jeffersonianum, the method of induction of the stress response (ACTH injection or a confinement-agitation [CA] protocol). As has been documented for other anurans, baseline corticosterone content of R. sylvatica increased close to metamorphic climax in all rearing locations, although the absolute level varied with rearing location. Baseline corticosterone content of A. jeffersonianum increased gradually over development, and the increase in corticosterone content following CA mirrored the increase in baseline levels, although the absolute magnitude of the increase with CA varied based on rearing location. In larvae of A. jeffersonianum, both the CA method and ACTH injection significantly increased corticosterone content, with 30 min eliciting the maximum hormonal response level. Our results suggest that rearing location can influence corticosterone levels and the response to a standardized CA protocol, and that care should be taken in extrapolating results from laboratory studies to free-living amphibian populations. E NDOCRINE pathways play important roles in many developmental processes, including metamorphosis. For organisms with complex life cycles, metamor- phosis represents a major developmental transition, and endocrine signals coordinate many of the morphological and physiological changes that occur during metamorpho- sis. Metamorphosis is common in many taxa including insects, crustaceans, echinoderms, cnidarians, and fish. However, amphibian metamorphosis is one of the most dramatic among all organisms (Shi, 2000). Larval develop- ment in anurans (frogs and toads) has three general phases: pre-metamorphosis, pro-metamorphosis, and metamorphic climax (Etkin, 1968). Pre-metamorphosis embodies embryo- genesis and early growth stages. Pro-metamorphosis is comprised of morphogenesis, specifically limb develop- ment. Metamorphic climax consists of rapid growth, development, and differentiation in preparation for adult- hood. Caudate (salamander) development is not as dramatic as anuran development in terms of morphological changes (Just et al., 1981), but is comprised of similar general developmental phases. The endocrine control of amphibian larval development and metamorphosis has been well studied from a mecha- nistic standpoint in numerous laboratory studies (reviewed in Denver, 2009). Several hormones have integral roles in amphibian development and metamorphosis, including prolactin, thyroid hormones, gonadal steroids, and gluco- corticoid hormones (Kikuyama et al., 1993; Shi, 2000; Denver, 2009). Corticosterone is the main glucocorticoid in amphibians (Idler, 1972), and is produced following activation of the hypothalamic–pituitary–interrenal (HPI) axis (Denver, 2009). During anuran development, tail fin absorption, hepatic enzyme activity, and cell differentiation are all mediated by corticosterone (Shi, 2000). Depending on the developmental stage of individuals, experimental eleva- tions of corticosterone in tadpoles can either accelerate or decelerate metamorphosis (Hayes, 1997). Fewer studies have addressed changes in corticosterone during caudate devel- opment as compared to anurans. Carr and Norris (1988) examined corticosterone changes throughout larval devel- opment in the Tiger Salamander (Ambystoma tigrinum). They observed corticosterone levels at the lowest level during pre- metamorphosis, significantly increasing towards mid-meta- morphosis and metamorphic climax, followed by a signif- icant decrease upon completing metamorphosis. Similar patterns of peak corticosterone levels occurring at meta- morphic climax have been seen in laboratory studies of anurans (Wada, 2008), such as Rana catesbeiana (Wright et al., 2003), R. pipiens (Glennemeier and Denver, 2002), and Xenopus laevis (Kloas et al., 1997). There are approximately 6500 named species of amphib- ians. Despite this, the majority of work done on the endocrine control of amphibian development and meta- morphosis has focused on just a handful of species, with the majority of studies completed on just two species: Bullfrogs, Rana catesbeiana, and African Clawed Frogs, Xenopus laevis. Even for these two species, there are differences in the activity of some key enzymes during development (Denver, 2009). We clearly still have much to learn from a comparative standpoint regarding the function of the endocrine system during amphibian development. Our primary objective was to expand our knowledge of the comparative endocrinology of amphibians by examining corticosterone level changes and interrenal responsiveness 1 Department of Biological Sciences, 2119 Derring Hall, Virginia Tech, Blacksburg, Virginia 24061; E-mail: (LKB) belden@vt.edu. Send reprint requests to LKB. 2 Present address: Department of Natural Sciences, 1 College Avenue, The University of Virginia’s College at Wise, Wise, Virginia 24293 E-mail: chambers@uvawise.edu. 3 Department of Biology, P.O. Box 6931, Radford University, Radford, Virginia 24142; E-mail: jmwojdak@radford.edu. 4 Department of Statistics, 406-A Hutcheson Hall, Virginia Tech, Blacksburg, Virginia 24061; E-mail: pangdu@vt.edu. Submitted: 23 September 2009. Accepted: 23 August 2011. Associate Editor: K. Martin. F 2011 by the American Society of Ichthyologists and Herpetologists DOI: 10.1643/CP-09-180 Copeia 2011, No. 4, 530–538