Kati Reed and Christopher K. Beachy Department of Biology and Amphibian Growth Project, Minot State University,Minot,ND Effects of Cadmium and Larval Density on Metamorphosis in Axolotl: a Model for Bioaccumulation of a Heavy Metal in North Dakota Foodwebs Acknowledgments. Aspects of this project were supported by NIH Grant Number P20RR from the INBRE program of NCRR, and a grant from the North Dakota Game and Fish Department. Assistance in animal care was provided by Nicole Schroeder, Ashley Aaland and Joshua Sweet. Cadmium exposure is a significant environmental human health risk. It is essential that relevant and effective biomonitoring systems are developed. We are using salamanders as a model system for (1) assessment of heavy metals in North Dakota foodwebs and (2) for experimental analysis of interactions between cadmium exposure and relevant environmental variation experienced by real organisms in real foodwebs. In this development, we use Ambystoma mavortium (western tiger salamander) and Ambystoma mexicanum (axolotl) as our model salamanders. A. mavortium is ubiquitous in North Dakota wetlands and surrounding agricultural areas. It is likely to be the keystone predator in temporary (i.e., fishless) wetlands and represents a huge fraction of the vertebrate biomass throughout the northern Great Plains. In contrast, the axolotl, while not a native to North Dakota, is a sister species to the tiger salamander and has a maturing, model organism infrastructure. Genomic, bioinformatic and living stock resources are available and a networking infrastructure is under development (Smith et al., 2005). The axolotl has recently been used for important genomic and genetic advances in thyroid-control in vertebrate development and in limb and spinal cord regeneration (Page et al., 2007, 2008; Monaghan et al., 2008, 2009). INTRODUCTION METHODS AND PROTOCOL REFERENCES Cabarle, K.C. R. Winburn, R.B. Page, S.R. Voss and C.K. Beachy. In preparation. Survivorship and transcriptional response of embryonic and larval amybstomatid salamanders to cadmium exposure. Monaghan, J.R., L.G. Epp, S.Putta, R.B. Page, J.A. Walker, C.K. Beachy, W. Zhu, G.M. Pao, I.M. Verma, T. Hunter, S.V. Bryant, D.M. Gardiner, T.T. Harkins, and S.R. Voss Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration. BMC Biology 7:1. Monaghan, J.R., J.A. Walker, R. Page, S. Putta, C.K. Beachy, and S.R. Voss Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum. Journal of Neurochemistry 101: Page, R. B., S.R. Voss, A. K. Samuels, J. J. Smith, S. Putta, and C. K. Beachy Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma). BMC Genomics 9:78. Page, R. B., J. R. Monaghan, A. K. Samuels, J. J. Smith, C. K. Beachy, and S. R. Voss Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum. Comparative Biochemistry and Physiology: Part C: Toxicology and Pharmacology 145: Richter, J., L. Martin, and C.K. Beachy Increased larval density induces accelerated metamorphosis independently of growth rate in the frog Rana sphenocephala. Journal of Herpetology 43: Smith, J.J., S. Putta, J.A. Walker, D.K. Kump, A.K. Samuels, J.R. Monaghan, D.W. Weisrock, C. Staben, and S.R. Voss Sal-site: integrating new and existing ambystomatid salamander research and informational resources. BMC Genomics 6:181. RESULTS Salamanders under stress hyperaccumulate cadmium Due to their permeable skin, salamanders are hypersensitive to environmental insult. Axolotls under density-induced stress accumulated significantly higher amounts of cadmium than those growing in isolation. What this indicates is that salamanders exposed to any dosage of cadmium are likely to accumulate more cadmium during any stress response. Given the abundance of naturally-occurring stressors in wetland ponds (high larval population size, decreasing pond size due to desiccation, arrival of predators), it is highly likely that stress can account for bioaccumulation and storage of environmental cadmium. Tiger salamanders are significant bioaccumulators of cadmium Cadmium is not- detected in water samples gathered from wetlands that host larval tiger salamanders, breeding adult tiger salamanders, and summer visiting tiger salamanders in northwest North Dakota. However, cadmium is detectable in larval salamanders occupying these wetland. Soil samples from areas adjacent to these wetlands have significant cadmium content. Adult salamanders collected from these terrestrial areas have significant cadmium loads that exceed the environment. (These data are from Cabarle et al., in prep.) We obtained axolotl embryos from the a wild-type cross at the growth facility of the Amphibian Growth Project at Minot State University. Upon hatching we placed 48 larvae in individual containers and fed them ad libitum. We fed brine shrimp (upon hatching) until the axolotls were large enough to consume california blackworms. At this point, axololts were assigned to one of four treatments: 1 axolotl 0 cadmium 1 axolotl 1 ppb cadmium 3 axolotls 0 cadmium 3 axolotl 1 ppb cadmium We then fed isolated axolotl ad libitum blackworms; axolotl at high density received the same amount of blackworms as those in isolation. Cadmium treatment was administered by adding an aliquot of cadmium chloride solution such that the aliquot brought the cadmium dosage in the container holding the axolotl(s) to 1 ppb. The EPA drinking water limit for cadmium is 5 ppb. Axolotls were grown until repeated measurements of axolotl mass indicated that growth was reduced at high density. In addition, axolotls at high density exhibited other signs of stress including limb loss due to aggression towards chamber-mates. When axolotls showed signs of treatment effects on growth, we then treated all axolotls with 50 nM thyroxine (TH) solution in order to induce metamorphosis. Untreated axolotls do not undergo metamorphosis and are described as “obligately paedomorphic.” We terminated the experiment after 33 days of TH treatment. The axolotls were give a developmental score from 0 (non-metamorphic) to 3 (metamorphosis complete) (see Fig. A). The western tiger salamander, Ambystoma mavortium The axolotl, Ambystoma mexicanum Axolotls grown at high density grew more slowly, as predicated by experimental design (Fig. B). Following treatment with the thyroid hormone, thyroxine, axololts are high density were similarly affected, I.e., final size differences due to density were the same as prior to TH-treatment (compare Fig. B to Fig. C). Interestingly, density did not affect metamorphic development. In larval amphibians that naturally metamorphose, the effect of density is known to accelerate development while reduced growth will retard development (e.g., Richter et al., 2009). Perhaps the ability of obligately paedomorphic salamanders to respond developmentally to density stress is reduced or lost. In no cases did cadmium exposure influence growth, post-TH growth, or metamorphic development. However, axolotls are high density did accumulate more cadmium than any other treatment group (see below). A BC environmental sample salamander liver sample 1ppb cadmium