Transcript PPT - Department of Biology

Effects of Cadmium and Larval Density on Metamorphosis in Axolotl: a
Model for Bioaccumulation of a Heavy Metal in North Dakota Foodwebs
Kati Reed and Christopher K. Beachy
Department of Biology and Amphibian Growth Project,
Minot State University,Minot,ND 58707
INTRODUCTION
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.
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The western tiger salamander,
Ambystoma mavortium
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A
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3
B
control
5 ppb Cd
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2
1
pre-TH mass (g)
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).
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C
80
control
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control
5 ppb Cd
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5 ppb Cd
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size (mm SVL)
0
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).
metamorphic score
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.
RESULTS
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0
60
10
1 larva
3 larvae
1 larva
3 larvae
1 larva
3 larvae
The axolotl, Ambystoma mexicanum
environmental sample
salamander liver sample
Tiger salamanders are significant
bioaccumulators of cadmium
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).
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.
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).
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.)
Salamanders under stress hyperaccumulate cadmium
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METHODS AND PROTOCOL
mg Cd/kg liver
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
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3 axolotls
0 cadmium
control
1ppb 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.
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6
4
2
1 axolotl
1 ppb cadmium
0
3 axolotl
1 ppb cadmium
1
3
axolotl density
REFERENCES
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.
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. 2009. 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. 2007. Early gene expression during natural spinal cord regeneration in the salamander
Ambystoma mexicanum. Journal of Neurochemistry 101:27-40.
Page, R. B., S.R. Voss, A. K. Samuels, J. J. Smith, S. Putta, and C. K. Beachy. 2008. 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. 2007. 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 145:15-27.
Acknowledgments. Aspects of this project were supported by NIH Grant
Number P20RR-016741 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.
Richter, J., L. Martin, and C.K. Beachy. 2009. Increased larval density induces accelerated metamorphosis independently of growth rate in the frog Rana
sphenocephala. Journal of Herpetology 43:551-554.
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. 2005. Sal-site: integrating new and existing
ambystomatid salamander research and informational resources. BMC Genomics 6:181.