Analysis of Developmental Exposure to Imidacloprid on Blood Glucose and Lipid Metabolism in African Clawed Frogs (Xenopus laevis)
Hannah R. Dean, Rosemary G. Myers, Leah V. Marshall, Kelsey L. Gustafson, Grascen I. Shidemantle, Zoey I. Campbell, Miranda J. S. Falso, Paul G. Falso
Department of Biology, Slippery Rock University, Slippery Rock, PA 16057

Introduction
• Globally there has been a decline in amphibian
populations and over one-third of amphibian species
are threatened with extinction.1
• While many studies focus on the toxicity of other high
use chemicals, information on the impacts of widely
used neonicotinoid insecticides on amphibian life is
scarce. 2,3
• Neonicotinoids mimic nicotine and consequently alter
the function of the central nervous system. 4
• As a result of increased neonicotinoid use to control
invasive forest insects, aquatic habitats show levels of
neonicotinoid contamination that are above what the
US EPA deems safe to aquatic life. 5,6 (Fig. 1)
• Chemical contamination in aquatic environments has
been shown to disrupt the endocrine system and
stress response in amphibians.7

Figure 1. Estimated 20-year imidacloprid use in the US in
1997 (A), 2007 (B) and 2014 (C) (adapted from
water.usgs.gov).

• The purpose of this study is to examine the impacts of
environmentally relevant levels of imidacloprid
exposure on blood glucose and lipid metabolism of
African Clawed Frog (Xenopus laevis) (Fig.2). It is
hypothesized that imidacloprid negatively alters
metabolism in these amphibians.

Figure 2. Adult male Xenopus laevis from the laboratory of
Dr. P. Falso (photo courtesy of P. Falso)

Materials & Methods
• African clawed frogs (Xenopus laevis) were exposed to four
environmentally relevant concentrations of the neonicotinoid
insecticide, imidacloprid, throughout development from
hatching through metamorphosis. (Fig.3)
• A subset of the animals was maintained through maturity in
the appropriate imidacloprid dose following metamorphosis.
(Fig.4)
• Nutrient metabolism will be examined by measuring plasma
glucose, triglycerides, and free glycerol.

Results
We validated the parallelism (Fig. 5) and accuracy (Fig.
6) of the Sigma glucose assay kit with plasma of X.
laevis. From our standard curve and standard curves of
previous researchers in this study, we found that a
minimum of 6 μL of plasma was needed for accuracy.
The kit accurately measures glucose in X. laevis plasma
without interference.
Figure 5. Validation of Parallelism shows that we could use 6
uL of plasma to get accurate glucose readings.

• Treatment groups:
• ethanol vehicle (0.2%)
• 0.3 parts per billion (ppb) imidacloprid
• 3.0 ppb imidacloprid
• 30 ppb imidacloprid
• 300 ppb imidacloprid

Figure 3 . Group housed X. laevis
larvae were exposed to five different
environmentally relevant treatment
groups throughout metamorphosis.
(image courtesy of P. Falso)

Figure 4. Twelve frogs from each
treatment were housed individually
through sexual maturity in continual
exposure to the above levels of
imidacloprid.(image courtesy of P.
Falso)

• Blood samples were previously collected from these animals
at sexual maturity to examine plasma concentrations of
endocrine hormones and glucose metabolism.
• Plasma Glucose Assay: The glucose assay has been
previously validated in the lab of P. Falso and scaled down to
accommodate a 96 well plate.
• Validation of Parallelism: A standard curve was generated
with known concentrations of glucose and dilution of plasma
produced a linear relationship relative to the standard curve.
• Validation of Accuracy: The accuracy of the glucose assay
was tested through the addition of 60 mg/mL glucose to male
and female X. laevis plasma samples. This kit accurately
measures glucose in X. laevis.
• Plate Reading and Analysis: 96 well plates were incubated
at 37 degrees C and absorbances were read at 450 nm by
the PerkinElmer Scientific EnSpire plate reader. This data
was accordingly analyzed.

Figure 6. Validation of Accuracy shows that the added male
plasma and spike amount to the expected spike. The female
plasma with spike is slightly higher than expected and we plan
to continue to monitor this difference.

References
1. S. N. Stuart et al., Science 306, 1783 (2004).
2. S. L. Feng, Z. M. Kong, X. M. Wang, L. R. Zhao, P. G. Peng, Chemosphere 56, 457 (2004).
3. C. M. Ade, M. D. Boone, H. J. Puglis, Journal of Herpetology 44, 591 (2010).
4. Imidacloprid Technical Fact Sheet, edited by National Pesticide Information Center
(Oregon State University, 2005).
5. M.A. Churchel, J.L. Hanula, C.W. Berisford, J.M. Vose, M.J. Dalusky. Southern Journal of
Applied Forestry 35, 26 (2011).
6. K. Starner, K.S. Goh. Bulletin of Environmental Contamination and Toxicology 88, 316
(2012).
7. T. B. Hayes, P. Falso, S. Gallipeau, M. Stice, Journal of Experimental Biology 213, 921
(2010).

Acknowledgements
• Previous Co-investigators: Rosemary G. Myers, Leah V. Marshall, Kelsey L. Gustafson,
Grascen I. Shidemantle, Zoey I. Campbell, Miranda J. S. Falso, Paul G. Falso
• All those funding the Student Research, Scholarship, and Creative Activity grant and the
Selection Committee