3. Background
Secondary metabolites are a common defense in plants but many
herbivorous insects have evolved the ability to detoxify or
sequester these toxic compounds.
Milkweed plants (Asclepias) produce cardenolides (Fig. 1) which
inhibit Na+
/K+
-ATPase, an important enzyme in animals.
The red milkweed beetle (Tetraopes tetrophthalmus) and swamp
milkweed beetle (Labidomera clivicollis) (Fig. 2) feed on milkweed
plants and sequester cardenolides.
Figure 2. Redmilkweedbeetle (left) andswampmilkweedbeetle
(right)Figure 1. Structure of a Cardenolide
4. Hypothesis
A previous study (Zhen et al., 2012) has found convergent
mutations at cardenolides-interacting sites on Na+
/K+
-ATPase (Fig. 3).
One hypothesis is the more mutations an insect has the more resistant
it will be to cardenolides. With a mutation originating from two
ancestral mutations (represented by an H) at position 122 I speculate
the swamp milkweed beetle will possess more resistance than the red
milkweed beetle.
Figure 3. Above isan abbreviatedaminoacidsequence showingthe four
mostimportantsitesinvolvedwiththe Na+
/K+
-ATPase.
5. Methods
Na+
/K+
-ATPase assay: measure the activity of Na+
/K+
-ATPase under
a series of toxin levels
Materials: heads from 1) red milkweed beetles 2) swamp milkweed
beetles 3) fruit fly (Drosophila melanogaster) as negative control
Procedure: Collects samples from the field Freeze samples in
liquid nitrogen Dissect off the head Freeze dry the head
Resuspend the sample in water Break up sample tissue by
sonification and grinding Expose the sample to a series of 8
buffers each composed of ouabain (the primary cardenolide) and
ions Stop the reaction Use a photospectrometer to run the
plate at 660 nm.
7. References
Petshenka, Georg and Dobler, Susanne. (2009). Target site sensitivity
in a specialized herbivore towards major toxic compounds of its
host plant: The Na+K+-ATPase of the oleander hawk moth (Daphnis
nerii ) is highly susceptible to cardenolides. Journal of
Chemoecology. 19. 235-239.
Zhen, Y., Aardema, M. L., Medina, E. M., Schumer, M., and Andolfatto,
P. (2012). Parallel molecular evolution in an herbivore community.
Journal of Science. 337. 1634-1637.
8. Conclusion
This protocol is successful when using Drosophila. The s-curve
produced by this assay behaves as anticipated. Further, the s-curve
produced aligns with other published results as shown below. The
thick lines are from this assay, while the other lines are data from an
ATPase on milkweed butterflies where Drosophila were used as the
control (Petshenka, 2009).
9. Results
The results of the ATPase assay are plotted above, with each data
point read as the percent of enzyme activity (y-axis) as a result of the
concentration of the ouabain in the buffer. The vertical line represents
the inhibition concentration at 50%. This represents that at an ouabain
concentration of 10-6 , 50% of the Na+/K+-ATPase stopped functioning.
10. Discussion and Future Plans
After several inconsistent assays on the red milkweed beetle head, several
major improvements to the protocol and the sample had to be made. Firstly,
after each addition to the plate, each well was to be mixed thoroughly. Ensuring
the proper pipette techniques as well as functional pipettes were being used
was also important. A multichannel pipette was to be used for the time sensitive
steps such as stopping the reaction and adding the chromogenic solution. These
three changes greatly improved the reproducibility of our results.
However, at this time, the original hypothesis cannot be accepted or denied.
Further analysis must be done. Future improvement may include using pure
neurons from the red milkweed beetle, or neurons centered in the body instead
of in the brain. The procedure could also be improved by shortening the
incubation period of the tissue in the buffers, or using sodium dodecyl sulfate, a
stronger substance, instead of trichlorocetic acid to stop the reaction.
Eventually, this assay can be used to accept or deny this hypothesis once the red
milkweed beetle and swamp milkweed beetle have been analyzed.
