Na+-K+-ATPase Assay on Cardenolide Sequestering Insects

Regina ViscontiPrinceton University

NSF REU Molecular Biophysics 2016

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KeyFly Head Data50%IC 50

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 1. Structure of a Cardenolide

Figure 2. Red milkweed beetle (left) and swamp milkweed beetle (right)

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 is an abbreviated amino acid sequence showing the four most important sites involved with the Na+/K+-ATPase.

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.

Acknowledgements

Peter Andolfatto

Lu Yang

Mariana Wu

Mathew Aardema

Thomas Pisano

National Science Foundation

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.

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).

ResultsThe 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.

Discussion and Future PlansAfter 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.