The Effect of Novel Animal Models on the Sexual Reproductive Pathways of Asclepias syriaca

One of the most beloved insect species alive today is the Danaus plexippus, or the monarch butterfly.  Ominously, they have seen concerning population declines which have been strongly linked to a decline in milkweed, specifically Asclepias syriaca (Common Milkweed) populations, the host plant of the Monarch caterpillar.  Between 1999 and 2010, the milkweed and monarch populations decreased by 58% and 81% respectively (Pleasants and Oberhauser 2012), highlighting the need for conservation.  While we know a great deal about monarch and milkweed interactions, we know comparatively less about the milkweed interactions with other specialist insects.  In addition, much of the literature deals with the milkweed’s ability to propagate itself through asexual budding, while the sexual pathways necessary for long term survival remains receive less attention.  By learning more about the milkweed’s sexual pathways, conservation efforts can be more targeted to increasing genetic diversity more efficiently, raising the likelihood that milkweeds can make a comeback.

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They Heard It Through the Grapevine: How Asclepias syriaca Communicates

Asclepias syriaca, or common milkweed, is a tall, herbaceous plant that reproduces both sexually, through pollination, and asexually, through producing multiple shoots from a single root system. It has strong chemical and physical defenses against herbivory, and when the plant senses that tissue is being disrupted by insects, it accordingly increases its production of latex (a gummy liquid) and cardenolides (toxic compounds). Previous studies have shown that other clonal plants with shoot or root connections are able to share resources such as water and nutrients between stalks. My research will investigate whether stalks of A. syriaca experiencing herbivory can share the signals that induce increased defenses with their clonal relatives; in effect, can milkweed stems “talk” to their fellow clones to warn them about present dangers?

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Conclusion

The goal for this summer was to have sampling, DNA extraction, Polymerase Chain Reaction (PCR), and fragment analysis sent off (to be later analyzed) completed on all 400 of the subsamples. Sampling and DNA extraction were finished. PCR unfortunately was not, delaying fragment analysis as well. Though the PCR protocol was developed during spring semester of 2017, new primers and a host of other problems made this step tricky. The first batch sent off for analysis came back blank, requiring adjusting of the protocol and the second had nebulous results. A delay in shipment meant that only 3 of the 7 primers were available for much of the summer. However, all extractions were completed successfully, and in the last remaining weeks of July, PCR was done on all 400 of the plants for 3 of the primers. All PCR products were shipped off for fragment analysis. Training began on using a fragment analysis software, so once results do come back, analysis will be smoother.

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Labwork (PCR)

Following DNA extractions, it was time for amplification of DNA through Polymerase Chain Reaction (PCR). PCR amplifies the DNA by denaturing, copying, and synthesizing over and over again. Denaturing occurs via temperature, first during the DNA extraction process, and then again using the Thermocline machine (or PCR machine). The thermocline helps to regulate temperature allowing for PCR to go through its different steps, such as synthesis or denaturing. Synthesis occurs using a special polymerase Taq.

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Labwork

Following the collection of data and samples, it was time for lab work to begin. The first step of my procedure was DNA extractions. With around 800 plant samples, I quickly realized financially and time wise doing all 800 would not be practical. This led to many discussions on how to subsample. Was it better to do more transects, with fewer plants from each transect? More plants, with fewer coverage of overall transects? How do you account for the difference in densities between transects? Overtime, with more and more discussions, it became clear that sampling more transects would be a better option, even if that meant fewer plants per transect. Additionally, for any transect with ~30 or fewer plants, the entire transect would be sampled. For any plant over that, a subsample would be done using a random number generator to randomly select which plants should be extracted.

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