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effect of Nicotiana species on the growth rate of Manduca sexta

Allie Conner, Biochemistry and Molecular Biology; Dr. Johannes Stratmann, Biological Sciences

Research Question: How does differential responsiveness of various Nicotiana species to FACs affect the
growth rate and mortality of Manduca sexta?

Project Goals and Objectives: The main objective of this project is to quantify how plants with a
different sensitivity to FACs, including Nicotiana benthamiana, Nicotiana sylvestris, Nicotiana tobaccum,
and Nicotiana knightiana affect the growth rate and mortality of the herbivore Manduca sexta.

Project Impact: Plants are sessile organisms and cannot produce the fight or flight response of mobile
organisms, therefore many species evolved chemical defense responses as means of combating
herbivory, pathogenic insult, and other forms of stress. For example, N. sylvestris, N. knightiana, N.
tobaccum and N. benthamiana are members of the tobacco family. Some of them can recognize herbivores
by fatty acid-amino acid conjugates (FACs) present in the salivary secretions of herbivorous insects,
including the tobacco hornworm M. sexta,. As a consequence they mount a defense response, e.g. by
synthesizing the toxin nicotine. The study of this defense system is of potential benefit to the agricultural
industry. It s possible that plants can be generated with proliferating resistance to herbivory, thus
reducing the global use of pesticides. The USDA reported 516 million pounds of pesticide were used in
the United States in 2008 while the North Carolina Department of Health and Human Services Division of
Public Health reported in 2012 that pesticide-related incidents had a national cost of around $200 million
US dollars in the form of emergency department visits, hospitalizations, and deaths (Langley et al., 2012;
Fernandez-Cornejo et al., 2014). Experiments that expand the current knowledge of biological plant
defense systems, like the one described in this proposal, have the potential to aide plant breeders to
cultivate more resistant crop plants and thus reduce overall pesticide usage.

Background: In a number of plant species, a signaling pathway is activated in response to herbivory, or
the consumption of plant tissue. In the tobacco genus Nicotiana (family Solanaceae), this signaling
pathway is the MAPK6 pathway which is activated by the FACs found in the salivary secretions of insects.
M. sexta, or the tobacco hornworm, is an insect that specializes in Solanaecae herbivory during its larval
stage (Halitschke et al., 2001). This pathway induces the production of jasmonic acid, a plant hormone
that serves as a long distance wound signal to induce a systemic defense response (Kandoth et al., 2007).
Of interest to our group is the direct defense response, which involves the production of toxic chemicals
like nicotine and anti-digestive proteins. These proteins prevent the absorption of essential amino acids
from the leaf into the intestines of the insect and thus have a detrimental effect on the growth and
development of the insect. (Chen et al., 1995; Constabel et al., 1995; Ryan, 2000) This increases the rate
of mortality and decreases the rate of growth in the insect (Baldwin et al., 2007). A previous study has
investigated FAC sensitivity in 4 Nicotiana species when treated with oral secretions from 2 different
caterpillar species: M. sexta and Spodoptera littoralis, a caterpillar that does not specialize in any plant
species. It concluded that FAC-induced resistance differed among the Nicotiana species in that M. sexta
resistance is species-specific and S. littoralis resistance is not. S. littoralis induced resistance was
displayed across every Nicotiana species tested, but only two showed induced resistance to M. sexta (Xu
et al., 2015). Testing four new Nicotiana species will extend the research into which species display an
induced resistance and which do not, and how plant resistance affects the growth rate of M. sexta. Among
the species in question (, N. tabacum and N. benthamiana were shown to be sensitive to FACs, whereas N.
knightiana and N. sylvestris were insensitive to FACs (Grissett, 2016).

Methodology: N. sylvestris, N. benthamiana, N. knightiana, and N. tobaccum will be sowed and grown in a
growth chamber for five weeks until leaves are large enough to sustain herbivory by M. sexta. M. sexta
eggs will be purchased from Carolina Biological Supply. Every plant will have two larvae placed on it
after the M. sexta eggs hatch. For each experiment, ten plants of each species will be tested (= 20
larvae/species or 80 larvae for all four species). The experiment will be repeated three times. Larvae will
be allowed to feed ad libitum at standard conditions at 27C for 14 days (17 hours light and 6 hours
dark). The growth rate will be determined by measuring the weight and length of each caterpillar over a
period of 14 days after which the larvae pupate. Growth will be plotted against time to obtain a growth
curve. In addition, it is expected that some larvae will die early in the experiment due to the plant
defenses. The relation of live individuals to dead individuals is defined as mortality. The larval growth
rate and mortality will be measured each day and a final average growth rate and mortality will be
determined for each species. The growth rate will be correlated to the sensitivity of the four Nicotiana
species to FACs. Measuring this sensitivity tests the hypothesis that species, which are less sensitive (or
not sensitive at all) to FACs, are not as well defended against M. sexta as the species that are sensitive to
FACs. This loss of sensitivity supports faster growth and reduced mortality in the insect.

1: Mid-January: Sow plant and further background research
3: Mid February- Late March: Larvae exposure and measurements
4: April: Analysis of correlation between M. sexta growth across Nicotiana species, Discover USC
5:Past April: Further dissemination and preparation for publication

Mid Late Early Mid Late Early Mid Late April Past
January January February February February March March March April

Anticipated Results and Dissemination: N. benthamiana and N. tobaccum are known to be sensitive to
FACs while N. sylvestris and N. knightiana are not sensitive, based on current research (CITE LAQUITA).
M. sexta larvae on N. benthamiana and N. tobaccum are expected to be smaller due the plants ability to
detect FACs as danger signals and then produce anti-digestive proteins that inhibit growth. Inversely, we
expect M. sexta larvae on N. sylvestris and N. knightiana to be larger and more fully developed as they lack
sensitivity to FACs and thus a means to induce a defense response. Additionally, the mortality of M. sexta
should be higher on individuals placed on N. benthamiana and N. tobaccum than on N. sylvestris and N.
I will present my findings at the University of South Carolinas Discover USC. In addition, I am a
member of the American Society of Biochemistry and Molecular Biology University of South Carolina
chapter and I will present this research at the annual meeting in April 2017 (part of the Experimental
Biology conference).

Personal Statement: My experiences with this project will continue my previous work in this lab on
related projects, adding to my skillset and strengthening my grasp on previously learned skills. The
results I will obtain from this experiment will also afford me the opportunity to present my research to
the scientific community, allowing for networking opportunities in the process and giving me the
opportunity to disseminate my research through various media including poster presentations and peer
review journals. Additionally, this experiment and the experiences I will gain from it will make me a
stronger applicant for acceptance into a Ph.D. program, which I hope to enter into after completing my
undergraduate degree. It is my hope that the results from this experiment will contribute to a future
publication by our lab group.

Baldwin, I., Meldau, S., Hettenhausen, H., Wu, J. (2007): Herbivory Rapidly Activates MAPK Signaling in
Attacked and Unattacked Leaf Regions but Not between Leaves of Nicotiana attenuata. The Plant
Cell 19:1096-122
Chen, H., Wilkerson, C., Kuchar, J., Phinney, B., Howe, G. (2005) Jasmonate-inducible plant enzymes
degrade essential amino acids in the herbivore midgut. Proceedings of the National Academy of
Sciences USA, 102:1923719242.
Constabel, C. P., Bergey, D.R., Ryan, C.A. (1995) Systemin activates synthesis of wound-inducible tomato
leaf polyphenol oxidase via the octadecanoid defense signaling pathway. Proceedings of National
Academy of Sciences USA 92:407 411.
Fernandez-Cornejo, J., Nehring, R., Osteen, C., Wechsler, S., Martin, A., Vialou, A. (2014) Pesticide Use in
U.S. Agriculture: 21 Selected Crops, 1960-2008. Economic Research Service 104:1-86.
Grissett, L.(2016) Sensitivity of species within the plant family Solanaceae to fatty Acid-Amino Acid
Conjugates from herbivore oral secretions. South Carolina Honors College Senior Thesis, Scholar
Halitschke, R., Schittko, U., Pohnert, G., Boland, W., Baldwin, I. (2001) Molecular Interactions between the
Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana
attenuata. III. Fatty Acid-Amino Acid Conjugates in Herbivore Oral Secretions Are Necessary and
Sufficient for Herbivore-Specific Plant Responses. Plant Physiology 125:711-717.
Kandoth, P., Lincoln, D., Howe, G., Miller, W., Walla, M., Jayanty, S., Pancholi, S., Ranf, S., Stratmann, J.
(2007) Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3function in the systemin-mediated defense
response against herbivorous insects. Proceedings of the National Academy of Sciences USA 104.
Langley, R., Mort, S. (2012) Human exposures to pesticides in the United States. Journal of Agromedicine
Ryan, C.A. (2000) The systemin signaling pathway: differential activation of plant defensive genes.
Biochimica et Biophysica Acta 1477:112121.
Xu, S., Zhou, W., Pottinger, S., Baldwin, I. (2015) Herbivore associated elicitor-induced defenses are highly
specific among closely related Nicotiana species. BMC Plant Biology 15:2.

For instructions on completing this form:

Magellan Scholar BUDGET FORM

Students Name: Allie Conner

Double-click on table to enter data
Student salary Hours Rate Subtotal
Estimated number of hours
Enter the hourly wage
student will work
Research hours during semesters
150 $10.00
when enrolled in classes $1,500.00
Research hours during semesters
when NOT enrolled in classes $0.00

Fringe: Student salary * student fringe rate 1 (what is fringe? See budget instructions or guidebook)
Enrolled in classes $1,500.00 0.37% $5.55
Not enrolled in classes $0.00 8.03% $0.00

Materials/Supplies Enter sub-total from below: $480.00

Travel Enter sub-total from below: $1,000.00

TOTAL: $2,985.55

Amount requested for Scholar award: $2,985.55

Budget Justification/Description
NOTE: Magellan Scholar awards are processed through E funds. All expenditures MUST remain compliant with E fund
procurement requirements. All budgets must be reviewed by department business managers prior to submission.

Student Salary: Indicate estimated number of student research hours per week and hourly rate separated by semesters when student is enrolled in
classes or not enrolled in classes (generally fall or spring vs summer semesters). Time during breaks (Fall, Winter or Spring break) are still hours
during semesters of enrolled classes.
I will work from January 16th until April 28th (=15 weeks) for 10 hours per week at a rate of $10/hour. This
amounts to a total of $1,500. Since I will graduate in May, I will only need support for one academic semester
(Spring 2017).

Materials/Supplies: Indicate items, quantity, and estimated price. Be sure to include taxes on all purchases.
Are you requesting funds for participant incentives? You must attach an approval memo from business manager see
I will grow Manduca sexta larvae from eggs, which will be purchased from Carolina Biological Supply Company
at $30/~40 eggs. For three experiments with four species and 20 eggs/species, I will need 240 eggs however,
the hatch rate is variable so 480 eggs will be purchased. This amounts to $360. I need growing supplied such
as soil at a total of $20. The poster for Discover USC will cost $100.

Travel: Indicate location, purpose of travel, provide itemized costs (list out each cost separately: transportation, lodging, registration, etc). For
conferences, provide name of conference, dates, and explain why this conference is most appropriate. No more than $1000 is permitted for
conference travel.
To attend the American Society for Biochemistry and Molecular Biology Annual Meeting, $400 is allocated for
plane ticket and $600 is allocated for lodging.