METHODS Biological Materials There were two organisms used in this experiment. The first being the tobacco hornworm, Manduca sexta larvae. The second being the aztec tobacco plant, Nicotiana rustica. The aztec tobacco plant uses nicotine as a defense mechanism which is normally used constitutively but wounding or herbivore attack induces the production of more nicotine. The tobacco hornworm has evolved to overcome the defense mechanisms of the tobacco plant, making it a specialist herbivore that primarily feeds on the Solanaceae family. M. sexta is an agricultural pest found in central America and the warmer parts of north America. As the name Manduca suggests, they eat very quickly and in large quantities. M. sexta eggs were acquired from Carolina Biology Supply Company (North Carolina, USA), and maintained at 22+2?, with 18 hours of light/day. We reared them on wheat germ growth medium (also from Carolina Biology Supply Company) until the majority had reached the 3rd instar larval stage (3 weeks). The environmental controls were set to mimic the environment in which M. sexta would be moved from their growth container to their host plants. The host, N. rustica (seeds) was obtained from Elana's Organic Garden (Lowbanks, ON). The seeds were planted in Pro-Mix potting soil ~4 weeks before the experiment was conducted. They were grown at 22+2? with 18hrs of light/day with consistent watering, and 20-20-20 fertilizer was applied in two equal intervals during that time. To reduce confounding variables the plants were kept in consistent and even light conditions. Plants were grouped as close to the same age as possible. Herbivory Experiment To test abiotic stress on the production of defence mechanisms in N. rustica, different concentrations of saline solution were fed to three different groups of the plant. Group one was treated as the control group and was given non-saline water, group two was given a low concentration saline solution, and group three a high concentration saline solution (50mL of 300mM). Then, three different M. sexta were selected and placed on each group of tobacco plant. Hornworms of approximately the same size were selected in the hope of minimizing the difference in eating patterns across the three groups. Clear plastic cups were also placed on top of each pot to minimize the exchange of airborne signals between each individual plant (phytosemiotics). The hornworms were left on their respective N. rustica host for 5 days before they were removed and measured. Measures were as follows: To measure if a plants defence increased or decreased each hornworm was weighed on day zero and day 5. The hornworms were taken from their housing tub and placed directly into a cup that was teared previously on the scale on day 0. On day 5 they were taken from their pot
and brushed lightly with a fine brush to remove any dirt that may contribute weight and appear greater than it should. Day zero observations consisted of the worm's reaction to touching the leaf, their behavior once they were settled, and their eating patterns.