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An ecology laboratory experiment conducted at morehouse college, focusing on the induction of secondary chemical defenses in tobacco plants (nicotiana alata or n. Tabacum) in response to herbivore damage. The study uses a brine shrimp bioassay to evaluate the toxicity of leaf extracts and investigates the difference between herbivore damage and physical damage on the concentration of toxic compounds in tobacco leaves. Students will perform pre-treatments on tobacco plants, prepare leaf extracts, and conduct brine shrimp bioassays to analyze the data using an analysis of variance (anova) test.
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Laboratory 6
Objectives
nutrients (particularly nitrogen) in many of these compounds has long suggested that chemical defenses are costly for plants to produce and maintain (Karban and Baldwin 1997). Such defense expenses could be minimized if plants could produce expensive chemical defenses only when they were needed (Baldwin 1998). Experimental evidence for rapidly inducible chemical defenses, producing or increasing chemical defenses in response to an initial herbivore attack, is very clear in a wide variety of plants (Karban and Baldwin 1997). The interactions between herbivores and plants occur in both ecological (the life span of a given organism) and evolutionary time. The production of an effective chemical defense may be overcome by a specialist herbivore that selectively feeds on the least defended parts of a plant, or evolves detoxification mechanisms that permit it to overcome a particular defensive chemical. The occurrence this kind of plant-herbivore arms race and the resulting co-evolution of chemical defenses and herbivore specializations has produced some of the most unusual and interesting plant-animal interactions (Futuyma and Keese 1992). Some specialist herbivores even use the toxins of their host plant for their own protection (Harborne 1993, Sotka, Wares and Hay 2003). In this study, we will use a bioassay to evaluate the toxicity of leaves from tobacco plants ( Nicotiana alata or N. tabacum ) and determine whether herbivore and herbivore-like damage will induce an increase in toxic secondary compounds. A specialist herbivore on tobacco, larval tobacco hornworms, Manduca sexta , will be used as natural herbivores in your experiment (Villanueva 1998). We will employ a Brine Shrimp Bioassay (Winnett-Murray, Hertel, and Murray 1997) to evaluate the toxicity of leaf extracts. Brine shrimp ( Artemia salina ) larvae, or nauplii, are commonly used in toxicological studies as a humane and inexpensive proxy for vertebrate animals. The 24-hour brine shrimp nauplii bioassay will provide a fairly rapid measure of leaf toxicity that would not be possible using the natural herbivores of tobacco. However, tobacco hornworms could be used in a more natural bioassay. How might tobacco hornworms be used to evaluate the toxicity of the tobacco plants in this study? Design a protocol for a tobacco hornworm bioassay. We will address three questions in this study:
Brine Shrimp Survival You will be using 8ml sample vials to conduct your brine shrimp bioassays. Each and every vial will have a final volume of 5ml including any volume of liquid used to transfer brine shrimp nauplii to the vial. Each vial can reasonably hold a total of 10 brine shrimp nauplii for a 24 - hour period to evaluate survival. Past experience has indicated that 1.0ml, 2.0ml, and 4.0ml of tobacco leaf extracts, prepared using boiling 1% NaCl, in final volumes of 5ml are appropriate for evaluating toxicity. Start by pipetting the appropriate volume of leaf extract to each vial (make sure your vial is marked with the extract source and concentration). Keep your vials in a vial rack to keep them from spilling. Using a dissection microscope, carefully isolate groups of 10 vigorously swimming brine shrimp nauplii in small volumes of 1% NaCl. Use glass Pasteur pipettes to isolate and count the nauplii. To each of your bioassay vials, transfer 10 vigorously swimming brine shrimp nauplii. Do not transfer dead animals or unhatched eggs. After you transfer the animals to a vial, examine the vial to be sure there are 10 nauplii present. Bring the volume of each vial up to 5ml with 1% NaCl. Use a vial containing a measured volume of 5ml as a measurement standard. Screw the cap loosely on each vial. The brine shrimp nauplii require oxygen like any other aerobic organism. After 24 hours, count all brine shrimp nauplii in each vial. Record the number alive and the number dead. Be sure to account for all 10 individuals who went into each vial, animals may get stuck to the vial when it is emptied. Data Analysis Start by calculating the mean number of brine shrimp alive and dead (at each concentration) that were from the same individual plant source. This will be the mean of your vial replicates for a given plant source and concentration. Different plants are the independent cases in this experiment.
Now, prepare an Excel spreadsheet file with your mean values for brine shrimp survival. This spreadsheet data file should have the following column headings: Your Name, Extract Source (Plant Treatment), Plant Replicate Number, Extract Concentration, Number Alive, and Number Dead. You will enter the mean number of alive and dead brine shrimp from each category of plant source and concentration. Using the spreadsheet equation function, calculate the percentage of brine shrimp nauplii surviving in each vial for 24-hours. Data will be analyzed using an analysis of variance (ANOVA) test to compare the brine shrimp survival at different concentrations of extract (for a given plant treatment) and to compare brine shrimp survival between plant treatments (at a given extract concentration). After everyone has entered their data, you will be provided with a complete data file (Induction Raw Data) containing the means (that you entered) of brine shrimp that were alive and dead after 24 hours exposure to each treatment. The output of the ANOVA test also will be provided to you. Keep in mind that the null hypothesis for this test is no relationship between the concentrations or treatments being compared and the number of brine shrimp alive after 24 hours. A p value of less than 0.05 means you can reject the null hypothesis of no difference between the treatments being compared. Literature Cited Baldwin, I.T. 1998. Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences USA 95:8113-
Billings, J. and P.W. Sherman. 1998. Antimicrobial functions of spices: why some like it hot. Quarterly Review of Biology 73:3-49. Feeney, P. 1992. The evolution of chemical ecology: Contributions from the study of herbivorous insects. Pages 1-44, in Herbivores: Their Interactions with Secondary Plant Metabolites. Volume 2, Ecological and Evolutionary Processes. 2 nd Edition. (Rosenthal, G.A. and M.R. Berenbaum eds.). Academic Press, New York, NY, 493 pages.
This secondary chemical defense induction protocol was developed by Blumer, L.S., Denton, M.K., and Brooks, L.E. 2007. Induction of Secondary Chemical Defenses. Pages 1-16, in Tested Studies for Laboratory Teaching, Volume 28 (M.A. O’Donnell, Editor). Proceedings of the 28th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 403 pages. This bioassay was modified in 2007 by J. Sarver to replace methanol extraction with a boiling NaCl extraction.