Lab 04 BIOL 1510 Lab Manual Plant Defenses F2017

Lab 04: Testing Plant Defensive Compounds

Plants face many challenges in their environment from abiotic stressors such as light, desiccation, and soil
chemistry to biotic stressors such as herbivores and pathogens. However, plants deal with environmental
challenges much differently than we do. One of the largest fundamental differences between plants and
animals is that plants cannot move and therefore, in the case of herbivory, must evolved various ways defend
themselves.

There are two main groups of animals that perform the

majority of herbivory of plants and each requires different
types of defenses. For larger herbivores such as mammals,
plants often produce structural defenses such as thorns, or
spines as a deterrent. This is quite effective as a cactus is
essentially a miniature oasis in a low precipitation
environment however cacti are well protected by modified
leaves serving as spines. There are also various examples of
structural defenses in the Mesozoic garden such as the
thorns of acacia trees or rose bushes. Structural defenses
however do not deter all herbivores. Insects and other small
herbivores can readily move around these mechanical
defenses, and may even be protected themselves from
predators when resides on the plants. In these scenarios,
plants use an alternative approach, chemical defenses.

Plant chemical defenses take the form of toxins, or allelochemicals, that cause harm to attacker. These toxins
are secondary metabolites produced by the host plant that serve to disrupt the behavior, growth/development,
or overall survival of the herbivore. For example, tannins, which are named for their color properties (also make
up teas for human consumption), bind to proteins within the gut to inhibit their digestion. Thus, the herbivores
that consume these plants are essentially starving while eating. Other compounds such as cardenolides from
milkweed causes cardiac arrest those that consume it.

The most common plant toxins are the Alkaloids. Alkaloids are naturally occurring compounds comprised of
amino acids and include substances such as caffeine, nicotine, cocaine, atropine, as well as the opiates
(morphine, heroine, etc.). Alkaloids vary in their effects; for example, some may be stimulants, analgesics,
depressants, and even psychotropic. While we know many of these toxins with regards to their effects on
humans, the adaptive value of these compounds resides in their effects on insects.

In this laboratory exercise you are going to investigate the effects of plant defensive compounds on small
organisms. You will be given various plant defensive compounds and measure their effect on the heart rate of a
small invertebrate.

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Common plants we may test, and their effects

Kava-kava (Piper methysticum) is a pepper plant used by

various cultures in the Pacific Ocean. The plant is revered for
its sedative properties and often part of cultural events within
the region. Various strains of Kava exist with various effects
from psychoactive properties to promoting relaxation. These
are effects are largely attributed to kavalactones which are
more concentrated in the roots. Preparation involved
grinding up the roots to steep in water.

Coffee (Coffee arabica) is the genus of plants made famous

for its effect as a stimulant. The seeds (coffee beans) of the
fruits are roasted, ground, and then steeped in hot water to
produce coffee. The active ingredient in coffee is caffeine,
widely used throughout the world and a major export for
Central and South American countries. The plants are shade-
tolerant and thrive in the forest understory.

Tobacco (Nicotiana tabacum) is a plant of the nightshade

family known for its stimulant and highly addictive compound
nicotine. Tobacco is an extremely important plant, and served
as a major industry of European countries and their colonies.
The production and exportation of tobacco was the dominant
industry in the early United States, and still holds a vast
influence culturally here as well as around the world. To
achieve the desired effect, leaves are dried and then smoked,
snuffed, or chewed.

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Chili peppers (Capsicum spp.) are also members of the

nightshade family. Chili peppers are peppers but not from the
Piper genus. The substance to gives peppers their burning
sensation when consumed is known as capsaicin. Peppers are
often ranked in intensity due to their relative concentration
of capsaicin present in their fruits. The use of chili peppers is
has been part of the human diet in the Americas only a few
thousand years after the advent of agriculture and various
cultivated strains have since spread and became staples in
the diets of various human cultures globally. Although the
intense burning associated with the consumption of peppers
is considered an adaptation to deter mammals, birds do not
suffer negative consequences from consumption. This is
considered as a mechanism for plants to choose birds and
their vector of seed dispersal over mammals.

Atropine is a compound from the Belladonna plant (Atropa

belladonna), also known as deadly nightshade, and is also a
member of the nightshade family. Belladonna means
beautiful lady because an extract of the plant was used in
eye drops by women, as it dilated their pupils to in effort
increase their perceived attractiveness. Atropine is still used
today to dilate pupils for eye exams. The plant, and others
from the nightshade family, are common but quite toxic so
should be handled with caution. The active compound is an
anticholinergic agent, which blocks the neurotransmitter
acetylcholine in the nervous system. Of its many effects, it is
known to increase heart rate. It does not have ill effects on
cattle, sheep, goats, and rabbits, but highly toxic to other
animals, which hints at possible seed dispersal vectors.

Hypothesis

For the 2017 version of this laboratory exercise, you will test the effect of two plant extracts, coffee and kava,
on the heart rate of Daphnia magna (water fleas). Based on the above information on plant chemical defenses,
develop hypotheses for how you expect this organism to respond to the exposure to each of the plant extracts.
Remember, a hypothesis is a statement, not a question.

What are some hypotheses you can think of?

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Procedure
For a video overview of the procedure, please see http://www.carolina.com/teacher-resources/Video/daphnia-
heart-rate-experiment-video/tr11197.tr

Using a plastic pipette cut to accommodate the large size of the specimen, place a single Daphnia magna
specimen into the well of a concave slide and remove excess (i.e. overflowing) water by using a tissue. Do not
use a coverslip. The specimen should have enough water to allow its gills to function but not so much that it is
actively swimming; the specimen should be somewhat immobilized. Locate the specimen using the light
microscope starting at the lowest magnification. If necessary, use higher magnification to locate the heart,
which is at the anterior dorsal end of the body, behind the eyespot (Figure 1).

Figure 1: Location of heart in Daphnia magna

Wait one minute for the specimen to acclimate to the slide. To establish a baseline heart rate, count the
number of heartbeats in 15 seconds using a timer. If the heart rate is too fast to count easily, use a pen/marker
to make a dot on a sheet of paper for every heartbeat you see and count the dots after 15 seconds (ask a
groupmate to serve as timer). After counting, turn off the light of the microscope to avoid heating the specimen
(may influence heart rate for future tests). Multiply that number by 4 to get heart rate in heartbeats/minute
and fill in the table for baseline heart rate in the worksheet. Now, to perform the control, place a drop of spring
water on to the specimen, wait one minute, and then obtain the heart rate in the same manner as before when
collecting the baseline heart rate. Place this specimen in the recovery beaker for used Daphnia. Repeat this
measurement twice more, each time with a new specimen, for a total of three replicates for this treatment. As
you complete each replicate, place the used Daphnia in the recovery tank as indicated by your instructor.
Please do not rinse live animals directly into the drain or into the wild.

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To test the effects of the coffee plant extract, acquire a new specimen and place on the slide as described
previously. First, determine baseline heart rate as before. Second, instead of spring water, place one drop of the
coffee plant extract solution onto the specimen, wait one minute, and obtain the heart rate in the same
manner as before. Topic for discussion: why are we measuring baseline heart rate instead of only comparing the
heart rate between controls and experimental groups after the application of the solution? As with controls,
replicate each test three times, each time using a new Daphnia, and retire old Daphnia to the recovery tank.

Repeat the above procedure for the kava plant extract.

Once you have collected 3 replicate measures of the change in heart rate produced by each treatment (control,
caffeine, kava), work with your instructor to have your lab groups data added to the master Excel file
maintained by your lab instructor for your section. Your lab instructor will make this file available to you on
Blackboard, and it is this master file that will be the basis for your lab report. You must write your report based
on the Excel file produced by your section. Do not write your report based only on your lab groups data, or on
the data from a different section.

Analyzing your sections results

When we must report the results of an experiment, we usually begin by calculating and comparing the mean (a
measure of central tendency; its also called the average) and variance (a measure of distribution or spread
around the mean) for each treatment (control, caffeine, kava). We may detect some differences in the mean
heart rate of D. magna between those exposed to plant extracts and the controls, but how do we know if these
results are real? Statistics provide insights to whether an observed mean difference is real- or if it is merely a
result of random chance.

Before going further, we need to understand the concept of hypothesis testing in statistical analysis. Most
comparisons are set up to test a null hypothesis about the differences in the groups under comparison. An
example of a null hypothesis is there is no difference the body length of water strider males and females
Obviously, an alternative to the null hypothesis is there is a difference in the body length of males and females
If our test allows us to reject the null hypothesis, then we can accept (for now) the alternative hypothesis that
there is a size difference.

Remember that statistics are performed on samples. If you measured the body length of all the water striders in
a pond on campus, that is only a small sample compared to the rest of the population you did not measure!
Therefore, your statistical test is really a test to determine whether the difference between males and females
in your sample really reflects the true difference that exists in the total population.

In statistics, we often refer to the P-value of a statistical test, which ranges from 0.00 to 1.00. The P-value is the
probability that we would measure the observed difference in our small sample when there is no real difference
in the overall population. For instance, if we find that females are 5mm longer than the males in one pond
campus, what is the probability that we would see a difference of this magnitude in our sample if in fact there is
no real difference in average male and female length in the population? In other words, what is the probability
we would collect such an odd sample by chance? If the probability is less than 0.05, we can read it as the
probability that we would see such a large difference in males and females in our sample, if there was no real
difference in the overall population, is very small (less than 1 in 20); therefore, the difference must truly exist in
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the population as a whole. Conversely, if the P-value was large, say 0.4, we would conclude that the probability
that we could see a 5mm difference in males and females when indeed there really wasnt a difference in the
population is high. We should not be surprised to see such a difference in our small sample; if we collected such
samples, we could expect to see such a difference in about 40 of them.

In a way, P-values reflect the probability that a null hypothesis is false. By convention If the P-value is less than
or equal to 0.05, we reject the null hypothesis, and accept (for now) the alternative hypothesis.

Some statistical tests take a simpler route: they start by stating what their cutoff value is for the P-value (by
convention we almost always use P=0.05) for a result to be significant. They then calculate a test statistic and
compare that statistic to a critical value; if the test statistic is larger than the critical value, we reject the null
hypothesis for that test. The Students t-test we will be using in this lab is just such a test.

Students t-test for differences between two means

A two-tailed t-test is a statistical procedure to determine whether the observed difference between two sample
means ( ) is meaningful, given the spread (or variance) around those means. For instance, we might use a t-
test to determine whether there is a difference in average height between men and women in our class, or to
compare the gas mileage between two kinds of car. If we wanted to see whether any difference existed (i.e.,
whether men were taller than women or vice versa), we would use a 2-tailed t-test; if we wanted to test the
specific prediction that men are taller than women, we would use a 1-tailed test. For the sake of this lab, we are
using a 2-tailed t-test.

To perform the t-test, youll need to calculate the test statistic, which in this case is the t-statistic or T. You will
then need to compare this number to the critical value for that statistic (which varies depending on P, which in
this case is 0.05, and the number of samples involved in each test; see below for more information).

Using your sections data from the experiment, calculate T for each statistical comparison (control vs caffeine,
control vs kava, caffeine vs kava) by following the procedure below.

How to use the t-test formula to calculate the t-value

The two-tailed t-test is a relatively simple to perform. The first step is to calculate the difference
between the means (
): calculate the average value of the variable tested (change in heart rate,
1 ) for one group (control), and compare it to the average value of the variable tested (change in heart
rate, 2 ) for the other group (the tested plant extract). You now have the information for the numerator
of the equation (you will use the absolute value of the difference between these means; see formula
below).

We then calculate the pooled variance of the two samples being compared. This is important because it
allows us to consider the spread of the groups when considering the difference between means. A high
variance means that individuals are generally spread out from the average for the variable in question.
For example, what if we find that females have a higher IQ than males in this room on average, but all
males scored higher than all females except for one female that scored dramatically higher than
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everyone. There is a difference in the means but it is misleading. The average for female IQ may be
higher than males in this room, but most males also have a higher IQ than most females in here as well.
Variance allows us a lens to peer through when comparing means. In the figure below there are two
scenarios in which the means do not change, but variance is vastly different. Under high variance, there
is plenty of overlap between the populations such that it is unlikely that these populations significantly
differ in the trait in question. With low variance, the means have not changed, but there is very little
overlap between the populations. In this case of low variability, the populations appear to differ
substantially. As an aside: hopefully now when reading articles, scientific or otherwise, you should
always pay attention to the spread of the populations and not only the means!

Simply put, the variance is the average distance an individual is from the group mean. Subtract each
observation from the group mean, sum those numbers, and then square the sum. You then divide by
the number of observations minus 1. To determine variance, use the equation below. Calculate variance
for your two groups (control and experimental). It should also be noted that standard deviation (another
measure of spread) is simply the square root of the variance.

2
( )2
= =
1

= Mean
= Individual value
= Total number of values

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You now have the two information you need to calculate T.

|1 2 |
=
2 2
1+ 2
1 2

1 = Mean of first set of values

2 = Mean of second set of values
12 = variance of sample 1
22 = variance of sample 2
n1 = Total number of values in sample 1
n2 = Total number of values in sample 2

(The denominator is the pooled variance for both samples)

Determining the critical value.

To determine the critical value for each test, you need to know the cutoff P-value (as explained above,
by convention we are using p=0.05), as well as the degrees of freedom (DF) of the test. For t-tests, DF is
the sum of the sample size involved in the test minus 2:
df = n1 + n2 2
n1 = Total number of values in sample 1
n2 = Total number of values in sample 2

You can use several resources to determine the critical

value. You can use Excel, by selecting a blank cell and
entering the formula =T.INV.2T as shown in the image to
the right.

There are also free online critical value calculators, such as

http://www.danielsoper.com/statcalc/calculator.aspx?id=10

Interpretation of the test is simple. If your T statistic is equal to or greater than the critical value, you
reject the null hypothesis and conclude that the observed difference in means is meaningful, or
significant.

Remember that you need to perform three t-tests, one for each possible comparison among the three
treatments: control vs caffeine, control vs kava, and caffeine vs kava.

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General structure of lab report

For an excellent overview of how to write a formal lab report visit
http://writing2.richmond.edu/training/project/biology/biology.html

Additional resources are posted to the Blackboard page.

Formal lab reports in biology follow the standard format of scientific publications. You
are required to detail the results of a biological science experiment in a report with the
following sections:
1. A title and abstract page: a single page with a clear title that explains in a single
sentence the main point of the paper, followed by an abstract (= a formal summary)
describing the objective of the experiment, the approach used, and key results, in
between 100 and 250 words. This page should also have your name, the course number,
and the date of submission.
2. An introduction (typically 1-2 pages): the introduction has two functions- to explain to
purpose of the experiment, and to give the reader enough background on the subject so
that they understand why the study was done. For instance, a paper on enzymes might
explain what enzymes are; briefly explain how they work and why they are important;
to give an overview of the research objectives of the report, and list the hypotheses.
3. Methods section (typically 1-2 pages): a concise description of the various methods
used in the experiment. In biology, this section is NOT in point form, and the materials
are not listed (this is different than what you may encounter in chemistry). Give enough
detail to allow someone who has never done the experiment (nor seen it done) to be
able to replicate your work. On the other hand, don't give too much detail- the kind of
spectrophotometer used and the wavelength you monitored are both important
information; the brand of Sharpie you used to label your tubes is not. Give enough
information to allow replication of the experiment- and no more.
4. A results section (typically 1-2 pages): a description of the results of the experiments
outlined in the methods section, presented in the same order as they were given in the
methods. The results section has two components. First, a description in narrative (NOT
point form) of the results. If the experiment involved calculating slopes, this is where
you give those slopes. If the experiment involved calculating unknowns, this is where
you give the unknowns. In scientific publications, calculations are typically not included.
However, for the purpose of this lab report please include your calculations at the end
of the report. These calculations can be done by hand or using excel. If the calculations
were done in excel, take a screenshot of the spreadsheet in two ways: once with the
values and the other with the equations (ask your instructor on how to do this). Second,
any tables or figures you have decided to include. In general, it is best to keep these to a
bare minimum, and make them as informative as possible; give each table or figure a
title (Table 1. -------------, Figure 1.------------------) and be sure to label all axes using the
correct units. The title for a table goes on top, while for a figure the title goes on the
bottom. ALL TABLES OR FIGURES MUST BE REFERENCED IN THE NARRATIVE PART OF
THE RESULTS (or elsewhere) USING THEIR NUMBER. DO NOT INCLUDE RAW DATA. I
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generally prefer that students insert graphs and tables after the first paragraph in which
they are mentioned. An example:

Analysis of student attentiveness over time indicated that student attentiveness peaked
within 30 min, and decreased steadily thereafter (Figure 1).
25

Student attentiveness
20

15

10

0
0 20 40 60 80 100 120
Time since beginning of lecture

Figure 1. Student attentiveness in lecture over time.

5. A discussion section (1-2 pages): this is your chance to explain whether the experiment
went as expected. Were the hypotheses you outlined in the introduction supported or
rejected? If your hypothesis was supported (i.e. you rejected H0), what are the
implications for the system you are studying? Conversely, if your hypothesis was
rejected (i.e. you failed to reject H0), what are the implications? Try to explain these
results. What other phenomena could have produced the results you obtained? Make
comparisons to other studies. This is also where you list how you would improve the
experiment, what other follow-up experiments could be done, and conclude with the
overall significance of your results.
6. A references / work cited section (at least 7 references total): cite any sources you used
for information. You should certainly cite the lab manual and I strongly advise using the
textbook (the invertebrate and animal behavior chapters would be a good place to get
info) as well. You may also cite peer-reviewed, published literature (perhaps collected
from Google Scholar or the NSU Find Articles page). ANY REFERENCES LISTED HERE
MUST BE CITED IN TEXT AND VICE VERSA. The list of references goes at the end of your
report and should be listed in alphabetical order with consistent formatting. In-text
citations go at the end of each sentence where you are referencing the material as
(Author, year).

General formatting: reports should be written on a word processor. Margins should be

normal and the font should be Times New Roman, size 12 pt. The line spacing should be
set at 1.5 and pages should be numbered. The various sections (intro, methods, results
etc.) should be labelled.

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Peer-review session

Scientific papers go through a rigorous review process in which their peers evaluate contributions to
provide credibility to scientific reports before being distributed to the community in scholarly journals.
This form of self-regulation and quality control is what differentiates reputable scientific reports from
other formats such as websites that are not scrutinized and held to a high standard before
dissemination. Imagine the consequences if your textbook was comprised of information from blogs as
opposed to scholarly reviewed reports.

To introduce students to the review process, you must bring in two copies of your report to lab without
your name, or identifying information, on it to be evaluated by your peers. The only identifier on your
work will be a number assigned to you by your instructor. Each student will review at least two papers
and anonymously provide comments on the structure, grammar, and scientific validity of the report.
After this process, each student will have the benefit of editing their manuscript before submitting their
final draft on the designated due date. No report is perfect, so please do your part in improving your
peers contributions, as you will benefit from their efforts as well.

Please note: Student peer assessments of your paper will not be factored into its grade. They are meant
to provide you with valuable feedback, while introducing you to the joys of the peer review process.
However, your assessment of your peers work will be factored into your grade, so please take this
process seriously. Review your peers work diligently and do your best to provide helpful, constructive
criticism.

A sample of a peer review assessment sheet, and a rubric explaining how we would like you to score
your peers, is on the following page.

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Peer Review Assessment Author #_______ Reviewer #______

For the peer review assessment you will receive two reports from your class-mates, and
you will grade them based on the following grading system.
General Clarity of the report
1 point Below average: The report is difficult to understand throughout.
2 points - Average: The report contains sections that are difficult to understand.
3 points- Above average: The report is concise and contains little clutter.
General Organization of the report
1 point Below average: The report is disorganized; the flow of ideas is confusing and chaotic. The specified sections
are not followed, or they are mixed.
2 points - Average: The report jumps around a bit; and the flow of ideas is confusing in some sections.
3 points- Above average: The report is well organized; the flow of ideas is logical and orderly.
Abstract
1 point Below average: abstract is excessive long and the aim, experiment, results and conclusions are not clear.
2 points - Average: The abstract contains some of the specified subjects: aim or experiments, but it does not have all of
them, or they are not clear.
3 points- Above average: abstract include aim, experiments, results and conclusions in less than 200 words, and they
are clear.
Introduction
1 point Below average: introduction does not include specific ideas: scientific background, limitations and gaps of the
experiment and purpose statement (= objectives), and paragraphs are disorganized.
2 points Average: introduction contains some of the specified ideas, but it does not have all, or they are not clear.
3 points- Above average: introduction include specific ideas: scientific background, limitations and gaps of the
experiment and purpose statement (= objectives), paragraphs are clear and organized.
Methods
1 point Below average: methods are not clear about what was done, what hypothesis was tested, what was used in the
lab (experiment design), how many samples were used, what analyses were done.
2 points Average: methods lack of some explanation about what was done, or what was tested, or about experimental
design in the lab and analyses.
3 points- Above average: methods are clear about what was done, what hypothesis was tested, what was used in the
lab, how many samples were used, what analyses were done.
Results
1 point Below average: results are not clear about the acceptance or rejection of the hypothesis and t-test values.
Tables and figures do not include analyzed data and are not referred in the text.
2 points Average: results lack of some explanation about the acceptance or rejection of the hypothesis and t-test
values. Tables and figures are not clear or referred in the text.
3 points- Above average: results are clear about the acceptance or rejection of the hypothesis and t-test values. Tables
and figures include analyzed data and referred in the text.
Discussion
1 point Below average: discussion is poor, and do not include comparisons with theoretical values or background.
Trends and patterns of the data are not discussed, conclusion was not included.
2 points Average: discussion lack of some background to compare with the findings, trends and patterns of data are
poorly discussed, and conclusion is not clear.
3 points- Above average: discussion is clear and includes comparisons with theoretical values or background. Trends
and patterns of the data are discussed, conclusion is included.
References
1 point Below average: references are not presented in the text, or they are disorganized in the text and list of
references is not present.
2 pointsAverage: some references are missing in the text or in the list of references.
3 points- Above average: references are included in the text, and all of them are organized in the list of references.

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