[name redacted], Pan 1

[name redacted] and Kate Pan

BIOL 428 A
Instructor: Prof. Jeffrey A. Riffell
6 June 2014
Final Research Proposal
Preamble: The self-medication of the caterpillar Grammia incorrupta in response to infection by
tachinid flies has been thoroughly documented from a behavioral perspective. We seek to
understand the sensory mechanism behind this behavior by asking whether increased
sensitivity of G. incorruptas pyrrolizidine alkaloid (PA) taste receptors in response to infection
triggers self-medication. By linking basic physiological changes in sensory systems with
responses to environmental hazards, we reveal possible mechanisms by which caterpillars are
capable of behaviors traditionally considered unique to organisms possessing higher-level
cognitive processing and learning abilities.
A. SPECIFIC AIMS
Aim 1: Determine the effect of knocking out medial phagostimulatory cell 9 on the selfmedicating behavior of Grammia incorrupta in response to parasitic infection.
Experiments in this section will focus on examining the relationship between the pyrrolizidinesensitive medial phagostimulatory cell 9 (MPC9) and self-medicating behavior in Grammia
incorrupta. Work on this aim will rely on previous experiments performed in G. incorrupta by
other researchers.
Hypothesis 1: Medial phagostimulatory cell 9 is necessary for induction of selfmedication in Grammia incorrupta. MPC9 is known to be sensitive to alkaloid compounds.1
However, its influence on behavior in G. incorrupta has not been critically evaluated. Using a
combination of artificial feeding experiments and laser ablation techniques, we will test the
hypothesis that MPC9 is necessary to G. incorruptas ability to self-medicate.
Aim 2: Determine the effect of parasitic infection on the sensitivity of medial
phagostimulatory cell 9.
Experiments in this section will examine whether MPC9 is responsive to parasitic infection. Aim
2 focuses on a molecular mechanism for the behavior examined in Aim 1.
Hypothesis 2: The sensitivity of medial phagostimulatory cell 9 increases in response to
parasitization. It is known that sensitivity of the MPC9 changes in response to alkaloid
consumption.2 High sensitivity of the MPC9 is associated with consumption of alkaloids by G.
incorrupta. However, this association has not been rigorously described or tested. We
hypothesize that increased sensitivity of the MPC9 is responsible for inducing self-medication in

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Grammia incorrupta in cases of parasitic infection. We will test our hypothesis by comparing the
sensitivity of the MPC9 in uninfected and infected caterpillars.
B. BACKGROUND
B.1. Introduction. When afflicted by parasitic infection, Grammia incorrupta increases its
consumption of pyrrolizidine alkaloid (PA) compounds.6 Increased consumption of PAs raises
the caterpillars chances of surviving the infection and killing off the parasites.6 However,
increased consumption of PAs also decreases growth and survival rate in uninfected
caterpillars7, making self-medication a balancing act between maintaining overall health and
destruction of parasites. Though studies in other caterpillar species suggested a negative
relationship between immune responsiveness and PA consumption, recent studies performed in
G. incorrupta show no correlation between the two.7
Potential Involvement of an Alkaloid-Sensitive Receptor. Several investigations into
this behavior have revealed an alkaloid-sensitive receptor in the brain of G. incorrupta.
Sensitivity of this receptor, termed medial phagostimulatory cell 9 (MPC9), is associated with
increased consumption of alkaloid compounds in G. incorrupta, revealing that alkaloid
compounds are phagostimulants rather than deterrents.1 Additionally, recent ingestion of
alkaloids results in decreased sensitivity of the receptor.2 MPC9 has both the potential to
increase consumption of alkaloids in G. incorrupta as well as the ability to change in response to
recent ingestion of alkaloids. These observations are consistent with the hypothesis that MPC9
has a role in regulating the self-medicating behavior of G. incorrupta. Both of the above
described characteristics are necessary for regulating self-medication. G. incorrupta must be
driven to eat alkaloid compounds to self-medicate, but it must not consume too many or it will
suffer detrimental health effects.
C. RESEARCH DESIGN AND METHODS
C.1. Experimental Methods
C.1.1. Stimulation
Immune System Stimulation: We will stimulate the immune system through infection
by Exorista mella eggs. This method has been used successfully by other researchers and is
well-described in the literature.
Egg Injection: Eggs will be injected by live female flies using a method of exposure
described by Singer.6 A captive colony Exorista mella flies will be used as parasites. Lab-reared
caterpillars will be individually placed into a container with a female fly. The fly will be allowed to
place one, two, or three eggs onto the caterpillar and then will be removed from the container.

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Gustatory Stimulation: We will deliver chemical stimuli of known concentration and
composition to the taste receptors of G. incorrupta at controlled intervals. We will use methods
found effective and described by experts in G. incorrupta studies.1
Stimulant Delivery: We will use stock solutions of seneciphylline n-oxide dissolved in
ethanol and then diluted with 0.1 M KCl solution to give a final alkaloid concentration of 5 x 10^3 M solution containing 5% ethanol. This stock solution will be diluted to various working
concentrations. The seneciphylline n-oxide will be dissolved in ethanol first because of its
insolubility in water. It is a known phagostimulant of MPC9 in G. incorrupta.1 The solution will be
applied drop by drop to the mouthparts of G. incorrupta using a glass pipette.
C.1.2. Behavioral Tests
Feeding Observations: We will examine the feeding behavior of G. incorrupta for selfmedication. Specifically, we will look for changes in consumption of PA compounds.
No-Choice Experiment: We will test feeding behavior by subjecting the caterpillars to
no-choice feeding experiments. Caterpillars will be individually isolated in a clear cup at room
temperature (23C) and presented with a glass fiber disc soaked in 0.01 mM monocrotaline, a
PA compound. Caterpillars will remain in this set-up for 24 hours. The glass fiber discs will be
presented with pins stuck through them so that the entire disc is accessible to the caterpillar for
consumption. As a control, some caterpillars will be put in the same conditions but presented
with a glass fiber disc with 0.01 mM sucrose in distilled water instead of monocrotaline. All discs
will have been weighed beforehand, and after the 24-hr period feeding period, the discs will be
retrieved and dried for another 24 hours. They will then be reweighed. The difference between
the initial weight and the final weight will be recorded as an indication of the mass consumed by
the caterpillars.
Two-Choice Experiment: We will test the feeding behavior of the caterpillars prior to
infection by conducting two-choice experiments. The caterpillars will each be separated into
their own cup where there will be two different food blocks to choose from. The first will be an
artificially made nutritional block composed of 22.4% protein (casein), 15.2% digestible
carbohydrate (sucrose), 2.2% Wesson's salt mix, 11.5% agar, and 48.5% alpha-cellulose, the
last of which is indigestible. The second block will be 0.1% monocrotaline and the rest made up
of alpha-cellulose. This experiment will last for five days and new blocks will replace the old
ones every 24 hours. All blocks will be pre-weighed, and then after the five days are up, they will
be dried and reweighed, the difference between the initial and final masses giving us the
amount that the caterpillar consumed.6
C.1.3. Neuronal Recording

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Intracellular recording: We will make electrophysiology recordings with the tiprecording method5 using live G. incorrupta. The caterpillars will be prepared according to the
same procedure followed by Bernays 2002.1 Each insect will be immobilized in a vial of 0.1M
KCl with a rubber gasket around its neck so that its head is exposed. The indifferent electrode
will be placed in contact with the KCl solution. Before and after stimulation, the mouthparts of
the insect and the recording electrode tip will be dried with absorbent paper to ensure that
concentration does not increase with evaporation. After each stimulation, the insects
mouthparts will be rinsed with distilled water.
C.1.4. Statistical Analysis of Behavioral Changes and Neuronal Sensitivity
Feeding Observations: We will quantify changes in feeding behavior by comparing the
masses of disks eaten by the caterpillars before and after infection. Averages from each
experimental group will be taken and then compared using t-tests to detect statistically
significant differences.
For the no-choice experiments, we will compare the data of the infected caterpillars
consumption of sucrose versus consumption of PA, of the uninfected versus infected
caterpillars consumption of PA, of the uninfected versus infected caterpillars consumption of
sucrose, and then compare those last two trends with each other. For the two-choice
experiments, we will compare the data of how much of the nutritious food blocks the caterpillars
ate versus how much of the PA food blocks they ate and compare that ratio between infected
and uninfected caterpillars.
Receptor Adaptation Speed: We will analyze receptor sensitivity by measuring
adaptation time. Using a constant concentration of stimuli, we will apply stimuli at 1 minute
intervals until the firing rate decreases to baseline. The firing rate will be analyzed using Spike
Train Software.3 Sensitivity will be quantified as the period of time from the first stimulation to the
end of activation. Average values for time will be taken for each experimental group and
compared with a two tailed t-test.
C.1.5. Blocking Receptor Activity: MPC9 will be destroyed using laser ablation techniques
described by Fang-Yen.4 A laser microbeam will be focused through the objective of a
microscope and can be adjusted in three dimensions to specifically target MPC9.
C.2. Research Designs and Experimental Strategies
Aim 1: Determine the effect of knocking out the medial phagostimulatory cell 9 (MPC9)
on self-medication in parasitized Grammia incorrupta.
Rationale: While the sensitivity of the MPC9 to PAs has been characterized in G.
incorrupta, its effect on behavior has not been observed. We seek to answer whether the PA-

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sensitive receptor has an actual effect of the behavior of G. incorrupta, providing evidence for its
role in self-medicating behavior.
Experimental Plan: We will test the effect of MPC9 knockout in G. incorrupta by
destroying the receptor using laser ablation (C.1.5) and then conducting feeding experiments
(C.1.2). Four groups of caterpillars will be used. One group of caterpillars put into the laser
ablation apparatus but not actually operated on will be used as controls. These caterpillars will
be further split into two groups, infected and not infected. A second, experimental group will
have their MPC9 receptors destroyed. This second group will also be split into two subgroups,
infected and not infected. All groups will be subject to the no-choice and two-choice feeding
experiments described in (C.1.2).
Potential pitfalls and alternative strategies: Laser ablation is known to have the risk of
destroying or damaging cells close to its target. If our laser ablation fails in such a way, we will
use a more accurate method with a femtosecond laser and a compound fluorescence
microscope as described by Steinmeyer.8 It is also possible that MPC9 plays minor roles other
than its major one of responding to the pyrrolizidine stimulus and that ablating it will thus have
unforeseen collateral effects on other cells. Additionally, the trauma of laser ablation may also
cause physiological responses in the two groups that undergo it which the two that do not would
not have.
The feeding experiments take place in unnatural environments and the caterpillars are
trapped within the cups, which does not accurately reflect the way they would find food in the
real world. This may lead to different feeding behavior than would be observed in nature. Also,
both the blocks and the discs are artificially made and do not look like the normal food that
caterpillars would consume. If this changes the caterpillars feeding behavior, an alternative
strategy would be to use natural food and outdoor cages rather than synthesized food and
indoor cups.
Interpretations: If MPC9 is regulating self-medication in G. incorrupta, we will see no
difference in the feeding behavior between the infected group with MPC9 knocked out versus
the feeding behavior of the uninfected control group that does not have MPC9 knocked out. This
would indicate that because a critical alkaloid receptor is not being activated, the caterpillars
cannot taste the alkaloids and so are not stimulated to eat alkaloid-containing foods. For our
controls, we would expect infected caterpillars with functioning MPC9 receptors to eat more of
alkaloid-containing foods than healthy caterpillars with functioning MPC9 receptors simply as a
result of previously established self-medicative behavior in G. incorrupta.

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Table 1: Hypothetical Results of Aim 1s No-Choice Feeding Experiment
Infected Caterpillars
Disc soaked with sucrose
Disc soaked with alkaloid
Healthy Caterpillars
Disc soaked with sucrose
Disc soaked with alkaloid

Destroyed MPC9
.05 grams eaten
.01 grams eaten
Destroyed MPC9
.05 grams eaten
.01 grams eaten

Functioning MPC9
.05 grams eaten
.09 grams eaten
Functioning MPC9
.05 grams eaten
.05 grams eaten

Aim 2: Determine the effect of parasitic infection on the sensitivity of the PA-receptor.
Rationale: Following Hypothesis 1, we seek to explore a molecular mechanism for how
MPC9 regulates the self-medicating behavior of G. incorrupta. Knowing that MPC9 is key, we
can look at why.
Experimental Plan: We will measure the sensitivity of the PA receptor (C.1.3) in infected
and uninfected caterpillars by measuring the adaptation speed of the caterpillars receptors.
Infected and uninfected caterpillars alike will be stimulated with 0.1mM solution of seneciphylline
n-oxide. We will record the length of time required for the receptor to adapt to the stimulus and
stop responding according to the analytical methods described in (C.1.4.).
Potential pitfalls and alternative strategies: One potential difficulty we anticipate in
this experiment is calibrating the concentration of seneciphylline n-oxide to obtain a clear signal
from an activated receptor. There is also variation in sensitivity between different individual
caterpillars, but by using a large number of them, these differences will likely average out.
Interpretations: If sensitivity of MPC9 is responsible for mediating self-medication, we
expect to see increased adaptation speed in infected caterpillars receptors versus those of
uninfected caterpillars, supporting Hypothesis 2. If we do not see any significant differences in
the adaptation speed of the receptors between infected and uninfected caterpillars, we will
conclude that our hypothesis is flawed.

D. LITERATURE CITED
1.

Bernays, E.A., Chapman, R.F., and Hartmann, T. (2002). A taste

receptor neurone dedicated to the perception of pyrrolizidine alkaloids in the medial

galeal sensillum of two polyphagous arctiid caterpillars. Physiological Entomology
27, 312-321.
2.

Bernays, E.A., Rodrigues, D., Chapman, R.F., Singer, M.S., and

Hartmann, T. (2003). Loss of gustatory responses to pyrrolizidine alkaloids after their

[name redacted], Pan 7

extensive ingestion in the polyphagous caterpillar Estigmene acrea. The Journal of
Experimental Biology 206, 4487-4496.
3.
Cajigas, I., Malik, W.Q., Brown, E.N. (2012). nSTAT: Open-source
neural spike train analysis toolbox for Matlab. J. Neurosci. Methods 211, 245264.
4.
Fang-Yen, C., Gabel, C.V., Samuel, A.D., Bargmann, C.I., Avery,
L. (2012). Laser microsurgery in Caenorhabditis elegans. Methods Cell Biol
107,177206.
5.

Hodgson, E.S., Lettvin, J.Y. and Roeder, K.D. (1955) Physiology

of a primary chemoreceptor unit. Science 122, 417-418.

6.
Singer, M.S., Mace, K., and Bernays, E. (2009) Self-medication as
Adaptive Plasticity: Increased Ingestion of Plant Toxins by Parasitized Caterpillars.
PLoS ONE 4(3), Article Number e 4796.
7.
Smilanich, A., Vargas, J., Dyer, L., Bowers, M. (2011). Effects of
Ingested Secondary Metabolites on the Immune Response of a Polyphagous
Caterpillar Grammia incorrupta. Journal of Chemical Ecology. 1-7.
8.
Steinmeyer, J. D., Gilleland, C. L., Pardo-Martin, C., Angel, M.,
Rohde, C. B., Scott, M. A., & Yanik, M. F. (2010). Construction of a femtosecond
laser microsurgery system. Nature protocols, 5(3), 395-407.