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Biochemical Analysis of Insect Camouflage inManduca sexta,

the Tobacco Hornworm

Honors Thesis

Carroll College Department of Natural Sciences

Helena, Montana

Kevin P. Semmens2012


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Acknowledgments

This project was supported by an award from the Murdock Charitable Trust

to Dan Gretch, Ph.D., and with support from the Cargill Foundation. I would like to

thank Dan Gretch for his continued dedication, support, and patience as a mentor

and teacher. I would also like to thank Dr. Gerald Shields for his assistance with the

writing process, and my readers Dr. Kyle Strode and Mr. Jack Oberweiser for their

constructive comments. Thanks to my family for their constant love and support

during my time at Carroll, and all others who have helped me in reaching this stage

of my life.


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Table of Contents

Acknowledgments..iii

Abstract........v

List of Figures.vi

Introduction..1

Materials and Methods.....3

Results..9

Discussion..12

Literature Cited..15


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Abstract

Manduca sexta(Tobacco Hornworm) larvae exhibit diet-induced adaptation,

demonstrating green coloration in their natural environment but becoming a pale blue

when placed on a lab diet lacking plant material. I hypothesized that the color change

observed in the Tobacco Hornworm was induced by the dietary intake of a plant pigment

of orange or yellow color (specifically -carotene). The pigment -carotene is highly

hydrophobic, so I hypothesized that it was traveling to the skin via a lipoprotein transport

pathway. Hornworms were reared and then bled to extract hemolymph.

Ultracentrifugation was used to float out the lipoproteins from the hemolymph.

Lipoprotein isolation was confirmed by gel electrophoresis. The lipoproteins isolated

from plant-fed insects did indeed contain a bright yellow pigment. UV-visible

spectroscopy supported -carotene as the pigment being carried by the lipoproteins and

responsible for inducing the insects color change.


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List of Figures

Figure 1: Growth Cycle of Tobacco Hornworms4

Figure 2: Hornworm External Anatomy........6

Figure 3: Diet Induced Color Differences.9

Figure 4: Extracted Hemolymph.......9

Figure 5: Ultracentrifuge Separation...10

Figure 6: Top and Bottom Layers..10

Figure 7: Gel Electrophoresis.11

Figure 8: Comparison of UV-visible Spectra.

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Introduction

Animal camouflage, specifically in insects, has been studied extensively and has

been a source of great fascination. The mechanisms underlying camouflage vary greatly,

from genetically programmed patterning of coloration that has proved valuable in

survival (Carroll, 2011), to dietary influences that change the actual pigmentation of the

animal (Greene, 1989).

Heliconiusbutterflies exemplify this as well-studied animals that genetically

adapt to survive (Carroll, 2011). These butterflies engage in mimicry, with wing patterns

enabling them to resemble species that could be potentially poisonous or harmful

(Carroll, 2011). Genetically distant species of these butterflies evolve in the same way

despite their geographical and genetic differences, converging on the same phenotypic

expression (Reed, et al., 2011). A single gene (optix) was identified as the cause of the

convergent striking color and wing patterns shared by all of these butterflies (Reed, et al.,

2011). This illustrates how genetics can contribute to butterfly patterning and coloration

and how an adaptive trait can be maintained.

The larvae of the emerald moth,Nemoria arizonaria, are also well documented in

their ability to adapt, specifically through polymorphism (Greene, 1989). These larvae

adopt variable morphologies, sparked by changes in their diet (Greene, 1989). Greene

(1989) investigated several different changes in environment for theNemoriacaterpillar,

including temperature, photoperiod, and diet, and discovered that diet was the only

significant factor that induced morphological alterations. Based on what the larvae eat,

they develop with incredible specificity into a morphology best suited for their

environment (Greene, 1989). For example, when they are fed oak catkins, the caterpillars


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become near perfect copies of a catkin, and when fed oak leaves they are nearly

indistinguishable from a small oak twig (Greene, 1989). Clearly a dietary factor causes a

developmental phenotypic change, giving them ideal camouflage (Greene, 1989).

Manduca sexta(tobacco hornworm) larvae also exhibit diet-induced adaptation.

In their natural habitat, the hornworms are green, blending nicely into the leaves of the

tobacco plant on which they feed. However, when placed on a laboratory diet devoid of

plant material, the hornworms never develop the green coloration, instead showing a pale

blue exterior (personal observation, Dr. Daniel G. Gretch). In my current study I have

sought to identify the causative dietary agent and investigate the pathway by which this

agent makes its way to the skin of the animal.

Since green is a secondary color formed by the mixing of blue and yellow, my

initial idea was that a plant pigment of orange or yellow color would be responsible for

the insect color change. -carotene is such a pigment, and so I investigated if it, or some

other pigment, was playing a role in the process. Not only does -carotene exhibit the

orange-yellow color, but it also has been documented in humans to cause skin

discoloration, known as carotenemia, when consumed in large amounts (Arya, et al.,

2003). As such, I hypothesized that the color change observed in the tobacco hornworm

was induced by a dietary intake of the pigment -carotene.

In my study, diet induced changes in the color of the insects blood (hemolymph)

were closely monitored, leading to the question of the mechanism by which absorbed

pigment molecules were transported through the circulatory system. Since pigments such

as -carotene are hydrophobic, I investigated whether insect lipoproteins (lipophorins)

were transporting the molecules through the hemolymph. I reasoned that if -carotene


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was present in the lipoproteins, then when extracted, these lipoproteins would give a UV-

visible spectrum that matched that of -carotene. This -carotene footprint would be

expected to peak around 445 nm (Misawa, et al., 1993). If this absorbance were the only

one observed then it would support my hypothesis of -carotene as the sole factor in the

diet-induced color change.

Materials and Methods

Rearing

Tobacco hornworms were purchased from Great Lakes Hornworms. Two

different diets were obtained, one being completely devoid of plant material. This

artificial lab diet was purchased from Carolina Biological specifically as a hornworm

diet, and was a bulk diet containing all the necessary components for growth and survival

but lacking any pigments. The alternative diet was mulberry based, coming from

Mulberry Farms as a goliath hornworm diet. Both diets were refrigerated.

The hornworms arrived in both egg stage and in the first instar of growth, very

recently hatched, when they were at their smallest and just beginning to feed. All eggs

were placed in a constructed hatching chamber, which consisted of a Petri dish with a

wire cross-netting over the top, with gaps large enough for newly hatched worms to crawl

through. Three apportioned cubes, each roughly 5.0g, were placed on top of the wire

mesh, providing a food source for the worms when they hatched. Eggs were scattered

evenly throughout the bottom of the Petri dish, and the entire apparatus was placed in a

plastic container with small holes poked in the lid to allow airflow. This hatching

chamber was placed on an open lab bench at a room temperature of roughly 24C, with


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heat lamps used if temperature dropped below 21C. Worms hatched in approximately

three days.

All newly hatched worms were split evenly into two groups and placed on the two

separate diets. Worms were split into groups of three in small 1 oz. plastic cups, with

holes poked in the lid, and approximately 2.0g of their appointed diet. As worms grew

into the third instar (Fig. 1), weighing around 50 mg, they were placed in larger

individual 9 oz. cups, still continuing with the same 2.0g serving of appointed diet per

cup. Cups were purchased from Educational Science. Worms were also kept at a room

temperature of approximately 24C, with heat lamps employed if temperature dropped

below 21C.

Figure 1. Growth Cycle of Tobacco Hornworms

Hemolymph Extraction and Removal of Cell s

Once worms reached the fifth instar of growth, on average weighing at least

1.25g, they were large enough for bleeding in order to extract the hemolymph. Typically

worms were not bled until they reached 3.0g, but always before reaching a pre-pupation


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stage characterized by a decrease in dietary intake and a physical contraction in body

length.

Preparation for hemolymph extraction required synthesis of a bleeding buffer.

The composition of the buffer included 0.05M potassium phosphate, monobasic, as well

as 0.05M potassium phosphate, dibasic, dissolved in 100mL of deionized (DI) water to a

pH of 6.5. Before bleeding, 20mL of this 100mL solution was aliquoted into a disposable

50mL centrifuge tube along with 0.02M of glutathione, which inhibits melanization.

Worms selected for bleeding from both the artificial and plant-based diet were

placed into an ice tub for ten minutes, until all motion had ceased and the hornworm body

became soft, feeling spongy. While the worms sat on ice, one milliliter of the prepared

bleeding buffer was pipetted into each of two 15mL centrifuge tubes sitting in ice. After

the worms ten minutes on ice, each was held over a labeled tube designating its diet, and

the second pro-leg of the worm was clipped(Fig. 2), being careful not to cut too closely to

the body area as this may result in breaching into the gut of the organism. Keeping the

clipped pro-leg above the tube, each worm was gently squeezed and pulled taut in an

accordion-like fashion, extracting as much hemolymph as possible. This process was

continued until a total of 5mL of hemolymph had been extracted into each of the

respective tubes. Bleeding buffer was then added until the total buffer volume matched

the total hemolymph volume, giving a total volume of 10mL.

Once the hemolymph-buffer solution was prepared, centrifuge tubes were

balanced using bleeding buffer to prepare for a centrifuge spin. The tubes were then spun

for five minutes on the high setting (1380 xg) in a clinical centrifuge to pellet and

remove hemocytes. Following the spin, the supernatant was removed and moved to a new


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15mL centrifuge tube, with the volume being adjusted to 8.3mL if necessary, using

bleeding buffer.

Figure 2.Hornworm External AnatomyPhoto from http://entomology.unl.edu/k12/caterpillars/hornworm/hornwormpage.html

L ipoprotein I solation

Lipoproteins were isolated from the hemolymph of the hornworms following the

procedure of Pattnaik et al.(1979), with some modifications. The density of the

hemolymph solution in each 15mL centrifuge tube was adjusted to 1.21 g/mL using

potassium bromide. Seven mL of each solution was then placed into two polycarbonate

ultracentrifuge tubes, saving the remaining sample in the refrigerator for later gel

electrophoresis. The ultracentrifuge tubes were then balanced against one another to

within 10 milligrams.

The tubes were spun in a MLA-80 rotor at 65,000 rpm for 19 hours using slow

acceleration and deceleration. One mL was taken from the top layer of each tube and

pipetted into labeled 1.5mL microfuge tubes. The lipoproteins should have floated to this

top layer during the spin. Then the rest of the remaining volume was carefully discarded

except for the bottom 1 mL of each tube, which contained the non-lipoprotein fraction of

Second Pro-leg


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proteins, and which was pipetted into additional labeled 1.5mL microfuge

Dialysis of l ipoprotein and non-l ipoprotein fr actions

In order to remove any residual potassium bromide prior to lipoprotein analysis,

dialysis was performed using 14,000 molecular weight cutoff dialysis tubing. Four

seven-inch segments of dialysis tubing were soaked in DI water for at least ten minutes

until softened. During this time, three liters of 0.01M Tris buffer at pH 7.0 were prepared.

Once the tubing was ready, one end of each strip was clamped and the four 1 mL samples

from the ultracentrifuge spin were pipetted into them separately. The open end of the

tubing was then clamped shut. The samples were dialyzed overnight in the Tris buffer at

4C, then placed into new 1.5mL microfuge tubes and stored in the refrigerator.

Confi rmation of L ipoprotein I solation Using Gel E lectrophoresis

In order to estimate how much sample should be loaded during the gel

electrophoresis, a Bradford (1976) assay was performed using bovine serum albumin as

the standard. Ten L of each sample were diluted with 90L of DI water, giving a 100L

experimental sample solution in glass centrifuge tubes. Using bovine serum albumin, a

standard concentration curve was established to compare against my unknown samples.

Then 1.5mL of dye was sequentially added to each of the 100L of experimental

samples, with vortexing after the dye was added. After the tubes had sat for roughly five

minutes, their absorbances at 595 nm were measured, with the auto zero being a mixture

of 100L DI water with 1.5 mL of dye. The absorbencies of the standard samples were

then plotted and given a line of best-fit estimating protein amount. This was used to

calculate the protein concentrations in the experimental samples.


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SDS-PAGE gel electrophoresis was performed using the protocol of Laemmli

(1970). A solution containing 350L of sample buffer and 20L of beta-mercapto-

ethanol (-me) was mixed. Then in microfuge tubes, 35L of each sample and 37L of

the buffer solution were mixed. Six samples were used, consisting of the two top layers

and two bottom layers extracted earlier from the ultracentrifuge, as well as two samples

of the hemolymph mixture that were not subjected to ultracentrifugation.

The six sample mixtures were placed in boiling water for approximately ten

minutes. They were then removed from the water and touch-spun to move all the liquid to

the bottom. Using a Bio-Rad (Tris-HCl) ready-gel with a 4-20% gradient, 5L of the

sample mixtures were added to the wells, in addition to 5L of molecular marker, and the

gel was run to completion at 190 volts, until the dye marker reached the end of the gel.

The gel was then removed and placed in a DI water rinse before staining, following the

Bio-Rad, Bio-Safe Coomassie G-250 stain and rinse procedure (Bio-Rad Laboratories,

Instruction Manual Bio-Safe Coomasie Stain).

L ipid Extr action and UV-Spectroscopy Analysis

The entire procedure for lipoprotein isolation was repeated, collecting the top and

bottom layers from the ultracentrifugation again. In order to extract out anything

associated with the lipoproteins, the Folch et al. (1957) method for lipid extraction was

used, with some alterations. Two mL of chloroform and 1.0mL of methanol were added

to 0.8mL of each sample, giving a 3.8mL mixture. This was periodically vortexed and

allowed to sit for one hour. Then for phase separation 3.0mL of chloroform and 0.75 mL

of 0.2M potassium chloride were added to the mixture, which was vortexed and then

spun at 1380 xgfor ten minutes. The bottom phase from each sample was collected and


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saved in 15mL glass screw cap tubes. These final sample mixtures were then analyzed

using ultraviolet-visible spectroscopy (UV-Vis).

Results

Worms that arrived already hatched reached the fifth instar and an appropriate

bleeding weight in just under two weeks. Worms placed on different diets by this time

showed extreme difference in coloration, easily visible to the eye (Fig. 3). This same

color difference was visible in the extracted hemolymph (Fig. 4), giving support to the

color-change inducer being carried in the blood. Both these results gave clear support to a

dietary influence in the external appearance of the worms.

Figure 3.Diet-Induced Color Differences

Figure 4.Extracted Hemolymph

The ultracentrifuge spin resulted in hemolymph lipoproteins floating (along with a

yellow pigment) to the top of the tube. The tube of spun hemolymph from worms reared

on a plant-based diet showed much greater coloration associated with the lipoproteins

(Fig. 5). This color difference was even more dramatic when the top 1mL lipoprotein

The blue worm (on the right) was fed the

lab diet lacking plant material. The green

worm (left) was fed a mulberry-based

diet. Their corresponding hemolymph

can be seen to the ri ht.

Fed Mulberry- Fed artificial

based diet diet


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containing layers and bottom 1mL non-lipoprotein containing layers were removed for

analysis (Fig. 6).

Figure 5. Ultracentrifuge Separation Figure 6. Top and Bottom Layers

Gel electrophoresis of the top and bottom layers isolated in ultracentrifugation

confirmed the presence of the lipoproteins in the top layers. Larval tobacco hornworm

lipoproteins consist of two apolipophorinsapolipophorin-I and apolipophorin-IIwith

their respective molar masses being 245,000 and 78,000 Daltons. Analysis of the stained

gel showed that these two apolipophorins, and thus the lipoprotein, were present in the

top layer isolated via ultracentrifugation and absent in the bottom layer, which contained

all other proteins from the hemolymph (Fig. 7). This confirmed that the ultracentrifuge

spin had been successful and the lipoproteins had been effectively separated.

The left tube was the result of an

ultracentrifuge spin of the hemolymph

from those worms fed an artificial diet

(blue worms). The tube on the right

was from the worms fed a mulberry-

based diet (green worms).

The tubes above are the bottom and top layers

isolated from the ultracentrifuge spin. They are, in

order from left to right, the bottom layer from green

worm hemolymph, the bottom layer from blue

worm hemolymph, the top layer from blue worm

hemolymph, and the top layer from green worm

hemolymph.


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Figure 7. Gel Electrophoresis

The results of the Folch et al.(1957) method for lipid extraction in conjunction

with UV-spectroscopy gave a footprint for both the blue worm hemolymph and green

worm hemolymph. The hemolymph extracted from green worms had a spectrum peaking

roughly at 450nmwith intensities for relative absorbance very closely matching that of

-carotene. The hemolymph extracted from the blue worms had a very low relative

absorbance, suggesting a difference in levels of the pigment in the hemolymph in the two

worm groups, and strongly supporting dietary -carotene as the cause of the color

difference in the two worm groups (Fig. 8). Additionally, only the one footprint was

observed, supporting -carotene as being the sole dietary pigment factoring into the color

change.


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Figure 8. Comparison of UV-visible Spectra

Discussion

In the rearing and then bleeding of the hornworms, the extreme differences in skin

and hemolymph coloration between worms fed different diets were influential in

formulating the hypothesis as well as later methodology. This color change from blue to

bright green, with no difference in rearing other than diet, shows clear support of a

yellow-orange pigment being a dietary factor in the hornworm camouflage.

When the ultracentrifuge spin of the 1.21 g/mL hemolymph solution floated a top

layer that was yellow in color, it was consistent with my supposition of a hydrophobic

pigment that would be yellow. The 1.21 g/mL density adjustment was designed taking

into account the previous work of Pattnaik et al. (1979), and was meant to float

everything associated with lipoproteins to the top layer. The yellow coloration of that top


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layer suggested at the least that the pigment instigating color change had been partly

isolated.

Using gel electrophoresis to ascertain that the floated layer definitely contained

the lipoproteins, I observed clear bands at 245,000 and 78,000 Daltons corresponding

directly to the expected molar weights of the apolipoproteins associated with hornworm

lipophorins (Pattnaik, et al., 1979). This confirmed not only that the colored pigment was

hydrophobic, but also that it was using lipophorins as a means of transport in the

hemolymph, as hypothesized.

UV-visible spectroscopy was the final key in gaining intimate knowledge about

the composition of the yellow-orange pigment causing color change in the hornworms.

The Folch method (Folch, et al., 1957) extracted everything associated with the

lipophorins, including the pigment of interest, and when the hemolymph extractions were

compared, they gave very different spectra for the green mulberry fed worms and blue

worms fed lab diet. The spectra given by the green worms showed an absorbance peaking

at 450 nm and a shape and intensity closely matching that of -carotene, as given by

Misawa et al. (1993). Collectively, these data suggest that -carotene is absorbed from

the plant diet, much as it could be in humans consuming large concentrations of -

carotene (Arya et al., 2003), and then distributed via lipoproteins, resulting in green

pigmentation in plant-fed tobacco hornworms.

However, in order for -carotene to be undeniable as the source of the color

change, further analysis must be done before being certain that the pigment causing the

color change is strictly -carotene. The first step in confirming this is exposing worms to

the original laboratory diet supplemented with pure -carotene, at a concentration similar


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to that of the mulberry-based diet. If this evokes the same response, it greatly strengthens

the support of -carotene as the sole color-change inducing agent. Additionally, -

carotene is not entirely unique in its characteristic as a hydrophobic carotenoid, or even

necessarily in its absorbance peak around 450 nm. Because of this, some other

hydrophobic carotenoidsespecially leutine, which is the most similar carotenoid to -

carotene in its UV spectra and hydrophobic characteristicmust also be examined as

possible agents aiding in the color shift.


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Literature Cited

Arya V., J. Grzybowski , R.A. Schwartz. 2003. Carotenemia. Cutis. 2003;71(6):441-

442, 448.

Bradford, M.M. 1976. "A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding". Anal.

Biochem.1976;72: 248254.

Carroll, S.B. 2011. How great wings can look alike. Science. 2011;333:1100-1101

Folch, J., M. Lees, G.H. Sloane Stanley. 1957. A simple method for the isolation and

purification of total lipids from animal tissues.Journal of Biological Chemistry.

1957;226:497509.

Greene, E. 1989. "A diet-induced developmental polymorphism in a caterpillar." Science.

1989;243:643-646 (3 February 1989)

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of

bacteriophage T4.Nature.1970;227:680-685.

Misawa, N., S. Yamano, H. Linden, M.R. de Felipe, M. Lucas, H. Ikenaga, G. Sandmann.

1993. Functional expression of theErwinia uredovoracarotenoid biosynthesis

gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis

activity and resistance to the bleaching herbicide norflurazon. The Plant Journal.

1993;4(5):833840.

Pattnaik, N.M., E.C. Mundall, B.G. Trambusti, J.H. Law, F. J. Kzdy. 1979. Isolation

and characterization of a larval lipoprotein from the hemolymph ofManduca

sexta. Comparative Biochemistry and Physiology Part B: Comparative

Biochemistry. 1979;63(4):469-476.


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Reed, R.D., R. Papa, A. Martin, H.M. Hines, B.A. Counterman, C. Pardo-Diaz, C.D.

Jiggins, N.L. Chamberlain, M.R. Kronforst, R. Chen, G. Halder, H.F. Nijhout,

W.O. McMillan.2011. optixdrives the repeated convergent evolution of butterfly

wing pattern mimicry. Science. 2011;333:1137-1140.

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