Abstract
The squid Loligo opalescens deposit their egg cases in conspicuous masses on the
floor of Monterey Bay. These egg masses remain largely undisturbed by predation
during the 30 days until embryo hatching. A reproductive gland of the adult
female squid contains bacteria of predominantly one species which the female
includes in the egg case during spawning. These bacteria are of the genus
Shewenella, closely related to Shewenella putrefaciens, a putrefying marine
bacteria which is characterized by the production of extremely large quantities of
the polyamine putrescine. In this study, bacteria isolated from the squid gland and
egg sheath were examined by HPLC to determine the quantity of putrescine. The
bacteria contained amounts of putrescine comparable to S. putrefaciens. The
possible role of the putrescine was then examined in a chemotactic response assay
utilizing the starfish Pisaster ochraceus, a common predator at the depth of the egg
masses. S. putrefaciens, the squid bacteria and a solution of putrescine were found
be significant deterrents to the P. ochraceus while a control strain, Vibrio harvevi.
with low levels of putrescine showed no deterrence. These results suggest a role
for putrescine in the protection of the L. opalescens egg cases from predation.
Introduction
Every spring the squid Loligo opalescens come to Monterey Bay to mate and
subsequently lay egg cases in large conspicuous masses on sandy shelves of the bay.
The adult squid die soon after mating. The egg masses incubate for 30 days until
the squid hatch suffering little attrition by predators, bacteria or fungus. The fact
that these egg cases are not eaten suggests they have some means of chemical
defense, a widespread survival strategy in ocean environments.
The sheath of the egg case contain large quantities of bacteria (Biggs and
Epel, 1991). These bacteria are presumably secreted into the egg case by the female
squid, who harbors the bacteria within her accessory nidamental (AN) gland. The
bacteria in the AN gland are primarily one species of the genus Shewenella, a close
relative of the bacteria Shewenella putrefaciens (Melissa Kaufman, unpublished
data). S. putrefaciens is a putrefying marine bacteria that synthesizes large
quantities of putrescine ranging from 3 umol/g cell weight (Hamana and
Matsuzaki, 1992) to 251.4 nmol/mg cell protein - approximately 10 umol/g cell
been done to elucidate the role of the release of polyamines. Polyamines are the
breakdown products of proteins (Cohen, 1971) so it is possible that the release of
polyamines is the bacteria's method for metabolizing a protein rich diet.
Polyamine interactions with ion channels are fascinating and have also
been researched as potential therapeutic agents and as tools to help elucidate
aspects of cationic pumps. Some spider toxins have been shown to be polyamines
(Scott et al, 1993). Spidamine and joramine were isolated from the venom of
Nephila clavata and shown to block neuromuscular glutamate channels
irreversibly and reversibly, respectively (Chiba et al, 1995). Polyamines may either
interact directly with the channel by competitively binding to the channel or
indirectly by binding with aspects of the cell membrane to affect voltage gated
channels (Scott et al, 1993).
The purpose of this project was to determine if the bacteria isolated from
the squid egg cases and AN gland were producing polyamines and whether these
polyamines deterred predation. The first priority was to determine whether these
bacteria contained polyamines in concentrations comparable to S. putrefaciens. To
détermine putrescine concentration a method developed by Taibi and Schaivo
(1992) for high performance liquid chromatography (HPLC) of polyamines was
used.
The second part of the project was to determine whether the putrescine, if
present, deterred starfish predation. Starfish have chemotactic tube feet that are
accurate indicators of repellent substances and have been used in previous
predator deterrence studies (McClintock et al, 1994). The starfish Pisaster
ochrasceus was chosen for this study because it is a common predator at the depth
of the egg masses.
Materials and Methods:
HPLC Determination of Bacteria Putrescine Content:
HPLC instrumentation:
An SCO high performance liquid chromatography machine with a 50 ul
injection loop and a variable wave-length UV detector was used for the
identification and quantification of polyamines in the squid bacteria. Separations
were achieved on a ODS2 column (15 cm x 0.46 cm I.D.; 3 um particle size). All
polyamine standards were obtained from Sigma and HPLC grade reagents were
obtained from Baker.
Bacteria growth conditions and harvest procedure:
Four bacteria were chosen for polyamine content determination. The
positive control was Shewenella putrefaciens (ATCC 8071) previously reported to
synthesize large quantities of putrescine (Hamana and Matsuzaki, 1992) . The
negative control was Vibrio harveyi (ATCC 14126), a bacteria known to have low
levels of putrescine (Yamamoto et al, 1990). Two strains isolated from the squid
gland (referred to as squid strain 10) and squid egg case (squid strain 26) were also
chosen. These bacteria had earlier been identified as Shewenella spp. (Melissa
Kaufman, unpublished work). All bacteria strains were cultured from glycerol
stocks stored at -80°C.
The bacteria were grown in Marine Broth (Difco). An overnight culture was
used to inoculate the larger volume used for the experiment. Cell concentration
(mg cell wet weight/ ml broth) was determined by weighing the bacteria in two ml
of bacteria culture. To do this, Ependorf tubes were weighed and then two ml of
culture were added, spun down and the supernatant was removed (one ml at a
time). The tube with the pellet was then weighed.
Polyamine extrations were adapted from the procedure of Yamamoto (1990).
To harvest bacteria for the derivatization, 20 mls of a stationary phase bacteria
culture were spun at 5000 x g at 4° C. The pellet was resuspended in 5 mls of an
ice-cold solution of 10 mM magnesium chloride with a sodium chloride
concentration equal to that of the growth medium, 19.5 g/L in the case of Marine
Broth. Five mls of 4% perchloric acid was added to the solution. This was
sonicated for 3 minutes in 10 second bursts then incubated for one hour at 50° C.
The solution was then centrifuged at 12,000 x g for 15 min. The supernatant was
removed and put in sterile, polypropylene tubes.
Three ml were removed from each sample for immediate derivatization,
and the rest of the solution was frozen at -20°C. The three mis were neutralized
with 5 M potassium hydroxide to a pH of approximately 9. Two one mi aliquots
were taken from the neutralized solution and derivatized as described below
before HPLC analysis.
HPLC standards:
Standards were run to determine the relationship between peak height and
polyamine concentration. Standard curves for putrescine and cadaverine were
used since these were the polyamines expected to be in the Shewenella spp. For
each polyamine standard to be quantified, three concentrations were chosen.
derivatizations were done at each concentration, then the sample were run on the
HPLC. Each concentration was done in duplicate and each set of concentrations
was repeated three times. Solutions of 62.5 uM, 125 uM, and 250 uM were made
from a stock solution of 250 mM. The solution was then benzoyl derivatized as
described below (Taibi, et al, 1992).
Derivatization:
All steps of the derivatization took place in acid-washed glassware to reduce
contamination. One ml of each sample was placed in an acid-washed test tube
and 1 ml of 2M sodium hydroxide was added. Five ul of benzoyl chloride were
added then the solution was gently vortexed and then incubated at room
temperature. After 20 minutes, 2 mls of saturated sodium chloride were added to
the test tube. Then a glass Pasteur pipet was used to add 2 mls of diethyl ether.
Glass was used in transferring all ether to avoid plastic contamination. The
mixture was gently vortexed to extract the derivatized compounds to the ether
layer. After centrifugation in a clinical centrifuge (1500 rpm) at low speed for four
minutes, the ether phase was removed and put in a clean tube. Two mls of O.1M
sodium hydroxide was added to the ether phase and again gently vortexed and
centrifuged. After the ether phase was removed to a new tube, a few milligrams of
anhydrous sodium sulfate were added to remove any residual water. The tubes
were centrifuged again and one ml of ether was removed from each tube and dried
under a nitrogen stream.
Chromatographic separation:
The dried sample was resuspended in 500 ul of 62% methanol and put
through a 2 uM filter to eliminate large contaminants. Fifty ul was injected into
the Spherisorb ODS2 column and run at room temperature. Derivatized
polyamines were eluted in 62% methanol at a flow rate of 0.75 ml/min. and the
UV absorption was detected at 254 nm. The HPLC was run at room temperature.
The UV absorption was recorded with a program written by Chris Patton that
recorded the peak height from the voltmeter every 0.02 seconds.
Starfish Tube Foot Assay:
The starfish assay was adapted from the protocol of McClintock (1994). It
employs the use of the chemotactic tube foot response to various stimuli to
determine the deterrence of a substance
Methanol extraction:
10 desalt and concentrate samples for the starfish assay, bacteria cultures.
Marine Broth media (Difco), homogenized mussel tissue and putrescine in sterile
sea water at 10 mM were methanol extracted. Two ml of each sample were used
and 4.7 ml of methanol were added to bring the final volume to 70% methanol.
The solution was vortexed and then left to extract at room temperature for about 3
hours then centrifuged for 20 minutes in a clinical centrifuge at 3000 rpm. The
supernatant was removed to new tubes and air dried overnight. The amount of
supernatant removed was recorded and the extractions were resuspended in 1:30
of that volume and refrigerated at 4°C.
Behavioral Assay:
The tips of glass rods were thinly coated with a 1:1 mixture of inert silicone
grease (Plumber's Silicone Lubricant) and the experimental (or control) treatment.
The starfish were placed on their aboral side in a small glass tank with enough UV
sterilized sea water to cover them. The glass rod was then placed next to their arm
so that the movement of their arm or the tube feet would bring the feet in contact
with the glass rod. The tube feet towards the tip of the arm were used since these
are known to be more chemosensory (Sloan, 1980). When the tube feet touched
the glass rod they would retract (exceptions being the mechanical and grease
control). Time from retraction to re-emergence of the tube feet was measured and
recorded up to a period of 30 seconds. Each treatment was tested at least 10 times
and starfish were exchanged after about 10 trials to ensure that the starfish was
naive to the experiment and not stressed by the conditions,
Three controls were done to test the conditions of the treatment. The first
control was an uncoated glass rod that served as the mechanical control for the
movement of the glass rod. The second was a control for the silicone grease which
was simply a glass rod thinly coated with the grease. The third control was a
positive control of a methanol extraction of homogenized mussel tissue,
The results of the starfish assay were analyzed by an ANOVA followed by
Tukey's test.
Results
HPLC results:
The standards from the HPLC were analyzed to determine the relationship
between polyamine concentration and UV absorption as recorded in peak height
so that the bacterial polyamine content could then be quantified. A typical chart
recording from a putrescine standard run is shown in Figure 2. The peak height
varied linearly with concentration of putrescine and the average elution time for
putrescine was found to be between 7 and 8 minutes. The standard peak heights
from the HPLC were plotted against concentration and least squares linear
regression was used to calculate an equation describing the relationship (Figure 3).
Peak height was used rather than peak area because both have been determined to
be linear with concentration (Taibi and Schaivo, 1992) and peak height was
considerably easier to calculate. Cadaverine results are not shown since they are
not applicable to these bacteria. When the equation is rearranged to calculate
concentration from peak height, bo (y intercept) is 12.5 and by(slope) is 598.8. The r
value of .938 is highly significant (p20.01) so this relationship can be used to
accurately determine concentration from peak height.
The results were then analyzed for the four bacteria. Figures 4 shows a
typical run of the negative control V. harveyi. All the bacteria charts contained
two peaks at about 7 minutes flanking the place where putrescine elutes. These
two peaks are unidentified, but they are consistent and distinct from the putrescine
peak. Figure 5 is an overlay of two chart recordings - V. harveyi control and the
positive control, S. putrefaciens. From this overlay it is clear that the large peak at
about 7 minutes between the two smaller peaks in the control V. harvevi is
putrescine. Figure 6 is an overlay of the V. harveyi and the squid bacteria strain 10.
It also contains a putrescine peak that matches the putrescine peak of the S.
putrefaciens, demonstrating that the squid bacteria does contain large quantities of
putrescine. A table of putrescine concentrations found in each bacteria species is
shown in table I and a graph of the interpreted results is shown in figure 7. The
control strain V. harveyi did not contain any putrescine down to our detection
level of .57 umol/g cell wet weight. The S. putrefaciens were found to contain
7.67 umol/g cell wet weight of putrescine and the squid bacteria also contained
large amounts of putrescine; strain 10 contained 4.72 umol/g cell wet weight and
strain 26 contained 4.37 umol/g cell wet weight.
Starfish Tube Foot Assay Results:
The average retraction time and standard error of the starfish tube feet assay
is shown in table II. Figure 8 is a graph of these results. The data was log
transformed to reduce the variability and normalize the deviations. An ANOVA
was then done and the results were analyzed by Tukey's test. This test is fairly
conservative and takes into account the possible interactions of data. All bacteria
extracts and the putrescine were compared with the media to see if they had a
significantly greater deterrence than the media. All the putrescine containing
extracts (squid strains 10 and 26, S. putrefaciens, and putrescine at 10 mM) deterred
significantly more than the media whereas V. harveyi did not. The p values were
as follows: S. putrefaciens, p-.002; putrescine, p-.018; strain 10, p-.019; strain 26,
pe.001; V. harveyi, p-.202.
Discussion
The HPLC analysis indicated that the squid bacteria contain putrescine in
large quantities similar to the putrefying bacteria Shewenella putrefaciens. The
starfish tube foot assay demonstrated that the Shewenella spp. bacteria extracts and
à solution of putrescine were more noxious to starfish than media whereas
bacteria without putrescine (V. harveyi) was not. Putrescine produced by the
bacteria might be deterring predation on squid egg cases.
The HPLC proved to be an excellent and relatively simple way to analyze
polyamine content of bacteria. The negative control of V. harveyi and the positive
control of S. putrefaciens allowed exact location of the elution point of putrescine.
Overlaying chart recording from S. putrefaciens and the squid bacteria confirmed
that the peak in the squid bacteria was putrescine. Although the HPLC had
contaminants, these were recognizable and consistent in elution time.
The starfish assay proved to be a bit more problematic, although the controls
were still consistent with literature (McClintock, 1994). The positive control of
müssel showed an average retraction time of about 7 seconds which was similar to
McClintock's finding that the tube foot retraction for positive control was generally
less than 10 seconds. Äfter retraction, which McClintock attributed to the gross
chemical change in environment, the tube feet re-emerge and the arm moves
towards the mussel extract. More puzzling, though, is that McClintock found that
toxins he was testing as deterrents caused tube foot retractions that lasted 40-60
seconds whereas retractions in this experiment were generally observed to be
around 20 seconds suggesting that the bacteria, while still feeding deterrents, were
10
less noxious than other toxins released by marine organisms. The lower toxicity is
possibly explained by the role of these compounds in protecting squid eggs as
opposed to adult organisms which are less sensitive to toxic compounds. The
other explanation for the longer retraction time in the McClintock study was the
temperature at which the assay was conducted. McClintock used Antarctic species
and had an assay temperature of 0°C, whereas the assay in this study was
conducted at about 15°C where biological actions such as retraction and re-
emergence would occur faster.
The starfish assay is a less definite assay than the HPLC because tube foot
retraction is part of a larger behavioral pattern with inherent variability. The
retraction times recorded were highly variable which was compensated for by a
relatively large number of samples for each treatment. The starfish also tended to
become stressed by the testing environment and this lengthened retraction times.
1o compensate for this, the best effort was made to test only starfish that did not
appear to be stressed and to exchange starfish as soon as the one being tested began
to appear stressed.
The other uncertainty in this study was the environment in vivo. The
putrescine in the starfish assay was used at a concentration of 10 mM because of
previously published data on S. putrefaciens. Later calculations showed that the
concentration of putrescine in the squid bacterial extracts was closer to 1 mM
suggesting there may be alternate compounds produced by the bacteria that also
worked to deter the starfish in the starfish assay.
Not enough research has been done on the system in vivo to determine the
amount of putrescine in the natural starfish/egg case interaction. The bacteria
might secrete the putrescine directly into the ocean and allow the soluble
compound to have long-range effects, although this possibility seems unlikely in
terms of the energy cost of maintaining a large concentration of putrescine close to
the egg cases. It is also possible that the bacteria sequester the putrescine in the egg
sheath so when a predator penetrates the egg case, a concentrated solution of
putrescine is released. Another possibility is that the level of putrescine in nature
is less than the amount necessary to deter starfish and so though this compound is
à deterrent in the test system, it is not the cause of predator deterrence in the real
world. In considering these possibilities, it is impossible to say what the
concentration of putrescine is in nature and this remains a fascinating and
important area to research.
Assuming that putrescine is the reason for predator deterrence in the ocean.
we are then left with the question of how the putrescine deters predators. The
three categories of roles attributed to polyamines - DNA/RNA interactions,
bacterial products, and ion channel interactions - suggests three different
hypotheses. The first hypothesis is that putrescine deters predators by an
interaction with the DNA or RNA of the predator. The only detrimental effect in
the interactions between DNA and RNA and polyamines is cell apoptosis which
can occur if cells are exposed to large quantities of polyamines during certain times
in their cell cycle. This hypothesis does not fit with the current data because cell
apoptosis would take place on a much longer time scale than the retraction of a
starfish tube foot.
The rejection of this first category leaves us with two viable categories of
polyamine function from which to form hypothesis. One hypothesis involves
putrescine affects on the ion channels of either sensory or other neurons in the
predator. The other hypothesis suggests that the putrescine may be part of a
scheme to imitate rotting food and thus evade predation.
The hypothesis that the putrescine interacts directly or indirectly with ion
channels is quite interesting. It has been shown that putrescine concentrations of 1
mM increased Ca¬ currents in neuroblastoma cells (Scott et al, 1993). All senses
12
depend on ion channels so it is possible that by interfering with the channels the
putrescine is interfering with the chemosensory abilities of the predators. The
interaction with the ion channels could also involve other neurons and be toxic
rather than simply affecting the chemosensation of the predator. Further study
would need to determine the effects of putrescine on various neurons in potential
predators of squid egg cases.
A final hypothesis is that the putrescine functions to disguise the squid egg
case as rotting material. Putrescine is found in many rotting materials (Yamanaka
and Masumoto, 1989) and a recent study has found that the squid egg case bacteria
release also hydrogen sulfide (Melissa Kaufman, unpublished work) which is part
of the smell of rotting eggs and other materials and could work cooperatively with
the putrescine to together convince the predators that the egg cases were rotting
tissue. Most marine organisms that live in the squid-laying area are predators that
will not scavenge putrefying substances (im Watanabe, personal communication)
The only organisms that have been found near the egg cases are Capitella ovincola
(a species of worm), hermit crabs, and sometimes bat stars. The worms Capitella
are often found in hydrogen sulfide rich environments and in sewage and cannery
run-off. Capitella ovincola is only found in the squid egg cases and does not affect
the eggs unless it multiplies rapidly and disrupts the eggs (Morris et al, 1980)
This study has shown that the symbiotic bacteria found in squid egg cases
synthesize large quantities of putrescine, which might be used to deter predation
on the eggs during the incubation period before hatching. The chemical defense
of squid eggs is not yet fully understood but the information collected thus far
gives à tantalizing glimpse of possible mechanisms of protection. Marine
chemical defenses are complex, fascinating systems and scientific research into
their mechanisms and possible applications to medicine is gaining momentum.
13
This study is a step towards a better understanding of how symbiotic bacteria may
contribute to the safeguarding of squid embryos and ultimately how marine
chemical defenses operate.
Conclusion
This study has elucidated two important facts concerning the bacteria that
inhabit the egg cases of the squid Loligo opalescens. High pressure liquid
chromatography of the squid bacteria determined that these bacteria produce
substantial quantities of the polyamine putrescine. A starfish chemotactic tube
foot assay demonstrated that putrescine is noxious to starfish and is a predator
deterrent. These results suggest that the bacteria of the squid egg case prevents
predation by their release of putrescine.
Acknowledgments
Tespecially wish to thank Melissa Kaufman for the time and patience she
gave me this quarter. Thank you also for the excellent guidance on real science,
medical school, and, of course, my project. I also greatly thank David Epel for his
wonderful advice and his insistence that I see all the options without making
assumptions. Thanks also goes to all the people in the Epel Lab - Nancy, Beth,
Lisa, and Chris - for all their support and helpful suggestions.
14
Literature Cited
Biggs, James and Epel, David. (1991) Egg Capsule Sheath of Loligo opalescens
Berry: Structure and Association with Bacteria. Journal of Experimental Zoology,
259. 263-267
Brooks, W. H. (1995) Polyamine Involvement in the Cell Cycle, Apoptosis, and
Autoimmunity. Medical Hypotheses. 44. 331-338
Chiba, Tadashige; Akizawa, Toshifumi; Mastukawa, Motmi; Pan-hou, Hidemitsu;
Kawai, Nobufumi; and Yoshioka, Masanori. (1995)
Large-Scale Purification and
Fürther Characterization of Spidamine and Joramine from Venom of Spider.
Nephila clavata. Chemical Pharmacology bulletin. 43. 2177-2181,
Cohen, Seymour S. (1971) Introduction to the Polyamines. Englewood Cliffs, New
Jersey: Prentice-Hall, Inc.
Hamana, Koei and Matsuzaki, Shigeru. (1992) Polyamine Distribution Patterns
Serve as a Phenotypic Marker in the Chemotaxonomy of the Proteobacteria.
Canadian Journal of Microbiology. 39. 304-310.
McClintock, J. B; Baker, B. J; Slattery, M; Hamann, M; Kopitzke, R; and Heine, I.
(1994) Chemotactic Tube-Foot Responses of a Spongivorous Sea Star Perknaster
fuscus To Organic Extracts From Antarctic Sponges. Journal of Chemical Ecology.
20. 859-870.
Morris, Robert; Abbott, Donald; Haderlie, Eugene. (1980) Intertidal Invertebrates
of California. Stanford, CA: Stanford University Press.
Sato, T; Ökuzui, M; Fuii, T. (1995) Evaluation of Polyamine of Common Mackerel
During Storage as Indicators of Decomposition. Journal of the Food Hygienic
Society of Japan. 36. 743-747.
Scott, Roderick; Sutton, Kathy; and Dolphin, Annette. (1993) Interactions of
Polyamines with Neuronal lon Channels. Trends in Neurosciences 16. 153-160.
Sloan, N. A. (1980) The Arm Curling and Terminal Tube-Foot Responses of the
Asteroid Crossaster papposus (L.). Journal of Natural History. 14: 469-482.
Tabor, Cecilia and Tabor, Herbert. (1984) Polyamines. Annual Review of
Biochemistry. 53. 749-790.
Taibi, Gennaro and Schaivo, Maria Rita. (1992) Simple High-Performance Liquid
Chromatographic Assay for Polyamines and Their Monoacetyl Derivatives,
Journal of Chromatography. 614. 153-158
Yamamoto, Shigeo; Chowdhury, M. A. R; Kuroda, Masako; Nakano, Takao:
Koumoto, Yasuyoshi; and Shinoda, Sumio. (1990) Further Study on Polvamine
compositions in Vibrionaceae. Canadian Journal of Microbiology. 37. 148-153.
Yamanaka, Hideaki and Masumoto, Misuzu. (1989) Simultaneous Determination
of Polyamines in Red Meat Fishes by High Performance Liquid Chromatography
and Evaluation of Freshness. Tokyo University of Fisheries. 30.396-400.
Tables
Table 1 Table of the Concentrations of Putrescine in the Bacteria
This table shows the experimentally determined values of putrescine in the four
bacteria that were tested. The number recorded for the Vibrio harveyi is a result of
the equation derived from the least squares regression not passing exactly through
zero. It can be considered the sensitivity level of the HPLC - the Vibrio is sure to
contain an amount less than that number, but the exact amount is impossible to
measure with the current HPLC system.
Species of Bacteria
Average Putrescine Content
Standard Error
(mmol/g cell wet weight)
Vibrio harveyi
0.57
0.055
6. putrefaciens
7.67
0.16
Squid 10
4.72
0.54
quid 26
437
0.26
Table II Table of the Starfish Tube Foot Assay Results
This table shows the mean response time and the standard error for each
treatment. The time recorded is the time from the retraction of the tube foot until
the re-emergence. A number less than about 10 seconds is normal for the positive
control as it represents the response to a rapid change in sensory environment,
An ANOVA and Tukey's test were performed on the log transformation of this
data. The four treatments with putrescine (putrescine, S. putrefaciens, squid strain
10 and squid strain 26) showed significant difference from the media control, but
V. harveyi did not.
Treatment
Mean Retraction Time
Standard Error
(seconds)
(seconds)
mechanical
0.76
grease
mussel
7.2
0.66
media
1.3,
V. harveyi
11.2
0.39
Putrescine
1.9
. putrefaciens
16.3
0.96
Strain 10
14.9
1.31
Strain 26
18.7
27
Figure Legends
Figure 1:
The Structure of Common Polyamines
This diagram shows the chemical composition and structure of the
common polyamines: putrescine, cadaverine, spermine, and spermidine.
These
compounds are simple carbon chains with between two and four nitrogens. Each
nitrogen carries a positive charge under physiological conditions, making
polyamines cationic molecules. Many of their roles in cells are a results of their
cationic nature.
Figure 2: HPLC Chart Recording of Putrescine Standard
This HPLC chart recording shows a typical chart for the putrescine standard at 125
mM. The putrescine peak is at approximately 7 minutes. The peaks about 3
minutes after injection are salt peaks that consistently occurred in all injections,
The height of the putrescine peak is relative to the concentration of putrescine in
the injection and standards were used to quantify the relationship between peak
height and concentration.
Figure 3: Standard Curve of the HPLC Standards
The results from the HPLC standards are graphed here and a least squares linear
regression was applied to quantify the relationship between peak height and
concentration. The equation and r value are shown.
Figure 4: HPLC Chart Recording of Vibrio harveyi
This HPLC chart recording shows a typical recording of the bacteria V. harveyi, a
bacterial species that contains no detectable putrescine. The peaks in the beginning
around 3 minutes are salt contaminants and are consistent in all charts. The two
peaks close to 7 minutes are peaks that appear approximately the same size in
every bacterial chart recording regardless of putrescine concentration.
Figure 5: HPLC Chart Recording of Vibrio harveyi and Shewenella putrefaciens
This HPLC chart recording overlays a typical S. putrefaciens chart on the V.
harveyi chart shown in figure 4. Overlaying the charts we can see the putrescine
elutes between the two small peaks that appear in all bacterial charts. By
measuring the height of this peak we are able to quantify the amount of putrescine
that was in the injection and therefore the amount of putrescine in the bacteria.
Figure 6: HPLC Chart Recording of Vibrio harveyi and Squid Strain 10
This HPLC chart recording overlays a typical squid bacteria chart onto the V.
harveyi chart. Here it is evident that the squid bacteria contain putrescine in
amounts comparable to the S. putrefaciens.
18
Figure 7: Bar Graph of HPLC Results
This graph is a visual representation of the numbers from figure 7. The errors bars
show the standard error. As is evident in this bar graph, S. putrefaciens have a
large amount of putrescine and the squid bacteria, although they don’t have as
much putrescine, contain substantial amounts.
Figure 8: Bar Graph of Starfish Tube Foot Assay Results
This is a graph of the results tabled in table III. The error bars depict the standard
error of the data. From this graph it is clear that the putrescine and the bacteria
containing putrescine deterred the starfish more than media
Figure 1:
Structure of common polyamine
HN-(CH2)-NH,
HaN-(CH)5-NH
Putrescine
Cadaverine
(1,4-Diaminobuane)
(1,5-Diaminopentane)
HN-(CH2)-NH-(CH2)-NH,
Spermidine
HN-(CH)-NH-(CH2)-NH-(CH.)-NH,
Spermine
Figure 2
Typical Putrescine Standard
at 125 uM concentration
0.3

50.25
--------------------------------------------------------

0.2
E
O
----------------------------------------------------------------------------------


Q 0.15
Wn





0.1

------------------------------------------------------
Q 0.05
10
20
Time (minutes)
0
Figure 3:
0.6
0.5
0.4
0.3
0.2
0.1
0.0*
-0.1
Putrescine Peak Heights at Varying Concentrations
=.00167X -0.021
7=.938
A
250
300
100 150 200
Putrescine Concentration (uM)
50
Figure 4
HPLC of Vibrio harveyi
O.3
025
.................-----------
--------------------

------------
—------------------------------------------------
0.2

...-------------
2 0.15

W

.
0.1 +
h 0.05
-----------------------------
0
4
O
2
10 12 14
Time (minutes)
Figure 5
HPLC of V. harveyi
and S. putrefaciens
0.3

—0.25
—
------------------------------------
---------------------------------------------

0.2




-------
0.15

-
O




---------------------
—

----------
0.1
+ 0.05 +
—
—-----------------------------------------------------
0
10 12 14
O
2
Time (minutes)
Figure 6
HPLC of
V. harveyi and Strain 10
U.3

S
0.25

-------------------------------------
+
0.2

----------- —
----------

30.15
I




0.1

Q 0.05 —
10 12 14
O
2
Time (minutes
Vibrio
harveyi
Figure 7:
Quantity of Putrescine in Bacteria as
Determined by HPLC
Squid 10
putrefaciens
Species of Bacteria
Squid 26
25
-10

Figure 8:
Starfish Tube Foot Retraction Times
Treatment