|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online December 14, 2005
Journal of Experimental Biology 209, 78-88 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.01972
Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica
1 Department of Biology, Center for Behavioral Neuroscience, and Brains and
Behavior Program, Georgia State University, Atlanta, GA 30303 USA
2 Department of Biology, University of Washington, Seattle, WA 98195
USA
3 Department of Physiology, University of Utah School of Medicine, Salt Lake
City, UT 84108 USA
* Author for correspondence (e-mail: cderby{at}gsu.edu)
Accepted 7 November 2005
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: chemical defense, ink gland, opaline gland, gastropod, sea hare, escapin, L-amino acid oxidase
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
; reviewed
by Carefoot, 1987;
Faulkner, 1992;
Yamada and Kigoshi, 1997;
Johnson and Willows, 1999;
Rogers et al., 2000;
Ginsburg and Paul, 2001).
Active chemical defenses are released only upon predatory attack, and they
include the secretions from two separate glands. One of these is ink,
generally a bright purple fluid that is released from the ink gland. The other
is opaline, a whitish and extremely viscous substance produced by the opaline
gland. These two secretions are released into the mantle cavity - usually, but
not always, simultaneously - and pumped out of the siphon toward the attacker
(Walters and Erickson, 1986;
reviewed by Johnson and Willows,
1999).
The ink gland of Aplysia californica is composed of three vesicle
types: red-purple, amber and clear (Prince
et al., 1998). Most vesicles appear to have pores leading to the
ventral surface of the gland. Attack by a predator stimulates motor neurons to
activate muscles around individual vesicles, which squeeze the contents out of
the vesicles and into the mantle cavity
(Prince et al., 1998) where
they are then directed by the siphon and pumped out of the mantle
(Walters and Erickson,
1986).
The ink gland and its purple ink contain a diversity of molecules, only
some of which have been investigated and their biological activity identified.
The purple color of ink comes from red-algal derived pigments, the most
abundant being phycoerythrobilin, a chromophore of the photosynthetic pigment
phycoerythrin; the biological activity of these pigments is uncertain
(Chapman and Fox, 1969;
MacColl et al., 1990;
Prince et al., 1998;
Coelho et al., 1998). Ink also
contains high concentrations of amino acids and related nitrogenous compounds
that act against crustacean predators through phagomimicry and/or sensory
disruption (Kicklighter et al.,
2005). Ink of many species of sea hares has been shown to contain
a protein of 
60 kDa (Yamazaki et al.,
1986; Kamiya et al.,
1989; Yamazaki,
1993; Nistratova et al.,
1993; Melo et al.,
2000; Rajaganapathi and
Kathiresan, 2002; Petzelt et
al., 2002; Yang et al.,
2005). In A. californica, this protein is called escapin
(Yang et al., 2005), and it
has a homologue in the albumen gland, called aplysianin-A
(Kamiya et al., 1986;
Cummins et al., 2004). Escapin,
aplysianin-A and their homologues have been shown to have antibacterial,
cytolytic, haemagglutinating, and/or anti-heparin activities (e.g.
Kamiya et al., 1986;
Cummins et al., 2004;
Yang et al., 2005), but we
believe it unlikely that this is the primary function of these proteins.
Rather, we believe that escapin and its homologues function as antipredatory
chemical defenses, though we are not aware of any reports of such activity.
Preliminary studies show that high concentrations of escapin, over a period of
many hours, will lyse sea anemone's tentacle tissue and symbiotic
zooxanthellae (Johnson, 2002);
however, adverse effects at natural concentrations and time courses have not
yet been demonstrated in sea anemones or spiny lobsters (C.E.K., unpublished
data). Thus, at present, we can assess escapin's biological activity only
using antimicrobial assays. Escapin's major substrates are L-lysine
and L-arginine, though the products of escapin's oxidation of
L-lysine and L-arginine have different antimicrobial
activities. Escapin's oxidation of both L-lysine and
L-arginine leads to bacteriostasis, probably due to production of
hydrogen peroxide; but oxidation of only L-lysine and not
L-arginine leads to bactericidal effects through mechanisms other
than cytolysis (Yang et al.,
2005).
Like ink, opaline contains many compounds but for only a few is the
biological significance known (Kicklighter
et al., 2005). High concentrations of amino acids, particularly
taurine, help defend sea hares from predatory spiny lobsters through
phagomimicry and/or sensory disruption. Opaline also contains unidentified
chemicals that inhibit ingestion. Opaline is a source of lysine, a substrate
for escapin. Additionally, given its viscous consistency, opaline has been
suggested to provide substance to the secretion, to bind ink and opaline
together or to adhere to the predators (for reviews, see
Johnson and Willows, 1999;
Kicklighter et al., 2005).
Experimental evidence supports this idea: ink has phagomimetic properties, but
when ink is released in the absence of opaline, it diffuses quickly and does
not protect sea hares from attack by spiny lobsters
(Kicklighter et al., 2005)
Given that the production of defensive chemicals in the ink-opaline
secretion of sea hares requires an enzymatic reaction between escapin and its
amino acid substrates, it would be interesting to know the distribution of
these components within the different types of vesicles of the ink gland, in
the opaline gland and in other tissues. The ink gland is known to have the
chromophore phycoerythrobilin packaged in the red-purple vesicles
(Prince et al., 1998). Clear
vesicles, thought to be devoid of ink, are especially abundant in sea hares
raised exclusively on lettuce or green algae and thus lacking the pigments to
produce purple ink (Prince et al.,
1998). Prince et al.
(1998) argued from microscopic
data that the 60 kDa protein of ink may be packed in either the
red-purple vesicles or amber vesicles.
Our study examines the location of the 60 kDa protein escapin and its substrate amino acids in the ink and opaline glands of A. californica. We used western blots, immunocytochemistry using antibodies against escapin and amino acids, amino acid analysis and bioassays to show that escapin is present exclusively and in high concentration in the ink gland and in no other tissues or secretions, and within the ink gland it is packaged exclusively in the amber vesicles and not the red-purple vesicles. The amino acid substrates of escapin, particularly L-lysine, are packaged in high concentration in the opaline gland and not in the ink gland.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Collection of pure secretions
Pure ink and opaline secretions were collected from A. californica
that were cold-anaesthetized by chilling for 3-4 h at 4°C and then
injected with MgCl2 to prevent inking. Ink and opaline glands were
removed by dissection. Ink glands were gently squeezed, releasing the ink.
Opaline glands were gently blotted dry and centrifuged at 30 000
g for 30 min at 4°C to separate opaline secretion from
gland tissue. These secretions were frozen at -80°C until needed. In
addition, to examine separately the contents of the two types of secretory
vesicles of the ink gland (red-purple vesicles and amber vesicles;
Prince et al., 1998),
microdissection was used to isolate separate populations of these
vesicles.
Escapin purification
Following the procedure described in Yang et al.
(2005), a preparative grade
Hi-Load Superdex 200 16/60 column (Amersham Pharmacia Biotech, Piscataway, NJ,
USA) or an in-house prepared Sephacryl 300 HR 26/60 column was used for
initial size separation of ink constituents. Fractions identified by bacterial
assay to have activity (see next section) were concentrated using a Biomax 5K
NMWL membrane Ultrafree Centrifugal Filter Device (Millipore: Billerica, MA,
USA). Active fractions were further purified on a cation exchange Mono S HR
5/5 column (Amersham Pharmacia Biotech) to yield pure escapin, which was
stored at -80°C until used.
Anti-escapin antiserum
Anti-escapin serum was obtained by injecting rabbits with denatured
recombinant escapin purified from an E. coli expression system, as
described by Yang et al.
(2005).
Western blots for identifying tissues in which escapin is expressed
Thirteen tissues and secretions (albumen gland, albumen gland complex,
mucus, parapodia, foot, gill, digestive gland, central nervous system, opaline
gland, ink gland, ink, crop and foregut) from three field-caught adult A.
californica (50-90 g) were analyzed by western blotting for expression of
escapin. In addition, to examine differences in reproductive tissues between
juvenile and adult animals, the albumen gland complex from two juveniles (5 g)
was also analyzed (the albumen gland of juveniles is so small that is too
difficult to dissect away only the albumen gland, so the entire albumen gland
complex was removed and analyzed). Purified escapin was used as a standard. To
remove tissues, animals were chilled for 3 h at 4°C and injected with
isotonic MgCl2 to anaesthetize them. Tissues from different
individuals were combined, frozen, lyophilized and ground to a fine powder.
0.1-0.2 g of dry tissue was placed in 1.5 ml microcentrifuge tubes and saline
solution containing 1% NP-40, and 2 mmol l-1 phenylmethylsulphonyl
fluoride (PMSF) was added at a volume of 4 ml solution g-1 tissue.
Tubes were then vortexed and spun at 25 000 g for 30 min. The
supernatant was drawn off and analyzed for protein content by Bradford
analysis (Bradford, 1976),
except for ink, since the purple pigment in pure ink interferes with
spectrophotometric readings.
Total protein (25 µg) of each tissue and escapin were separated by
SDS-polyacrylamide gel (12%) electrophoresis (PAGE) in a minigel apparatus
(Mighty Small II, Amersham Pharmacia Biotech) according to the method of
Laemmli (1970).
Electrophoresed proteins were transferred to polyvinylidene difluoride
membranes (Applied Biosystems Inc., Foster City, CA USA) and analyzed by
western blotting using standard procedures
(Harlow and Lane, 1988).
Proteins were then incubated in anti-escapin antiserum (1:5000 dilution).
Alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad: Hercules, CA,
USA) was used as the secondary antibody (1:3333 dilution). Antibody binding to
the immunoblot was visualized by developing in nitroblue tetrazolium (NBT) and
5-bromo-4-chloro-3-indolyl phosphate (BCIP) in alkaline phosphatase buffer
(2.5 mmol l-1 Tris-HCl, 100 mmol l-1 NaCl, 5 mmol
l-1 MgCl2, pH 9.8).
Western blots for identifying types of ink gland vesicles in which escapin is expressed
SDS-PAGE assays were run for ink, amber vesicles and red-purple vesicles.
Ink was collected as described above and purified using the procedure of Yang
et al. (2005). Individual
amber and red-purple vesicles were microdissected, under a light microscope,
from dissected ink glands. Vesicles of each type were separately pooled into
small centrifugation tubes in approximately the same frequency as they are
present in the glands. Vesicles from approximately one animal were placed on
each lane of each gel.
Immunocytochemical localization of escapin and amino acids in the ink and opaline glands
Vibratome sections
Ink glands were dissected from 5 g A. californica as described
above, pinned out in a Sylgard-covered dissecting dish, and fixed for 15
h at room temperature by immersion in 4% paraformaldehyde in 0.1 mol
l-1 Soerensen phosphate buffer (SPB) with 15% sucrose added to
adjust osmolarity. After rinsing with SPB, pieces of about 3x3 mm were
cut out of uniformly thick parts of the gland that contained numerous small
purple vesicles. These pieces were embedded in gelatin as described in detail
previously (Schmidt et al.,
1992). The gelatin blocks were hardened with 4% paraformaldehyde
overnight at 4°C, rinsed briefly with SPB, and cut into 50-200 µm thick
cross sections on a vibrating microtome (VT 1000 S, Leica: Wetzlar, Germany).
The free-floating sections were incubated overnight at room temperature in the
polyclonal anti-escapin antibody (ID-4;
Yang et al., 2005) at a
dilution of 1:1000 in SPB containing 0.3% Triton X-100 (TSPB). Afterwards the
sections were rinsed four times for 30 min each in TSPB, then incubated for 4
h at room temperature in secondary antibody (goat-anti-rabbit CY3-labeled;
Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:400 in TSPB. After a
further four rinses in SPB for 30 min each, the sections were covered with a
solution of 1:1 glycerol:SPB containing 5% DABCO (diazabicyclol[2.2.2]octane)
to prevent bleaching and a coverslip was placed over the top. Control
sections, obtained from the same pieces of tissue, were treated in parallel,
only they were incubated in pre-immune serum (diluted 1:1000 in TSPB) from the
rabbit that had produced the ID-4 antibody instead of the antibody itself.
The sections were viewed and imaged using a fluorescence microscope (Axiophot 2, Zeiss: Jena, Germany) equipped with a high-resolution digital camera (DC 500, Leica: Wetzlar, Germany). Images of vesicles were captured under bright-field illumination with the appropriate filter settings for CY3 fluorescence. Both digital images were overlayed by a graphics program (PaintShop Pro 5, Jasc Software: Eden Prairie, MN, USA) before they were arranged into the final figures using Adobe Illustrator software (San Jose, CA, USA).
Plastic sections
Ink and opaline glands were dissected from A. californica as
described above and fixed in 2.5% paraformaldehyde/1% glutaraldehyde in 0.1
mol l-1 phosphate buffer (PB) overnight, or for up to several days
at 4°C. The tissue was dehydrated through a graded methanol series (75%,
85%, 95%, 2x 100%, 30 min each solution) and two changes of absolute
acetone (30 min each). The tissue was transferred to a 50:50 mixture of
acetone and Eponate plastic and incubated overnight at room temperature.
Following transfer through two 1-h changes of fresh Eponate, the tissue was
embedded in sectioning blocks and cured overnight at 65°C. Individual,
consecutive 100-500 nm thick sections, obtained using a Leica Ultracut T
microtome (Leica Inc., Bannockburn, IL, USA) and diamond knife (Delaware
Diamond Knifes, Wilmington, DE, USA), were transferred to water drops in
separate wells of Teflon-coated spot slides (Erie Scientific, Portsmouth, NH,
USA) and allowed to dry. Previously described post-embedding
immunocytochemical procedures were used
(Marc et al., 1990). Briefly,
sections were deplasticized with 25% sodium ethoxide (one part saturated
sodium hydroxide in three parts absolute ethanol, 7 min), rinsed in 100%
methanol (3x 2 min each), rinsed in ultra-pure water (5 min) and dried.
Individual sections were incubated in a humidified chamber overnight at room
temperature with one of the following primary polyclonal rabbit antibodies
(with final dilution): anti-taurine (1:4000 dilution),
anti-L-alanine (1:5000 dilution), anti-L-cysteine
(1:1000 dilution), anti-L-arginine (1:500 dilution),
anti-L-aspartate (1:1000 dilution), anti-L-lysine
(1:1000 dilution), anti-glutathione (1:100; Signature Immunologics Inc., Salt
Lake City, UT, USA), anti-escapin (1:2000; ID-4;
Yang et al., 2005) and
pre-immune serum (1:2000) diluted in 0.1 mol l-1 PB containing 1%
goat serum and 0.05% thiomerosal (pH 7.4). Dot blot analysis indicates that
the aspartate and taurine antibodies are at least 1000-fold less
cross-reactive to other structurally related antigens (Marc et al.,
1990,
1995). Similarly low
cross-reactivity is reported by the manufacturer for cysteine, arginine and
alanine. Elimination of primary antibody, incubation of the tissues with only
the secondary antibody and incubation in pre-immune serum resulted in no
immunoreactivity. Following a rinse with 0.1 mol l-1 PB, sections
were incubated in nanogold-conjugated goat anti-rabbit secondary antibody (1
nm, 1:50 dilution; Amersham Corp., Arlington Heights, IL, USA) for 1 h at room
temperature, rinsed with 0.1 mol l-1 PB for 1 h and silver
intensified for 3 min at 32°C using 0.14% silver nitrate in a hydroquinone
(43 mmol l-1)/citrate buffer (64 mmol l-1, citric acid;
141 mmol l-1 sodium citrate) solution
(Kalloniatis and Fletcher,
1993). Following silver intensification, the slides were covered
with a cover glass and Eponate plastic, then cured at 65°C overnight.
Eight-bit digital images were captured on a Zeiss Axioplan 2 microscope using
an Axiocam camera and Axiovision acquisition software (Carl Zeiss Microimaging
Inc, Thornwood, NY, USA).
Determination of concentrations of amino acids in ink glands
The concentrations of amino acids in ink and opaline have been published
already (Kicklighter et al.,
2005). In that study, ink was collected by dissecting out the ink
gland and squeezing the ink out of the gland into a dish. This method of
collection allowed for escapin to be mixed with its major substrates,
L-lysine and L-arginine
(Yang et al., 2005), for a
period of at least several minutes and up to 1 h, during which the
concentrations of these amino acids are reduced through oxidation by escapin
(Yang et al., 2005); in fact,
no L-lysine or L-arginine were detected in ink collected
in this way (Kicklighter et al.,
2005) Since we wanted to determine the levels of these amino acids
in ink before oxidation, in this study we collected ink glands in a different
way. We dissected out ink glands from three animals (total mass of ink
glands=3.45 g) and immediately froze whole glands in liquid nitrogen. The
tissue was ground on liquid nitrogen and placed in methanol containing 1%
trifluoroacetic acid (TFA) to inactivate escapin. After removal of methanol by
lyophilization, a residue was dissolved in water and extracted with chloroform
to remove compounds of low polarity, and then lyophilized again. For a
control, we dissected out ink glands from three animals (total mass of ink
glands=3.39 g) and collected ink by squeezing the glands. The collected ink
and the squeezed ink glands of these controls were incubated at room
temperature for 30 min to allow the amino acid oxidase activity of escapin to
occur. These glands were then placed in liquid nitrogen and treated as for the
whole glands. Concentrations of lysine, arginine and other free amino acids in
ink and opaline were determined using an ion exchange, post-column ninhydrin
detection system (Beckman Model 6300/7300 Amino Acid Analyzer, Scientific
Research Consortium, Inc.:
www.aminoacids.com).
Concentration of escapin in ink
We quantified the concentration of escapin in the ink of 200-300 g
field-caught A. californica and of the homologue of escapin,
dactylomelin-P, in the ink of 200-300 g field-caught A. dactylomela.
Sea hares were kept in the lab and fed Gracilaria ferox ad libitum
for at least 5 days before their use. Escapin levels were determined in ink
collected in two ways. The first was from ink released by sea hares that are
attacked by `predators', thus representing the concentration of ink as it is
expelled from the glands. Sea hares were removed from the aquarium by a
glove-wearing human predator, blotted dry with a paper towel to prevent
dilution of the ink when released and induced to release their ink by vigorous
handling. A 200 µl pipette was used to suck up ink from the mantle cavity
as it was released. Ink from each individual was placed in a separate 1.5 ml
microcentrifuge tube and kept on ice. We used only animals that released ink
and not opaline. For A. californica, we collected ink twice, with the
collections separated by 1 h. Our second method of collection of ink was from
ink glands dissected from anaesthetized animals, thus representing the
concentration of ink as it is stored in the glands. Six animals were
anaesthetized by chilling for 3-4 h at 4°C and then injected with isotonic
MgCl2. Ink was obtained from ink glands as described above. In
total, ink was collected from 24 animals, 12 from each of the two species and
12 for each of type of ink collection. All ink samples were frozen at
-20°C until their use.
|
Assay of antibacterial activity
Escherichia coli (MC4100, DH5a) was plated on Petri dishes
containing LB medium. Contents of separately microdissected amber vesicles and
red-purple vesicles (see the procedure for collecting these samples described
above), as well as opaline and ink, were tested for antimicrobial activity.
Five µl of the samples were placed on the bacterial lawn, and the dishes
were incubated overnight at 37°C or room temperature. If microbial growth
was inhibited, it was identifiable as a clear spot on the plate while the rest
of the plate had a bacterial lawn. Antibacterial activity was also determined
by co-incubation of bacteria with samples of the test substance, followed by
measurement of turbidity or counting of number of viable colonies.
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
).
|
60 kDa, these levels correspond
to 25-30 µmol l-1. However, the concentration of escapin in
naturally released ink of A. californica was only 0.028±0.007
mg ml-1. The escapin concentration in naturally released ink
collected from these same six individual A. californica 1 h after the
first collection of ink was similarly low (0.025±0.007 mg
ml-1).
Distribution of escapin within the ink gland
We used three techniques, SDS-PAGE, immunocytochemistry and a bioassay, to
demonstrate that the expression of escapin in the ink gland is limited to only
one of the two types of vesicles: the amber vesicles.
First, by microdissecting amber vesicles from red-purple vesicles and analysing them by SDS-PAGE, we found that amber vesicles but not red-purple vesicles contain the 60 kDa protein escapin (Fig. 3).
|
Second, immunocytochemistry with the anti-escapin antibody shows that escapin-like immunoreactivity is present in amber vesicles but not in red-purple vesicles. On cross sections through the ink gland that were at least 100 µm thick, numerous red-purple and amber vesicles were clearly identified by their color, visible with transmitted light illumination (Fig. 4A). In addition, some structures of the same size and shape but without colored contents could also be identified. Red-purple vesicles usually had a regular spherical to elliptical shape and ranged from 100 to 200 µm maximum diameter (mean diameter = 157 µm). Amber vesicles often had more irregular shapes and ranged from 150 to 260 µm in maximum diameter (mean diameter = 191 µm). The anti-escapin antibody selectively labeled the amber vesicles (Fig. 4B,C). Purple vesicles and the structures with clear contents were never labeled. Control sections incubated in pre-immune serum instead of antibody were devoid of labeling. The antibody labeling was intense only at the surface of the sections, and this lack of penetration prevented the labeling of vesicles that were not cut through in the sectioning process. We believe that this is the reason for the occurrence of a small percentage of unlabeled amber vesicles. Frequently, we observed small areas of intense labeling among the larger labeled areas that could unequivocally be attributed to the presence of amber vesicles. We interpret these structures as sections through the bottom or top of amber vesicles with the main part of the vesicle remaining in the adjacent section and thereby preventing the identification of the vesicle type based on its color.
|
|
Immunocytochemical localization of amino acids within the ink and opaline glands
Knowing the distribution of amino acids in the ink and opaline glands is
important, for two reasons. First, one might expect that the basic
L-amino acids lysine and arginine, which are especially good
substrates for the L-amino acid oxidase, escapin
(Yang et al., 2005), would be
packaged separately from escapin in order to prevent premature generation of
bioactive compounds. Thus, we might expect that lysine and arginine are
packaged in the ink gland in vesicles different from those of escapin or in
the opaline gland. Second, given that certain amino acids (especially taurine,
lysine, aspartate, alanine, glutamate and histidine) are in high concentration
in ink and/or opaline and have an antipredatory function via phagomimicry
(Kicklighter et al., 2005), it
would be interesting to know their vesicular distribution in these glands.
We found that vesicles in the ink gland differ with respect to their amino
acid content, and they can be divided into at least three classes.
Fig. 6 shows several examples
from three ink glands. One class of vesicles (labeled `e' in
Fig. 6) is intensely and
specifically labeled for escapin, having low or no immunoreactivity for amino
acids and including arginine, cysteine, aspartate, taurine, alanine (shown in
Fig. 6) or lysine, or the
tripeptide glutathione (not shown). These are probably amber vesicles, given
our results described above showing that escapin is present in amber vesicles.
The second class of vesicles (labeled `a' in
Fig. 6) is immunopositive for
amino acids but with low or no immunoreactivity to escapin. Taurine, aspartate
and alanine tend to have the highest levels of immunoreactivity and lower
immunoreactivity for arginine and lysine. This is interesting since these
immunoreactivity levels are closely correlated with the relatively high
abundance of taurine, aspartate and alanine in ink
(Kicklighter et al., 2005). We
hypothesize that this second class of vesicles are the red-purple vesicles. A
third class of vesicles (labeled `c' in
Fig. 6) has low or no
immunoreactivity to most amino acid antibodies and for escapin, and usually
similar degree of immunolabeling as preimmune controls. The immunolabeling in
these vesicles is usually more intense than in the surrounding non-vesicular
tissue. This suggests no or low levels of amino acids in these vesicles,
without specificity. We hypothesize that these are the clear vesicles, which
are devoid of ink, either because they are not filled or they have already
released their ink contents.
|
|
). However, since ink but not opaline contains escapin, it is
possible that our finding that ink contained no lysine or arginine is because
escapin had oxidized it all before chemical analysis. Thus, we examined the
concentrations of lysine, arginine and other amino acids in ink glands frozen
in liquid nitrogen immediately after dissection from an animal and before ink
was released from the gland. We found lysine and arginine at concentrations of
14 and 38 µmol l-1, respectively. The total amino acid
concentration in this sample was approximately 10% of that in our measurements
for released ink (Kicklighter et al.,
2005), but this might be the result of the necessary treatment of
the frozen sample with TFA, methanol and CHCl3. Thus, if we assume
that the loss is a general one for all amino acids, then we can correct for
this by scaling up the values in the frozen sample by a constant factor. When
we do this, the values for lysine and arginine are 150 and 418 µmol
l-1 respectively, which we assume to be the upper limits to their
possible concentrations in the ink gland. Thus, we believe that the
concentrations of amino acids in the ink gland before escapin is mixed with
them is 14-150 µmol l-1 for lysine and 38-418 µmol
l-1 for arginine. If we compare these with the concentrations of
lysine and arginine in opaline - 65, 190 and 340 µmol l-1
respectively, this leads to two conclusions: (1) lysine and arginine together
are a relatively minor component of the amino acid content of ink (51-568
µmol l-1 out of 29 620 µmol l-1); and (2) lysine
is at much higher levels (over 200 times greater) than arginine in the mixture
of ink and opaline. Given that only lysine and not arginine mediates the
bactericidal effect, and lysine is effective only at high (>1 mmol
l-1) concentrations, and given that lysine and arginine mediate the
bacteriostatic effect only at doses >1 mmol l-1, we conclude
that it is the lysine in opaline that is the primary substrate for escapin's
antimicrobial effect. |
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The protein escapin has a highly restricted distribution across the tissues
of the sea hare A. californica. The only tissue containing escapin is
the ink gland; no other tissue or secretion contains escapin, not even those
previously shown to have defensive chemicals, such as the opaline gland
(Kicklighter et al., 2005) and
skin (Kinnel et al., 1979;
Fig. 1). In addition, escapin
is absent from the albumen gland of both sexually mature and immature animals.
This is especially interesting because it contains aplysianin-A, a protein
with 60% identity to escapin (Cummins et
al., 2004) and with antimicrobial function
(Kamiya et al., 1986).
Within the ink gland, escapin is present only in the amber vesicles and not
in the red-purple vesicles, as supported by four lines of evidence. First,
SDS-PAGE identified a 60 kDaprotein that had the molecular mass of escapin
(Yang et al., 2005), only in
amber vesicles and not in red-purple vesicles
(Fig. 2). Second, using an
antiserum generated against escapin, immunoreactivity occurred only in the ink
gland's amber vesicles (Fig.
3). Third, an antimicrobial assay showed that amber vesicles but
not red-purple vesicles have antimicrobial activity
(Fig. 4), which is a property
of escapin. Fourth, purified escapin is amber in color, because of the
presence of FAD (flavin-adenine dinucleotide;
Yang et al., 2005). Taken
together, these observations lead us to conclude that escapin is present in
the amber vesicles and not the red-purple vesicles.
The ink gland has high concentrations of escapin (in A.
californica) or dactylomelin-P (in A. dactylomela). When
squeezed from the ink gland of A. californica or A.
dactylomela, the concentrations of these proteins are 1.48 and 1.92 mg
ml-1, respectively, or 25-30 µmol l-1. The
concentration of this protein in ink as it is released from an attacked sea
hare is about the same as in the ink gland of A. dactylomela (1.58
vs 1.92 mg ml-1) but not in A. californica (0.028
vs 1.48 mg ml-1). The explanation for this difference in
two closely related species is not known, but it is possible that in A.
californica there is a disproportionately low release of escapin compared
with other fluids in the ink gland. In both Aplysia species, multiple
bouts of inking are typical (Nolen and
Johnson, 2001), so the ink store of each animal is not depleted in
the conditions of our experiments. The concentrations of this protein in
released ink are sufficiently high to have protective effects. The
concentration of released protein from both A. californica and A.
dactylomela is much higher than the minimum concentration of escapin that
is inhibitory against several bacterial species, which is 0.0003-0.005 mg
ml-1 (Yang et al.,
2005). Thus, the released protein represents a highly potent
effector at its natural concentrations.
Though it is known that the ink of A. californica is an effective
anti-feedant against the large predatory sea anemone Anthopleura
xanthogrammica (Nolen et al.,
1995), what role escapin may play has not been established.
Preliminary data suggest that escapin is capable of causing direct damage to
sea anemone tentacles and their symbiotic zooxanthellae
(Johnson, 2002); however, the
sea anemone behavioral response to ink occurs in a matter of seconds and yet
it takes hours for lysis by escapin at natural concentrations (C.E.K.,
unpublished data). Nonetheless, it is unlikely that natural selection would
maintain the production of such large quantities of a bioactive protein to be
secreted in a defensive mixture that is only released under predatory attack
if it did not serve an antipredatory role. It may have evolved from
antimicrobial proteins, as its activity suggests, but it is unlikely that sea
hares shower their predators with antibiotics after an attack in order to keep
the predators microbe free. Additionally, if sea hares were releasing escapin
to cover their own bodies with antibiotics, then it should be released at more
regular intervals. Along with the sea anemones, several species of sea stars,
crabs and fish are known to prey on sea hares
(Sarver, 1978;
Willan, 1979;
Johnson and Willows, 1999);
thus, it is plausible that escapin may deter one or more of these predators
through mechanisms, as yet, unknown.
The substrates for escapin are distributed in a way to enhance their
effectiveness. Lysine and arginine are the main substrates for escapin's
oxidase activity (Yang et al.,
2005). However, lysine probably plays a more important role in
this hypothesized defense than arginine, for two reasons. First, lysine is
both bacteriostatic and bactericidal, whereas, arginine is primarily only
bacteriostatic (Yang et al.,
2005). Secondly, lysine is present in the defensive secretion at a
much higher concentration than arginine (our results;
Kicklighter et al., 2005).
Significantly, lysine is packaged separately from escapin: lysine is in high
concentration in opaline but not ink, and escapin is only in ink. Thus, the
bioactive chemicals in this pathway are only produced when ink and opaline are
mixed. This occurs when the sea hare is attacked by a predator, after which
the ink gland and opaline gland often co-release their contents into the
mantle cavity, and then the ink-opaline mixture is squirted out via
the siphon and directed toward the site of attack. This separate packaging of
the enzyme from its substrates would aid in preventing autotoxicity within the
animal before the vesicular release of the ink. Whether other
L-amino acid oxidase toxins, such as those in venomous snakes
(Torii et al., 2000;
MacHeroux et al., 2001;
Du and Clemetson, 2002;
Lu et al., 2002;
Stábeli et al., 2004),
are packaged separately from substrates is, to our knowledge, not known.
The reason for a diversity of vesicle types in the opaline gland,
specifically why one class of vesicles is relatively high in taurine and
another class is relatively low in taurine
(Fig. 7), is not known. The
role of taurine in one type of chemical defense, phagomimicry, against
predatory spiny lobsters is known
(Kicklighter et al., 2005).
The opaline gland is also known to have feeding deterrents
(Kicklighter et al., 2005) and
toxins (Flury, 1915), but
whether they are distributed in different types of vesicles in this gland is
not known at present.
In our laboratory studies of attacks by crustacean predators on sea hares,
ink and opaline are typically both released more or less simultaneously
(Kicklighter et al., 2005).
Such co-release would obviously benefit the escapin-based defensive pathway by
allowing mixing of the enzyme and its substrate. However, ink and opaline
contain other antipredatory compounds that do not require enzyme activity or
mixing of the glandular secretions, so co-release of ink and opaline may not
always be functionally necessary. In fact, the neural pathways controlling the
release of ink and opaline are different, with each gland being innervated by
different sets of motor neurons located in different ganglia, even though some
of the sensory pathways to the motor neurons may be shared
(Tritt and Byrne, 1980;
Walters and Erickson, 1986).
We have seen some instances in which opaline is released at least several
seconds before ink (see supplemental figure in
Kicklighter et al., 2005; C.
E. Kicklighter, unpublished data), while others have noted that ink appears to
have a lower threshold to release than opaline
(Kandel, 1979;
Byrne, 1980;
Tritt and Byrne, 1980;
Walters and Erickson, 1986;
Nolen et al., 1995;
Nolen and Johnson, 2001). It
will be interesting to examine whether sea hares can regulate the thresholds
at which the two glands are released depending on the identity of the
attacking predator and thus which chemical defenses are more likely to be
needed.
The ink of most sea hares has its distinctive red-purple color because of
chromophores such as phycoerythrobilin. These pigments, which are derived from
consumption of red algae, are also differentially packed only in the
red-purple vesicles of the ink gland
(Prince et al., 1998). These
chromophores have not been definitively shown to have a protective function,
either antipredatory or antimicrobial. It is possible that they function
visually, as a chemical `smoke-screen' (see
Carefoot, 1987;
Johnson and Willows, 1999) or
as a visual mimic of sea hares during phagomimetic defense
(Kicklighter et al., 2005).
They have been speculated to be involved in chemical defenses, but the support
for this is incomplete (Carlson and Nolen,
1997). Why they are packaged in ink gland vesicles separately from
escapin is not known.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Byrne, J. H. (1980). Neural circuit for inking
behavior in Aplysia californica. J.
Neurophysiol. 43,896
-911.
Carefoot, T. H. (1987). Aplysia: its biology and ecology. J. Exp. Biol. 24,167 -284.
Carlson, B. and Nolen, T. G. (1997). The effect of Aplysia's defensive chemical ink on the dactyl chemoreceptors of predatory crabs (Cancer antennarius). Soc. Neurosci. Abstr. 23,188 .
Chapman, D. J. and Fox, D. L. (1969). Bile pigment metabolism in the seahare Aplysia. J. Exp. Mar. Biol. Ecol. 4,71 -78.[Medline]
Coelho, L., Prince, J. and Nolen, T. G. (1998).
Processing of defensive pigment in Aplysia californica: acquisition,
modification and mobilization of the red algal pigment r-phycoerythrin by the
digestive gland. J. Exp. Biol.
201,425
-438.
Cummins, S. F., Nichols, A. E., Amare, A., Hummon, A. B.,
Sweedler, J. V. and Nagle, G. T. (2004). Characterization of
Aplysia enticin and temptin, two novel water-borne protein pheromones
that act in concert with attractin to stimulate mate attraction. J.
Biol. Chem. 279,25614
-25622.
Du, X. Y. and Clemetson, K. J. (2002). Snake venom L-amino acid oxidases. Toxicon 40,659 -665.[Medline]
Faulkner, D. J. (1992). Chemical defenses in marine mollusks. In Ecological Roles of Marine Natural Products (ed. V. J. Paul), pp. 119-163. Ithaca (NY): Comstock.
Flury, F. (1915). Über das Aplysiengift. Arch. Exp. Pathol. Pharmakol. 79,250 -263.[CrossRef]
Ginsburg, D. W. and Paul, V. J. (2001). Chemical defenses in the sea hare Aplysia parvula: importance of diet and sequestration of algal secondary metabolites. Mar. Ecol. Prog. Series 215,261 -274.
Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Johnson, P. M. (2002). Multi-component chemical defense in seahares (Gastropoda: Opisthobranchia): antipredator compounds act as both honest and deceptive signals to multiple predator species. PhD dissertation, University of Washington, USA.
Johnson, P. M. and Willows, A. O. D. (1999). Defense in sea hares (Gastropoda, Opisthobranchia, Anaspidea): multiple layers of protection from egg to adult. Mar. Fresh. Behav. Physiol. 32,147 -180.
Kalloniatis, M. and Fletcher, E. L. (1993). Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina. J. Comp. Neurol. 336,174 -193.[CrossRef][Medline]
Kamiya, H., Muramoto, K. and Yamazaki, M. (1986). Aplysianin-A, an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare, Aplysia kurodai. Experientia 42,1065 -1067.[CrossRef][Medline]
Kamiya, H., Muramoto, K., Goto, R., Sakai, M., Endo, Y. and Yamazaki, M. (1989). Purification and characterization of an antibacterial and antineoplastic protein secretion of a sea hare, Aplysia juliana. Toxicon 27,1269 -1277.[Medline]
Kandel, E. R. (1979). Behavioral Biology of Aplysia. San Francisco, CA, USA: Freeman and Co.
Kicklighter, C. E., Shabani, S., Johnson, P. M. and Derby, C. D. (2005). Sea hares use novel antipredatory chemical defenses. Curr. Biol. 15,549 -554.[CrossRef][Medline]
Kinnel, R. B., Dieter, R. K., Meinwald, J., Van Engen, D. C. J.,
Eisner, T., Stallard, M. O. and Fenical, W. H. (1979).
Brasilenyne and cis-dihydrorhodophytin: antifeedant medium-ring
haloethers from a sea hare (Aplysia brasiliana). Proc.
Natl. Acad. Sci. USA 76,3576
-3579.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[CrossRef][Medline]
Lu, Q. M., Wei, Q., Jin, Y., Wei, J. F., Wang, W. Y. and Xiong, Y. L. (2002). L-amino acid oxidase from Trimeresurus jerdonii snake venom: purification, characterization, platelet aggregation-inducing and antibacterial effects. J. Nat. Toxins 11,345 -352.[Medline]
MacColl, R., Galivan, J., Berns, D. S., Zimec, Z., Guard-Friar, D. and Wagoner, D. (1990). The chromophore and polypeptide composition of Aplysia ink. Biol. Bull. 179,326 -331.[Abstract]
MacHeroux, P., Seth, O., Bollschweiler, C., Schwarz, M., Kurfurst, M., Au, L. C. and Ghisla, S. (2001). L-Amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme. Eur. J. Biochem. 268,1679 -1686.[Medline]
Marc, R. E., Wei-Ley, S. L., Kalloniatis, M., Raiguel, S. F. and Van Haesendonck, E. (1990). Patterns of glutamate immunoreactivity in the goldfish retina. J. Neurosci. 10,4006 -4034.[Abstract]
Marc, R. E., Murry, R. F. and Basinger, S. F. (1995). Pattern recognition of amino acid signatures in retinal neurons. J. Neurosci. 15,5106 -5129.[Abstract]
Melo, V. M. M., Duarte, A. B. G., Carvalho, A. F. F. U., Sebra, E. A. and Vasconcelos, I. M. (2000). Purification of a novel antibacterial and haemagglutinating protein from the purple gland of the sea hare, Aplysia dactylomela Rang, 1828. Toxicon 38,1415 -1427.[Medline]
Nistratova, S. N., Mitskevich, I. N., Mirabet-Regalado, M. E. and Josef, N. (1993). Inhibitory effect of the purple fluid produced by a mollusk Aplysia punctata on growth of gram-negative marine bacteria. Microbiology 61,630 -632.
Nolen, T. G. and Johnson, P. M. (2001). Defensive inking in Aplysia spp.: multiple episodes of ink secretion and the adaptive use of a limited chemical resource. J. Exp. Biol. 204,1257 -1268.[Abstract]
Nolen, T. G., Johnson, P. M., Kicklighter, C. E. and Capo, T. (1995). Ink secretion by the marine snail Aplysia californica enhances its ability to escape from a natural predator. J. Comp. Physiol. A 176,239 -254.
Petzelt, C., Joswig, G., Stammer, H. and Werner, D. (2002). Cytotoxic cyplasin of the sea hare, Aplysia punctata, cDNA cloning, and expression of bioactive recombinants in insect cells. Neoplasia 4, 49-59.[CrossRef][Medline]
Prince, J., Nolen, T. G. and Coelho, L. (1998). Defensive ink pigment processing and secretion in Aplysia californica: concentration and storage of phycoerythrobilin in the ink gland. J. Exp. Biol. 201,1595 -1613.[Abstract]
Rajaganapathi, J. and Kathiresan, K. (2002). Heparinase in purple fluid of the sea hare, Bursatella leachii. Curr. Sci. 82,264 -266.
Rogers, C. N., de Nys, R., Charlton, T. S. and Steinberg, P. D. (2000). Dynamics of algal secondary metabolites in two species of sea hare. J. Chem. Ecol. 26,721 -744.[CrossRef]
Sarver, D. J. (1978). The ecology and energetics of Aplysia juliana (Quoy and Qaimard, 1832). PhD dissertation, University of Hawaii, USA.
Schmidt, M, Van Ekeris, L. and Ache, B. W. (1992). Antennular projections to the midbrain of the spiny lobster. I. Sensory innervation of the lateral and medial antennular neuropils. J. Comp. Neurol. 318,277 -290.[CrossRef][Medline]
Stábeli, R. G., Marcussi, S., Carlos, G. B., Pietro, R. C. L. R., Selistre-de- Araújo, H. S., Giglio, J. R., Oliveira, E. B. and Soares, A. M. (2004). Platelet aggregation and antibacterial effects of an L-amino acid oxidase purified from Bothrops alternatus snake venom. Bioorgan. Med. Chem. 12,2881 -2886.[CrossRef][Medline]
Torii, S., Yamane, K., Mashima, T., Haga, N., Yamamoto, K., Fox. J. W., Naito, M. and Tsuruo, T. (2000). Molecular cloning and functional analysis of apoxin I, a snake venom-derived apoptosis-inducing factor with L-amino acid oxidase activity. Biochemistry 39,3197 -3205.[CrossRef][Medline]
Tritt, S. H. and Byrne, J. H. (1980). Motor
controls of opaline secretion in Aplysia californica. J.
Neurophysiol. 43,581
-594.
Walters, E. T. and Erickson, M. T. (1986). Directional control and the functional organization of defensive responses in Aplysia. J. Comp. Physiol. A 159,339 -351.[CrossRef][Medline]
Willan, R. C. (1979). The ecology of two New Zealand opisthobranch mollusk. PhD dissertation, University of Auckland, New Zealand.
Yamada, K. and Kigoshi, H. (1997). Bioactive compounds from the sea hares of two genera: Aplysia and Dolabella. Bull. Chem Soc. Jpn. 70,1479 -1489.[CrossRef]
Yamazaki, M. (1993). Antitumor and antimicrobial glycoproteins from sea hares. Comp. Biochem. Physiol. 105C,141 -146.[Medline]
Yamazaki, M., Kimura, K., Kisugi, J. and Hisao, K. (1986). Purification of a cytolytic factor from purple fluid of a sea hare. FEBS Lett. 198, 25-28.[CrossRef]
Yang, H., Johnson, P. M., Ko, K.-C., Kamio, M., Germann, M. W.,
Derby, C. D. and Tai, P. C. (2005). Cloning, characterization
and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid
oxidase from ink of the sea hare Aplysia californica. J.
Exp. Biol. 208,3609
-3622.
This article has been cited by other articles:
|
|
 |
|
  K.-C. Ko, B. Wang, P. C. Tai, and C. D. Derby Identification of Potent Bactericidal Compounds Produced by Escapin, an L-Amino Acid Oxidase in the Ink of the Sea Hare Aplysia californica Antimicrob. Agents Chemother., December 1, 2008; 52(12): 4455 - 4462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mai-Prochnow, P. Lucas-Elio, S. Egan, T. Thomas, J. S. Webb, A. Sanchez-Amat, and S. Kjelleberg Hydrogen Peroxide Linked to Lysine Oxidase Activity Facilitates Biofilm Differentiation and Dispersal in Several Gram-Negative Bacteria J. Bacteriol., August 1, 2008; 190(15): 5493 - 5501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. Derby Escape by Inking and Secreting: Marine Molluscs Avoid Predators Through a Rich Array of Chemicals and Mechanisms Biol. Bull., December 1, 2007; 213(3): 274 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Prince OPALINE GLAND ULTRASTRUCTURE IN APLYSIA CALIFORNICA (GASTROPODA: ANASPIDEA) J. Mollus. Stud., July 11, 2007; (2007) eym016v1. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||
