Abstract
Like other members of its genus, Conus californicus has a highly evolved venom
apparatus with which it injects paralyzing venom into its prey through a modified radula
tooth. Although much research is being conducted on the venom itself, relatively little is
known about the biology behind the snail’s ability to inject the venom. The venom duct,
radula sac and teeth were characterized in terms of anatomy, physiology and chemistry.
The passageway between the venom duct and proboscis is a complex branching structure
lined by a unique secretory cell type. Evidence from a combination of all methods used
suggests that there are similar active peptides in the teeth as in the duct, leading to the
possibility that the teeth are pre-loaded with venom.
Introduction
Snails of the genus Conus have long been of interest, both for their unusual shells
and unique predatory hunting strategies, which employ a battery of peptide neurotoxins
to paralyze their prey. A typical gastropod has a row of chitinous radular teeth that it
uses for scraping food. The cones have modified their teeth into a combination of
miniature harpoon and hypodermic needle that is used one at a time to inject venom into
the prey. Each species has a unique tooth structure with varying size, shape, and number
of barbs, and the differences in the teeth constitutes a basis for phylogenic classifications
of Conus (Kohn et al., 1999
Although there are several hundred tropical species, Conus californicus is the
only temperate species to inhabit the ocean off of the Pacific coast from the Farralone
Islands, California to Cabo San Lucas, Baja California (Hanna and Strong, 1949). C.
californicus, like all Conus species, is predatory, but it is unusually omnivorous
compared to many typical tropical species, many of which have highly specific feeding
habits (Kohn, 1966; Nybakken, 1970).
In all cones, the venom apparatus consists of a muscular bulb connected to a long,
highly convoluted venom duct (Fig.1). The teeth are made in the radula sac, which
consists of a short and long arm. The radula sac enters the proboscis slightly anterior
from the venom duct, i.e. towards the tip of the proboscis (Fig.1C). The teeth are formed
in the long arm of the radula sac, and mature teeth are thought to be stored in the short
arm until use (Marsh, 1977). The teeth of C. californicus are about 1 mm long with a
basal and an apical opening and five barbs (Kohn et al. 1999). When resting the cone
holds its proboscis inside of its mouth, but when prey is sensed, through chemoreceptors
in its osphradium, (Spengler and Kohn; 1995), it extends the proboscis and begins to
search for food. When the cone finds its prey, it will sting forcefully by inserting the
tooth and injecting the venom.
The anatomy of the venom apparatus has been characterized for several species.
and there is a general model for how the venom is made and injected through the tooth
(Endean and Duchemin, 1967; Songdahl, 1973; Hinegardner, 1958, Freeman et al.,
1974). Specific peptide components of the venom are thought to be made as pro-peptides
in the posterior region of the venom duct near the muscular bulb and then undergo
posttranslational modifications to form mature venom peptides as they move down the
duct (Bingham et al. 1996). It has been hypothesized that the muscular bulb provides the
force to move the venom through the duct into the proboscis (Olivera, 1997). This idea
has been questioned, however, because the muscular bulb does not appear to be capable
of providing sufficient pressure to inject the venom (Songdahl, 1973). It is more likely
that the proboscis itself provides the force to inject the venom (Kohn, et al. 1999). The
hollow tooth is presumably held at the tip of the proboscis by a sphincter muscle, and in
some species it has been reported that the tip of the tooth can be seen before the sting
(Kohn 1956). In some cases the cone continues to hold onto the tooth after the injection
and then uses it to pull the prey into its mouth.
The main contemporary thrust of Conus research is focused on purification and
characterization of specific peptides in the venom. These efforts have primarily
employed tropical Conus species, because they are large and their venoms, especially
those of the piscivorous species, are lethal to mammals including humans (Kohn,1998).
Several Conus peptides are of commercial pharmaceutical interest at the present time
(Well, 1998)
This study provides a description of the general anatomy of the venom apparatus
in C. californicus with the specific aim of elucidating the anatomical pathways by which
the venom and teeth enter into the proboscis. The study also examined the composition
of both the venom duct and the teeth using anatomical, physiological and chemical
means.
Material and Methods
Histology
Specimens of C. californicus were collected from Monterey Bay. Some were
kept in an aquarium with water at ambient temperature, and others were held at room
temperature. Three animals were relaxed in 2% magnesium chloride in order to dissect
out the venom apparatus. Tissue samples were immediately fixed in 2% glutaraldehyde,
80% filtered sea water and 100 mM HEPES, dehydrated in a graded ethanol series and
embedded in Spurs resin. Only two of the three samples infiltrated well, and sections
0.5u to 2 u thick were cut on an ultra-microtome with glass knives, stained with
methylene blue in 0.1 N NaÖH, and examined on an Olympus BH-2 microscope.
Physiology
Individual teeth were manually removed from the short arm of the radula sac in
filtered seawater. They were then broken up using a hand homogenizer in 50 uL of
filtered seawater, and added to 0.2 ml of external recording solution (see below) yielding
a final concentration of 0.022 teeth per uL of solution. Venom from the duct was
manually extruded onto a piece of parafilm, mixed with 0.5 ml of external recording
solution in a 1.Sml microcentrifuge tube, homogenized by quickly vortexing for 20
seconds and heated to 95° C for 5 minutes. Äfter a brief centrifugation (4 min at 13,000
rpm), the supernatant was diluted 1:5 with external recording solution for testing activity.
Voltage dependent ionic currents were recorded form squid giant fiber lobe
neurons using conventional single-electrode, whole-cell patch clamp. Linear (i.e. not
voltage-dependent) ionic and capacity currents were removed online with a standard P/4
technique. The holding potential was-80 mV. Temperature is indicated in the legend to
Fig. 7.
External and internal pipette solutions were designed to eliminate K+ currents by
eliminating K from both solutions. The external solution was composed of 480 mM
Nacl, 10 mM CaClz, 20 mM MgCl, 20 mM MgSO4, and 10 mM Hepes at pH 8. The
internal solution was composed of 100 mM sodium glutamate, 50 mM NaF, 50 mM
Nacl, 300 mM tetramethylammonium glutamate, 10 mM NazEGTA, 25 mM
tetraethylammonium chloride, and 10 mM Hepes at pH 7.8.
Matrix-assisted laser desorption-ionization time of flight mass spectrometry
(MALDI TOF
The venom apparatus was dissected out and shipped to University of Illinois on
ice. Individual portions of the tissues were dissected in isotonic NaCl, and placed in
2,5dihydroxybenzoic acid (DHB) on a MALDI target. Mass spectra were obtained using
’Physiology experiments were done by William F. Gilly at Hopkins Marine Station, Stanford University,
MALDI TOF MS was done by Jonathan Sweedler at the University of Illinois at Urbana-Champaign.
two mass spectrometers; a Voyager Elite and a Voyager DE STR (PE Biosystems,
Framingham, MA) equipped with delayed ion extraction. A pulsed nitrogen laser
(337nm) was used as the desorption/ionization source, and positive-ion mass spectra were
produced using both linear and reflectron mode. Mass calibration was preformed
externally using either bovine insulin (SIGMA, St. Louis, MO) or a previously calibrated
spectrum obtained from Aplysia bag cells.
Results
The Venom Duct
The venom duct is a thin convoluted tube connecting the muscular bulb and the
proboscis. Although the entire duct was not analyzed completely, it was examined in
several places as indicated in Fig. 20.
As the duct leaves the muscular bulb (Fig. 2A) it is an orange pink color, about
250u in diameter, and the wall is composed of 3 distinct layers. There is an outer
epithelium overlaying a muscular layer. A layer of relatively large cells (60u wide x 40u
tall) surrounds the lumen, which consist of some granular material as well some diffuse
non-cellular content (Fig. 2A).
About one third of the way to the proboscis the duct grows to about 375u in
diameter, changes color to creamy white, and also changes morphologically (Fig. 2B).
There is still an outer epithelium, although it is much thinner, and this covers an
extremely thin muscular layer (Fig. 2B). The cells of the inner layer are much larger,
about 75u tall and 60u wide, and are densely packed with granules of different kinds,
There is still a central lumen, and this is packed with granules. It is difficult to
unambiguously visualize the border between these cells, and between these cells and the
lumen. The anterior end of the venom duct looks similar to the middle section, except it
has grown to 475u in diameter.
At the site where the venom duct enters the proboscis musculature it narrows to
125u in diameter and is lined by cells about 25u tall with large basal nuclei (Fig. 2D).
Cells with visible boundaries that contain granules can be seen, and these granules often
appear to be segregated into different types within individual cells.
In addition, a very distinct type of cell appears in this region that is filled with tiny
granules stained dark purple by methylene blue. These cells surround the duct as it
passes through the proboscis (Fig. 3-4). As the duct crosses the proboscis, the lumen
becomes very narrow, about 20u wide, and is mostly filled with small granules that do
not stain with methylene blue. Light and dark blue granules from the lumen of the duct
outside of the proboscis can be seen entering into the passageway but seem to disappear
where the lumen branches (Fig. 3D,E). The white granules continue in the channel,
however.
The passageway branches about 250u into the proboscis (Fig. 3E, F) and the two
channels cross an ambiguous area before they exit the proboscis between folds of the
epithelium that lines the proboscis lumen (Fig. 4). These epithelial cells have well
defined basal nuclei. In the area where the venom duct exits, these folds extend further
into the proboscis wall (Fig. 4B), and the epithelium appears to merge here with the
purple cell layer that comprises the wall of the venom duct. There is a central finger that
has purple cells lining both sides, and two flanking fingers where purple cells only line
the side facing the duct.
The Radula Sac
The short arm of the radula sac consists of an outer epithelial layer about 7.5u
thick, a layer of connective tissue and an inner epithelial layer about 125u thick. The
ligament sac takes up a large part of the short arm. Another large portion is filled with
cells about 25u long, with basal or central nuclei. These cells have diffuse contents that
stain light blue with methylene blue (Fig. 5C), and form a thick layer that folds onto
itself. The teeth in the short arm are usually in close contact with this cell layer. Some
of the teeth are positioned in random directions, but a majority of them face with their
points towards the proboscis, and all of the teeth entering the proboscis point this way
(Fig. 5A). The space around the teeth contains a mixture of granular, cellular and
unidentifiable material, some of which appears to be similar to material in the venom
duct (Fig. 5E, F). Similar material also fills a majority of the teeth, especially those
entering the proboscis (Fig. 5A).
The teeth are enveloped by muscle as they travel through the proboscis. As the
connection forms, the sac and the proboscis are in close contact on many sides, and folds
of the proboscis wrap around the sac. The teeth enter into the proboscis tip first, and more
then one tooth in the passageway can be seen (Fig. 5A)
The long arm of the sac was studied not in detail, although individual teeth were
examined. Teeth at the blind end were less developed, and generally had less discrete
matter in their lumen than teeth from the short arm (Fig. 6).
Physiology
Contents of the venom duct and radula sac were also characterized in terms of
their neurotoxic activities. Previous work on in C. californicus has indicated the
existence of a peptide that specifically inhibits voltage-gated sodium channels in
cephalopods and not in gastropods (Bingham et al. 2000). Duct venom isolated without
any chemical extraction in the present study also inhibits sodium channel activity in squid
neurons (Fig. 7B). Fig 7A shows that both outward (top panel) and inward (middle
panel) sodium currents were inhibited by the tooth venom.
Mass Spectrometry
Results from the reflectron MALDI mass spectra, which requires more
concentrated samples and greater instrumental optimization, but provides greater
accuracy, are shown in Fig 8. Linear mode operation gave similar results, but these data
are not shown. Region 1, the most posterior region examined (level 1 in schematic of
Fig. 8), shows only one peptide, with a molecular weight of 2993-2995, that occurs
elsewhere in the duct and none that co-occur in the radula sac (Fig. 8A,B,C). Region 2
and region 3, the mid and anterior regions of the duct, have many peptides in common
(Fig. 8B,C). They also share at least three peptides with those found in the tooth (Fig.
8B,C,D).
Discussion
The Venom Duct
The anatomy and chemistry of the venom duct of C. californicus is in general
agreement with previous studies of other Conus species (Hermitte, 1946; Endean and
Duchemin 1967; Bingham et al. 1996). The posterior or muscular bulb end of the duct
has a distinct morphology, the peptides in this region are larger than those in more
anterior regions, and is not as active physiologically. This supports the current
hypothesis that a pro-peptides in the venom is manufactured in the posterior region and
then undergo post transitional modifications as they moves towards the anterior region of
the duct (Olivera, 1997; Endean and Duchemin, 1967,)
How the venom gets from the duct where it is made to the tip of the proboscis has
not been discovered. None of the current literature describes the passageway from the
duct into the lumen of the proboscis. The most prominent feature of this region is the
appearance of cells containing granules that stained purple. These cells appear in the
wall of the duct just before it enters the proboscis and line the passageway for the venom
through the proboscis wall.
Although the purple cells appear to be secretory, the identity of the putative
product is unknown. There are several possibilities. The cells may simply secrete some
sort of mucus, which might act as a carrier for the venom flow. Another possibility is
that some enzymatic material is secreted that could activate the venom peptides. Secreted
enzymes could also possibly help break down tissue at the injection site and allows the
venom to exert its effects faster or more efficiently.
10
A final possibility is that these purple cells could be secreting a unique venomous
peptide not found elsewhere in the system. Bingham et al. (1996) suggested the
existence of such a unique peptide in milked venom samples of C. striatus, which they
felt was not the result of a posttranslational modification of a peptide in the venom duct.
The location of these purple cells puts them in a position to be the source of such
peptides. Further studies on these purple granules need to be done in order to determine
what they are secreting and what role they play in the venom apparatus.
The branching of the passageway also raises some questions. It is difficult to
follow any one branch as it travels through the proboscis, leaving a gap in the
understanding of the passageway’s structure. However, significant insight has been
gained. The passageways after branching are very small, only about 1Ou across at its
widest point, yet this appearance of the passageway lasts through more than 100u of
sections. This suggests the passage is not a cylindrical duct, but rather a flat channel that
is much bigger in one dimension than the other. How many of these channel there are is
still uncertain.
The branching also could suggest that some sorting of granules goes on in the
passageway to ensure that only mature peptides are passed into the proboscis lumen. The
venom that the snail injects is much simpler than the venom found in the duct in regards
to the number of peptides. Perhaps the complex nature of the passageway acts like a
sieve to permit sorting of venom components on a basis of granular size or some other
feature of individual peptides.
The area between the branching point of the passageway branches and the
proboscis lumen is also nebulous. In my sections a large cavity-like area appears in this
gap, but it is unclear whether this is real cavity or an affect of the fixation process (Fig.
4). Small channels can be seen entering into the passageway around a finger of the
proboscis lumen and it is presumed that the venom exits through these (Fig. 4
Further studies need to be done in order to better determine the nature of this
complex passageway. Nevertheless, it is clear that the passageway through the proboscis
is not a straight large diameter tube.
The Radula Sac and Teeth
The literature to date characterizes the teeth as being hollow and empty when they
are ready for insertion to prey. (Kohn, 1998; Marsh 1977). Marsh (1971) reported that
the teeth in the short arm of the radula sac for several Australian species of Conus were
free from cellular debris, and speculated that the salivary glad duct could be responsible
for this clearing. The teeth of C. californicus, however, are full of different types of
granules and cellular debris. This is true for teeth in both arms of the sac, and especially
for the teeth entering into the proboscis. The content of the teeth looks very similar to the
content of the sac outside of the teeth. In some cases, as in Fig. 5D, clusters of granules
that stain solid blue with methylene blue are found in the wall of the radula sac, and
similar individual granules inside teeth as well. These granules are similar to ones in the
venom duct. Although the exact nature of these individual granules is unknown.
similarities in their morphology suggest that they may contain similar peptides.
Two other lines of evidence support this idea. First, examination of an individual
tooth using MALDI revealed that some of its peptides were indistinguishable from those
12
in the venom duct. The peaks in the 3131-3133 MW range in Fig. 8, are of particular
interest. Bingham et al. (2000) have characterized a venom duct peptide of this
molecular weight that is thought to be the peptide with sodium-channel blocking activity.
Physiological tests also indicate the presence of a specific sodium-channel blocker
in material isolated from both the venom duct and the teeth (Fig. 7). Although the effect
of the tooth venom is not as strong as the duct venom samples, it nevertheless exists. The
discrepancy is probably only due to the fact that the teeth sample was extracted from 5-6
teeth with an estimated total volume of several nL, where as the venom duct sample was
prepared from a whole duct with an estimated volume of several mL. Further
experiments are needed to quantitatively compare the potency of material from the two
source
Results from this study provide strong evidence that the radula teeth of C.
californicus contain some of the same peptides as those normally found in the venom
duct. Furthermore, one of these peptides acts to inhibit sodium channel activity. These
data are thus contrary to the widely held view that the teeth are completely empty tube
and act only as a passive delivery device. Whether or not these data are relevant to other
Conus species remains to be established. It is also unclear what role the seemingly small
amounts of toxins stored in the teeth might actually play in the hunting process.
Additional studies are needed to structurally identify the peptides in the teeth, to
determine the biological activity of these peptides and to elucidate the source of these
peptides and the mean by which they are packed into the teeth.
13
Acknowledgements
1 would like to thank William F. Gilly for being my advisor on this project and
being so patient through this entire learning experience, and for providing me with
physiology data for this project. I would also like to thank Jonathan Sweedler and his
group at the University of Illinois for doing the MALDI work. Many thanks go out to
Lisa Walling, and Gina Kang, and special thanks to Jim Watanabe for providing Cone
specimens. My gratitude is also extended to the following people: Jon-Paul Bingham,
Barbara Block, Chris Patton and William C. Pitts and the entire Gilly lab for their
support.
Literature Sited
Bingham, J. P., A. Jones, R. J. Lewis, P. R. Andrews, and P. F. Alewood. 1996.Conus Venom
Peptides (Conopeptides): Inter-Species, Intra-Species and Within Individual Variation
Revealed by lonspray Mass Spectrometry. pp. 13-27. in Biomedical Aspects of Marine
Pharmacology.
Bingham, J. P., A. Burlingame, E. Moczydlowski, and Wm. F. Gilly. 2000. A new highly selective
conotoxin from Conus californicus that targets voltage-gated neuronal Na' channels of squid.
Abstracts of Papers Presented at the 54th Annual Meeting of the Society of General
Physiologists.
Endean, R. and C. Duchemin. 1967. The venom apparatus of Conus magus. Toxicon. 4:275-284.
Freeman, S. E., R. J. Turner, S.R. Silva. 1974. The venom and venom apparatus of the marine
gastropod Conus striatus Linne. Toxicon. 12:587-592.
Hanna G. D., and A. M. Strong. 1949. West American Conus. Proceedings of the California
Academy of Science, 4th series. 26: 247-322.
Hermitte, L. C. D. 1946. Communications: Venomous marine molluscs of the genus Conus.
Transactions of the Royal Society of Tropical Medicine and Hygiene. 39: 485-512.
Hinegardener, R. T. 1958. The venom apparatus of the cone shell. Hawaii Medical Journal
7:533-536
Kohn, A. J. 1956. Picivorous gastropods of the genus Conus. PNAS. 42:168-172
Kohn, A. J. 1966. Food specialization in Conus in Hawaii and California. Ecology. 47:1041-1043.
Kohn, A. J. 1998. Family Conidae. pp in Beesley, P. L., Ross, G. J. B., and Wells, A. (eds)
Mollusca: The Southern Synthesis. Fauna of Australia. Vol. 5 CSIRO Publishing:
Melbourne.
Kohn, A. J., M. Nishi, and B. Pernet. 1999. Snail spears and scimitars: A character analysis of
Conus radular teeth. J. Moll. Stud. 65:461-481.
Marsh, H. 1971. The foregut glands of vermivorous Cone Shells. Australian Journal of Zoology.
19:313-326.
Marsh, H. 1977. The radular apparatus of Conus. J. moll. stud. 43:1-11
Nybakken, J. 1970. Correlation of radula tooth structure and food habits of three vermivorous
Species of Conus. The Veliger. 12:316-318.
Olivera, B. M. 1997. Conus venom peptides, receptor and ion channel targets, and drug design: 50
million years of neuropharmacology. Molecular Biology of the Cell. 8:2101-2109.
Songdahl, J. H., 1973. The venom and venom apparatus of the Atlantic cone, Conus spurius
atlanticus (Clench). Bulletin of Marine Science. 23:600-612
Spengler, H. A., and A. J. Kohn. 1995. Comparative external morphology of the Conus
osphradium. Journal of Zoology (London). 235:439-453
Wells, W. A., 1998. The snail companies. Chemistry and Biology.
:R235-R236
0
Fig. 1A.
Schematic representation of venom apparatus in C. californicus. This
figure, which is not to scale, shows the main parts of the venom apparatus
including the siphon, proboscis, muscular bulb, venom duct and radula
sac.
Fig. 1B.
Complete venom apparatus as it looks in a snail . The venom duct
changes color at about the same region it would in a live snail.
Fig. 1C.
Venom duct and radula sac connection to proboscis, showing that the
venom duct enters posterior to the radula sac
Abbreviations: es, esophagus; m, mouth; mb, muscular bulb; nr, nerve ring; p, proboscis;
rs, radula sac; sg, salivary gland; sgd, salivary gland duct; t, radula teeth.
Fig. 2
Micrographs of cross sections through the venom duct from the regions
indicated in panel C. Schematic representations, not to scale of two of the
regions are shown in panel 2A1 and 2B1. Scale bar is 50u throughout.
Fig. 2A.
Posterior or muscular bulb end of the venom duct, showing inner epithelial
cells surrounding a central lumen that contains some granules
Mid-region of venom duct after color change. Inner epithelial cells are
Fig. 2B.
larger and packed with granules. The central lumen also has more
granules.
Fig. 2C.
Diagram illustrating the different levels of the venom duct analyzed.
Fig. 2D.
Venom duct as it approaches the proboscis. Cells with larger basal nuclei
appear. Discrete bundles of granules can be seen in the inner epithelial
cells. Blue granules are localized in cells at the top; clear granules are in
cells at the bottom-left.
Fig. 3.
Longitudinal sections through the venom duct as in the proboscis wall.
Scale bars are 50u throughout.
Fig. 3A
Venom duct as it starts to enter proboscis wall. The purple cells first
appear here.
Fig. 3B.
Magnified view of A.
Fig. 3C.
The venom duct is pushing through proboscis wall with the purple cells
lining passage.
Fig. 3D.
Venom granules in channel in the proboscis wall. A narrow passage for
venom from the duct to enter into the proboscis is seen here.
Fig. 3E. & F
Two views of the channel branching. 3E shows the branches disappearing
on the right. The large layer of purple cells surround the central channel is
shown. 3E also shows that the blue granules which are seen before the
branching seem to disappear, but the clear granules continue.
Fig. 4.
Longitudinal sections through the venom duct crossing the proboscis wall
and entering proboscis lumen. Scale bar is 50u throughout.
Overall view of venom duct crossing the proboscis. The whole proboscis
Fig. 4A.
wall is shown.
Magnified view of channel exiting proboscis wall into proboscis lumen
Fig. 4B.
There are two exit channels visible. The proboscis lumen epithelium
merges with the venom duct epithelium in this region.
Fig. 4C.
Fig. 5.
Fig. 5A & B.
Fig. 5C&D
Fig. 5E& F.
Fig. 6.
Fig. 6A.
Fig. 6B.
Fig. 6C.
Fig. 7.
Fig. 8.
Schematic diagram of how the venom duct crosses the proboscis. Not to
scale.
Teeth in the radula sac. Three different views of teeth in the radula sac are
shown. Each panel on the right is a magnified image of the panel of the
left. Scale bar is 50u throughout.
Teeth entering proboscis wall from radula sac. The teeth are all pointing
towards the proboscis lumen and are all filled with material.
Comparison of radula sac content with tooth content. The basal opening
can be seen on two teeth in the short arm of the radula sac. Material inside
and outside teeth appears very similar
Blue granules in clusters can be seen in the radula sac wall and individual
granules can be seen in the tooth. These granules look similar to those
found in the venom duct, (see Fig. 2).
Teeth from different parts of the radula sac. Line drawing shows
approximately where teeth were taken from. Scale bar on all is 50u.
Living tooth taken from the short arm of the radula sac. It is more fully
developed, larger, and the lumen is packed with granular material in it.
Tooth from long arm fixed in gluteraldehyde, placed into agar to hold it,
embedded in Spurs resin, and stained with metheylene blue. The ligament
can be seen off the basal end. The lumen is not as full of material.
Living immature tooth from extreme end of long arm. The ligament sac is
coming off right end. The tooth is not fully developed.
Results of the tooth venom and venom duct extract experiments. The tot
panels shows the effect on a inward current, the middle panels shows the
effect on an outward currents and the bottom panels show the voltage
applied. The tooth venom experiment (panel A), showing that 0.002 tooth
units inhibits the sodium channel. The venom duct extracts (panel B)
show similar effects. The temperature of each experiment is indicated at
the bottom.
Results from the reflectron MALDI MS. Intensity is along the y-axis and
mass per charge is along the x-axis. Marked peaks of indicated molecular
weight are the same peptide in different samples. The sample locations
are shown at right.
Fig.1
S
siphon
proboscis
S
mouth
mb

sgd
es

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venom
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—
B
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A
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rS

C
Fig2
A
puisculature
epithelium



e

4

lumen
innerlg
granules
epithelium
B
musculature
inner
epithelium
granules

lumen
epithelium
mb
A2
2.
25
80
56
0
*
-
9


E
A
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Z
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