ABSTRACT
Similar to other members of its genus, Conus striatus has a highly evolved venom
apparatus that serves to inject paralyzing venom into its prey through a hollow,
tubular radular tooth. Although much research is currently being conducted on the
properties of the venom itself, little investigation has been done to elucidate the
biomechanics behind the snail’s ability to inject its venom. Studies were carried out
with individual radular teeth that defined the pressure-flow characteristics in order to
estimate the pressure used by a snail to eject venom during prey capture. Pressure
and flow were directly proportional over the range of investigated pressures (up to 4
cmHg) and flow rates (up to 2 ul s*). Behavioral observations of prey capture were
carried out with C. catus, a closely related species that employs similar toxins. Video
data revealed the approximate duration of venom ejection to be about 100 ms. The
duration of venom ejection by C. striatus is probably similar, and the volume of
ejected venom in this species can be 20 ul or more based on ’milking’ trials. Data
suggest that the pressure necessary to carry out such an ejection must range from 2-4
atm.
INTRODUCTION
Predatory marine snails of the genus Conus have long been of interest because of
their unique hunting strategies that employ peptide toxins to paralyze prey. In cone
snails, the chitinous radular teeth resemble barbed hypodermic needles that are used
to inject venom into the prey. Each species has a unique tooth structure, with varying
size, shape, and number of barbs. These differences are often used as a basis for
phylogenetic classifications (Kohn et al., 1999).
When resting, the snail holds its proboscis retracted inside the rostrum. When
prey is sensed by water-borne chemical signals (Spengler and Kohn, 1995), the
proboscis is extended with a tooth held in place near the tip (Greene and Kohn, 1989).
Searching behavior begins, and when contact with the prey is achieved, the snail
impales the prey, paralyzing it, and pulls it into the expanded rhynchodeum by
contraction of the proboscis (Kohn, 1956).
Delivery of Conus venom involves generation and storage of radular teeth, transfer
of a tooth to the proboscis, and final ejection of the venom. These processes are
carried out within a complex venom apparatus that is relatively invariable between
Conus species (Halstead, 1988). Marshall, et al. (in press) have detailed the
anatomical correlates of the venom apparatus of the cone snail C. californicus (Fig.
1). It is highly likely that similar structures are found in piscivorous feeders such as
C. striatus and C. catus.
In Conus, the radular sac consists of two arms. Teeth are manufactured in the long
arm and stored in the short arm (Marsh, 1977). Using scanning electron microscopy,
Kohn et al. (1972) verified that each of the radular teeth is basically a sheet,
presumably of chitin, rolled into a hollow tube. This tube has a large central lumen
through which venom from the venom duct passes during the injection into the prey.
In C. striatus, the venom enters the tooth via an adapical opening and leaves via an
apical opening (Freeman, 1974). In preparation for prey capture, a single tooth is
transported into the pharynx where it is surrounded by the relaxed proboscis and
grasped by the sphincter near the tip of the proboscis (Greene and Kohn, 1989;
Hermitte, 1946). Once at the tip of the proboscis, the lumen of the tooth is thought to
be empty (Marsh, 1977), but findings from Marshall, et al. (in press) suggest that
active peptides similar to those found in the anterior venom duct are present in the
tooth lumen as well. Finally, the proboscis extends the tooth held at the tip, impales
the prey, and injects the venom.
How the force for final venom ejection out of the tooth or for tooth penetration is
produced remains unknown. Although a muscular bulb posterior to the venom duct
has often been hypothesized to provide the needed force, there is no direct evidence
for this idea (Olivera, 1997). 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 for venom ejection (Kohn, et al. 1999).
This report provides a description of the processes involved in venom ejection in
C. striatus and C. catus, species that prey exclusively on fishes. Toxic components
exist in the venom and details can be found in studies by Freeman (1974), Ramilo, et
al. (1992), Cruz (1996), and Hahin, et al. (1991). Details of the mechanisms by which
venom enters the tooth and is ejected into the prey are lacking. We have used isolated
teeth to examine the biomechanical properties of ejection. First, we have found that
maximum flow rate is directly proportional to peak pressure. Second, we have
identified some previously unreported morphological characteristics of the C. striatus
radular tooth through video data and scanning electron microscopy. Video data of
venom ejection in C. catus revealed that the pressure that expels the venom also
seems to insert the tooth into the victim. Data also indicated the approximate
duration of venom ejection. Manual ’milking’ of C. striatus revealed the approximate
volume of venom released per ejection. From all of these data, we were able to
estimate the approximate pressure necessary for a living C. striatus to eject its venom.
MATERIALS AND METHODS
Specimens of C. striatus were obtained from American Samoa, and specimens of
C. catus were obtained from Hawaii.
Extraction of the teeth
The radular sacs of C. striatus were removed and dissected from the animal and
stored in -80 °C for several months prior to use. Teeth were dissected from the short
arm of the radular sac, rinsed and stored in distilled water, and kept in 4 °C up to 3
days prior to use in ejection studies. Teeth were kept for approximately 15 days prior
to use in electron microscopy.
Ejection studies
Teeth stored in distilled water were air-dried prior to use. Because we wanted to
determine differences in fluid flow with and without the ligament attached, some
teeth were left with the ligament intact, while the ligament and base of a number of
teeth were cut using a microknife prior to use. Each tooth was placed inside a
segment of clear plastic tubing. The tubing was positioned with the tip of the tooth
exposed, and the tooth was then sealed in place with epoxy and allowed to dry for 5-
10 minutes. The tooth and tubing were then mounted and connected to a
microinjection apparatus that served as the pressure source. A schematic diagram of
the setup is shown in Fig. 2. As pressure was applied to the tooth, blue fluid
consisting of distilled water and .03% Blue #1 dye flowed out of the microinjection
apparatus and filled the mounted tooth. Blue fluid from the tooth was ejected into a
separate capillary tube filled with clear distilled water. The set-up was connected to a
pressure transducer, which was connected to an oscilloscope that recorded the voltage
changes during each timed pressure pulse applied with an eppendorf microinjector
5242 apparatus. Calibration of pressure transducer is as follows:
cm H20 = 0.7529 + 0.5704 * mV (Fig. 3)
The experimental procedure was recorded using a Sony SSC-C374 color video
camera. Teeth were viewed through a Nikon SMZ-2T microscope. Video images of
the tooth were used to analyze how much fluid was being ejected with each pressure
pulse (Fig. 4). The volume of fluid released into the capillary tube was measured by
calibrating the length of fluid inside the capillary tube when a known volume of fluid
was added.
Scanning electron microscopy
Teeth were cleaned briefly in concentrated sodium hypochlorite. A separate set of
teeth was kept free of sodium hypochlorite. Teeth were immersed in 4%
glutaraldehyde and were dehydrated through increasing concentrations of ethanol.
Prior to scanning, teeth were air-dried, mounted on stubs with double-sided carbon
tape, and coated with a thin layer of gold-palladium in a vacuum evaporator. They
were examined with a Hitachi S-450 scanning electron microscope. No significant
differences in scanning electron microscopy were observed in teeth cleaned with
sodium hypochlorite and teeth kept free of sodium hypochlorite.
Video data from C. catus
Feeding behavior of C. catus was filmed with the same camera and microscope
used for the ejection experiment. A fish (guppy) was held with a pair of forceps and
presented to the C. catus for feeding.
Milking' studies of C. striatus
Part of the fin of a fish was attached to a 1.7 ml micro-centrifuge tube. The tube
was then sealed with a piece of latex. This target was presented to C. striatus to
induce venom ejection into the tube (Fig. 5). Volume of venom ejected was estimated
by comparison with known volumes in other tubes.
RESULTS
The radular tooth
The radular tooth of C. striatus is approximately 8 mm long with a diameter of
0.235 mm. The shaft is cylindrical and armed apically with a barb and a barbed blade
together with a long backward pointing process with a recurved tip. A ligament is
attached to the base of the tooth. A schematic diagram is shown in Fig. 6. Scanning
electron microscopy of adapical and apical opening are shown in Figs. 7 and 8,
respectively.
Video data from ejection studies revealed a second opening associated with the
large recurved barb. Fluid would flow out of this location, although exact site of fluid
exit was unclear (Fig. 9).
Ejection studies
Minimal differences in total volume ejected at various pressures applied for 100
ms pulses were found between teeth with ligament attached and those without (Fig.
10a). Almost no differences in the flow rate at a constant applied pressure of 400 hPa
were found in teeth with and without ligament (Fig. 10b). Öther measurements
showed that pressure sensed at the basal end of the tooth was directly proportional to
applied pressure.
Table I shows the different variables measured as pressure from 100 hPa to 1000
hPa was applied for 100 ms pulses. Fig. 1 la shows the voltage changes recorded on
the oscilloscope for 3 different pressure applications. Fig. 1 1b plots the volume of
fluid released following the 100 ms pressure pulses. Initial slopes over the first
second were calculated to determine fluid flow rate. Rate of flow of fluid ejection is
directly proportional to peak pressure recorded on the oscilloscope (Fig.12).
Venom ejection by C. catus
Video footage of C. catus venom ejection revealed that the radular tooth was not
located at the tip of the proboscis prior to ejection as previously observed in other
Conus species (Marsh, 1977). Rather, the tooth was located approximately one tooth
length away from the tip of the proboscis prior to ejection (Fig. 13). In every case, the
tooth remained in this position until the apparent moment of venom ejection. Within
one video frame, the tooth was forced out to the tip of the proboscis, presumably by
the same pressure that enables venom ejection. The basal end appeared to be grasped
by the tip of the proboscis, and the volume behind the tooth then swelled for several
frames. Although a donut shaped ring, presumably of muscle, is present at the base of
the proboscis (Fig. 15), no constriction or other obvious changes in the muscular ring
occurred prior to, during, or after venom ejection. Although it was difficult to
ascertain the actual duration of venom ejection, we based our estimate on the time
elapsed from when the tooth impales the prey to when the snail starts to pull back on
the tooth in preparation for swallowing (Fig. 16). In every case examined, this time
was 3-4 video frames (n=6).
DISCUSSION
An initial hypothesis regarding the role of the ligament was that it acts as a
protective barrier that prevents the contents of the tooth from seeping out of the base.
Given this potential role, we hypothesized that flow rates of teeth with the ligament
attached would be slower than that of teeth without the ligament. However, ejection
studies using the experimental set-up described revealed that no significant
differences in volume released were observed in teeth with and without ligament. In
both cases, volume, flow, and pressure are all directly proportional. Whether the
ligament indeed acts as a protective barrier remains to be seen. Future studies of the
structure of the ligament using techniques such as transmission electron microscopy
could be of help in bringing greater understanding of its role and function in venom
ejection in Conus species.
Ejection studies also revealed that a second opening located near the recurved barb
of the radular tooth is present. We were unable to locate this second opening through
scanning electron microscopy, mostly because of difficulties in achieving the correct
orientation of the tooth. Further investigation of the exact location, size, and function
of this opening would be helpful in understanding the mechanics behind venom
ejection in cone snails.
Video data from venom ejection by living Conus catus revealed that tooth
penetration into prey, paralysis of prey, and time prior to retraction of the proboscis
spans approximately 100 ms. We take this to be a minimum period for venom
ejection. It is highly likely that the duration of venom ejection in C. striatus is similar.
Manual milking of C. striatus revealed that approximately 20ul of venom is released
per ejection (Dr. Joseph Schulz, personal communication). Given these observations,
we were able to deduce that the rate of volume ejection by C. striatus would be 200
uls
Extrapolating from our pressure-flow data (Fig. 12), we were able to ascertain that
approximately 2-4 atm of pressure is required to inject 20 ul of fluid out of the C.
striatus radular tooth in 100 ms. However, our data were gathered using distilled
water as a venom replacement. Because venom is probably much more viscous, even
greater pressures would be required.
The literature to date characterizes the tooth to be located at the tip of the
proboscis prior to penetration into prey (Marsh, 1977). The finding that prior to
penetration, the tooth was actually located farther back into the proboscis is obviously
in contrast to that idea. The mechanism by which the tooth is propelled from its initial
location 5-8 mm away from the tip of the proboscis into the prey has yet to be
determined. It appears that the same force that propels the venom forward also
propels the tooth, although this observation has yet to be supported by conclusive
evidence. The anatomical structure that exerts this force remains to be elucidated.
ACKNOWLEDGEMENTS
1 would like to thank Dr. William F. Gilly for his steadfast guidance and unwavering
patience throughout my learning experience. This project would not have been
possible without his constant support and caring mentoring. I would also like to
extend my gratitude to Chris Patton for his help with SEM, Dr. Joseph Schulz for
providing advice, assistance, Conus specimens, and video data, and the entire Gilly
lab for opening their doors to a novice learner.
LITERATURE CITED:
Cruz, L.J. 1996. Neuroactive peptides of the marine snail Conus striatus. J. of
Natural Toxins. 5(1):122.
Freeman, S.E., R.J. Turner, S.R. Silva. (1974). The venom and venom apparatus of
the marine gastropod Conus striatus. Toxicon. 12(6):587-592.
Greene, J.L. and A.J. Kohn, 1989. Functional morphology of the Conus proboscis
(Mollusca: Gastropoda) J. Zool. Lond. 219:487-493.
Hahin, R., G.K. Wang, B.I. Shapiro, B. Strichartz. 1991. Alterations in sodium
channel gating produced by venom of the marine mollusk Conus striatus. Toxicon.
29(2):245-260.
Halstead, B.W. 1988. Poisonous and Venomous Marine Animals of the World.
Darwin Press, Princeton, NJ. Pp. 243-263.
Hermitte, L.C.D. 1946. Venomous marine mollusks of the genus Conus. Trans. Roy.
Soc. Trop. Med. Hygine 39: 485-512.
Kohn, A. J. 1956. Picivorous gastropods of the genus Conus. Proceedings of the
National Academy of Science. 42:168-172.
Kohn, A.J., J.W. Nybakken, and J.J. Van Mol. 1972. Radula tooth structure of the
gastropod Conus imperialis elucidated by scanning electron microscopy. Science.
176:49-51.
Kohn, A.J., M. Nishis, and B. Pernet. 1999. Snail spears and scimitars: A character
analysis of Conus radular teeth. J. Moll. Stud. 65:461-481.
Marsh, H. 1977. The radular apparatus of Conus. Journal of Molluscan Studies. 43:1-
11.
Marshall, J., W.P. Kelly, S.S. Rubakhin, J. Bingham, J.V. Sweedler, and W.F. Gilly.
2002. Biological Bulletin. In press. Anatomical Correlates of Venom Production in
Conus Californicus.
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.
11
Ramilo, C.A., G.C. Zafaralla, et al. 1992. Novel alpha- and omega- conotoxins from
Conus striatus venom. Biochem. 31(41): 9919-9926
Songdahl, J.H. 1973. The venom and venom apparatus of the Atlantic cone, Conus
spurious 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.
TABLES
TABLE I. MEASURED VARIABLES AS PRESSURE APPLIED VARIES
(100 ms pulses)
n=3 for 100-800 hPa pulses; n=1 for 1000 hPa pulse
Applied
Total vol
Mean peak
Mean Flow
Standard
Standard
Pressure
pressure
Deviation
Rate (uV/s)
Deviation
(ul)
(cm H20)
Pressure
Flow Rate
100 hPa
0.368
3.71
0.5154
0.009292
0.073
1.127
0.04949
200 hPa
9.854
0.214
0.344
400 hPa
2.195
19.69
0.654
0.895
0.008021
800 hPa
4.05
40.37
0.035119
1.387
0.568
5.965
1000 hPa
30.24
none
none
22
FIGURE LEGENDS:
Fig. 1. Diagram of venom apparatus of a typical cone snail. Modified from Marshall,
et al. (in press)
Fig. 2. Diagram of experimental set-up. Tooth was inserted into a clear plastic tubing
Tip of tubing was sealed with epoxy (yellow). Tooth was connected to a
microinjector apparatus that pumps blue fluid into the tooth. Blue fluid flows out of
the tooth and into a capillary tubing filled with clear distilled water. Set-up was
connected to a pressure transducer that was also connected to an oscilloscope.
Experiments were filmed using a color video camera (not shown).
Fig. 3. Graph of pressure transducer calibration.
Fig. 4. Diagram of video measurements. Measurements were made after every 3
frames (100 ms). Length of fluid released into the capillary tube filled with distilled
water was measured. Calibration of fluid volume in the capillary tubing enabled us to
determine how much volume was being released at each frame. See text for additional
details.
Fig. 5. Schematic diagram of milking procedure of C. striatus.
Fig. 6. Schematic diagram of a radular tooth from C. striatus. Not to scale.
Fig. 7. Scanning electron micrograph of adapical opening of the radular tooth from C.
striatus with ligament attached.
Fig. 8. Scanning electron micrograph of apical end of the radular tooth from C.
striatus.
Fig. 9. Scanning electron micrograph of barbed aperture of the radular tooth from C.
striatus. Second opening is presumed to be at an unspecified location within the
boxed region. Possible locations include the tip of the barbed aperture itself or an
opening located at the shaft of the tooth beneath the barbed aperture.
Fig. 10a. Graph of total volumes ejected as pressure applied with 100 ms pulses was
varied for teeth with and without ligament.
Fig. 1Ob. Graph of incremental volume ejected per 100 ms with an applied pressure of
400 hPa in teeth with and without ligament. Duration of pressure was 100 ms.
Fig. 11a. Pressure transients in a tooth with ligament as pressure was applied at 200
hPa, 400 hPa, and 800 hPa. Duration of pulse was 100 ms.
14
Fig. 11b. Graph of incremental volume ejected over time corresponding to the
pressure transients illustrated in Panel a.
Fig. 12. Graph of flow rate vs. pressure. Data points are means (+ 5.0) for pressure
pulses of 100 hPa, 200 hPa, 400 hPa, and 800 hPa (applied pressures, 100 ms pulses).
n=3 for all, except n=1 for 1000 hPa.
Fig. 13. Modified diagram of venom apparatus. Arrows indicate the observed location
of the tooth as well as the muscular ring inside the proboscis.
Fig. 14. Relevant frames depicting duration of venom ejection in C. catus. Arrow in
Frame 1 depicts the radular tooth located a few millimeters away from the tip of the
proboscis as observed prior to ejection. Frame II shows that the radular tooth has
already penetrated the prey. Frame III shows the radular tooth still impaled to the
prey. Arrow in Frame IV reveals that the cone snail has started pulling back on the
radular tooth out of the prey in preparation for swallowing. Estimated duration of
venom ejection is spread out over Frames II-IV. Time lapse is 1/3 seconds per frame.
Fig. 15. Extrapolated pressure-volume curve given a time pulse of 100 ms and a
volume of 20 ul.
e
FIGURES
radular sac
long arm
muscular bulb



short arm
proboscis
—
—

esophagus
k-
pharynx
anterior
Figure 1
)
venom duct
- posterior
Figure 2
H

Pressure
Transducer
Pressul
Source
(microinjec
apparatus
Oscilloscope
0
100 -
80-
60 -
20-

High-pressure transducer calibration
cm H20 = 0.7529 + 0.5704'mV
(n-3, bidirectional)
170
Pressure transducer out (mV)
Figure 3
Figure 4

1915.03
E
19.15.06
19:15:09
Figure 5

fishfin
latex
Figure 6
100um
Figure 7
Figure 8
50 um
Figure 9
500 um
wand wo lig PV
—
100 200 300 400 500 600 700 800 900
PrSSu (NPA)
Figure 10a

400HPA TV

fines
Figure 10b
12 14 16
4-0NPAI
A
Figure 1la
6.5 cm H20
Pressure
N
55
Figure 11b
10-
os-
8 00
2 3 4 5
ûme (s)
Figure 11a-b
100 ms Pressure Pulses
—
—
319.
10-
805
oo
012345
time (s)
o
012345
time (s)
C
e
Figure 12
Flow Rate vs Pressure

Mean Flow Rate ulls
proboscis


muscular
ring
Figure 13
radular sac
muscular bulb

oo

venom duct

sophagus

—
pharynx anterior
posterior

e
..
10000
1 atm 1000
Figure 15
2-4 atm pressure range

:
9

0.01
100
1000
Flow (ulis)
20 ul injected
in 100 ms