ANATOMICAL STUDY OFTHE HYPOBRANCHAL GLAND OE
CALLIOSTOMA CANALICULATUM AND THE NEURONALLY
CONTROLLED SECRETION OF YELLOW STUFE (YS
Jason Toranto
Bio 175H
Hopkins Marine Station
Research Advisor: Prof. William Gilly
6/98
Abstract
The channeled top snail, Calliostoma canaliculatum, releases a yellow mucous,
called yellow stuff or YS, in response to perturbation and especially attempted predation
from sea stars such as Pycnopodia hellantholdes (Bryan et al., 1997). The source of YS is
the hypobranchial gland. Therefore, anatomical studies of this gland as well as electrical
stimulation experiments, to visualize secretion from this organ, were performed. The
anatomical study divulges the existence of cells that contain numerous large vesicles that
appear to contain yellow stuff. Stimulation of the gland directly as well as other body sites
with an electrode reveals that a one second train of brief shocks with a repeat frequency of
20Hz is sufficient to cause secretion of YS. Stimulated secretion was reversibly abolished
by low Ca“ seawater. These stimulation experiments demonstrate that the secretion of YS
is under neuronal control. Finally, from the trends that were visualized in the anatomical
study and from a consideration of the time course of secretion, holocrine secretion is
proposed to be the method by which YS is released from the yellow cells of the
hypobranchial gland.
Introduction
The channeled top snail, Calliostoma canaliculatum, lives in the kelp forest
ecosystem of Monterey Bay, often found clinging to the fronds of Macrocystis. Like other
snails, C. canaliculatum flees into its shell upon attack, but unlike many other snails and
specifically other snails of this genus also found in Monterey Bay (C. ligatum and C.
annulatum), C. canaliculatum has an additional defense mechanism. Upon sufficien
disturbance, such as that caused by the predatory actions of the sea star Pycnopodia
hellantholdes, C. canaliculatum will secrete a yellow mucous (appropriately termed yelloy
stuff or YS) that causes the starfish to retract both its tube-feet and arm (Bryan et al.
1997). The active compounds in YS causing these reactions have not been chemically
identified.
Roller et al. (1995) studied the hypobranchial gland of Stramonita haemastoma
canaliculata to determine whether the toxin it uses to paralyze oysters comes from this
gland, but concluded that it was coming from the salivary gland. The defensive secretion
of Calliostoma canaliculatum comes from the hypobranchial gland, however (Denny,
1989). Little else is known about this gland, however, except that it is located within the
mantle cavity, adjacent to the gill and rectum. It appears coiled and twisted, with a right
and left side, one always more pronounced than the other. Its function in C. canaliculatum
is thought to be to secrete mucous, which is then used as a substrate to carry waste particles
out of the shell (Hyman, 1967; Fretter et al., 1962).
This study sought to physiologically characterize the hypobranchial gland and
specifically determine the location of the yellow secretory material within the gland and to
provide a preliminary characterization of the process of secretion.
Materials and Methods
Experimental Organisms
Some Calliostoma canaliculatum were collected in Monterey Bay in the Hopkins
Marine Wildlife Refuge. Others were obtained from Sea Life Supply, Sand City, CA. All
were kept in tanks with flow-through sea water at ambient temperature (13-15
Tissue Sectioning and Staining
One Calliostoma canaliculatum was placed in 350mM MgCl, for 5 minutes and then
removed from its shell. The snail was then fixed for 15 minutes at room temperature and
then for 7 hours at 4°C in 0.065M Phosphate buffer (pH 7.4) with 3% gluteraldehyde,
0.5% tannic acid, and 6% sucrose. The snail was then washed for 2 hours in O.065M
phosphate buffer with 6.0% sucrose and then placed overnight in a solution of O.065M
phosphate buffer with 6.0% sucrose and 1% gluteraldehyde. The sample was dehydrated
using a 15 minute per step graded ethanol series - 50%, 70%, 90%, 95%, and 100% (twe
times) - and infiltration was allowed to occur for 48 hours at room temperature using LR
White medium (London Resin Company). Äfter washing two times with fresh LR White
resin after the 48 hour period, the snail and LR White were placed in a gelatin capsule and
the resin was allowed to polymerize for 24 hours at 60°C. All sectioning was carried out
using glass knives and a Sorvall JB-4 microtome. The sections were cut at Zum thickness.
Sections were placed on silane coated slides (Polyscience) in a drop of water and allowed
to dry for 10 minutes on a slide heater at 50°C. Some slides were then stained with either
multiple stain (Polyscience) or toluidine blue (Polyscience). All slides were mounted using
Permount (Polyscience).
Electrical stimulation
Snails were removed from their shells without MgCl, sedation, and the mantle
sheath was cut and peeled back to expose the hypobranchial gland. A NE-100 concentric
bipolar electrode (Rhodes Medical Instruments) and a Grass Instruments SD9 stimulatoi
were used to stimulate to various regions of the snail, including the hypobranchial gland
and regions around the epipodial tentacles. Each stimulus was a 1 second train of 0.2 ms
shocks delivered at 20Hz. A voltage of 60V proved to be the most effective. Some
experiments were conducted in Ca“-free ASW (475mM NaCl, 5mM NaÖH, 1OmM KCl.
5OmM MgCl,, 1OmM HEPES).
RESULTS
Visualization of YS
Throughout all these experiments, small samples of the secreted yellow stuff were
taken and analyzed under a microscope. From this analysis, two important discoveries
were made. First, yellow stuff was full of intact vesicles, presumably containing the YS
(Fig. 1). Second, the yellow stuff was strongly fluorescent under both rhodamine and
fluorescein filters (Fig. 1). These results thus indicated that in the histological analysis of
the hypobranchial gland, a YS-secreting cell should be identifiable by its vesicles and
fluorescence.
Tissue Sectioning and Staining
Looking at the hypobranchial gland in this snail it was possible to discern very
yellow structures that looked like cells (Fig. 2). As a general trend, these structures
seemed to be located within the crevices of the folds of the organ. At higher magnification.
it was determined that these yellow structures were cells due to the fact that they were
bounded by a plasma membrane and contained a nucleus in their basal end (Fig. 3.1). The
apical end of the cells always faced the mantle cavity, with some of the cells actually
protruding into the cavity. Only the plasma membrane, nucleus, and secretory vesicles,
seemingly the same ones seen in the YS, were visible in these cells, which ranged in length
from 40-120um.
There were other cell types within this gland. The function and purpose of the
majority of these cells were unable to be determined. One of the undetermined cell types
was packed with white vesicles. Study of these cells was not conducted, and the
possibility remains that these cells also may secrete an active component(s) in YS. The
color of the exudate and the vesicles found in YS suggest that the yellow cells are the major
source of secretion, but a role of the white cells is plausible.
The other cell type examined closely was a ciliated variety. Looking at a freshly
removed hypobranchial gland under water-immersion optics, the cilia, which were found to
always be located on the exterior surface of the gland facing the mantle cavity, were readily
visible due to their motion. Obtaining a picture of these cilia on the "live" organ proved
difficult because of their rapid motion and was not accomplished. However, under high
powered oil-immersion optics it was possible to view the cilia in the tissue sections (Fig.
4).
Electrical stimulation
One of the key questions addressed in this study was whether YS secretion could
be induced by stimulation of another region of the body. Using an electrode and
stimulating various sites around the body of the snail, it was possible to demonstrate that
secretion of yellow stuff could be caused by delivering brief shocks at a frequency of 20Hz
for 1 second. Shocking the hypobranchial gland directly, even with single shocks, led to
secretion (Figs. 5.1 & 5.2). Stimulation of the cephalic tentacles did not cause secretion to
occur, however (Figs. 5.3 & 5.4). A shock in the region of the epipodial tentacles led
directly to the secretion of yellow stuff (Figs. 5.5 & 5.6). This demonstrated that secretion
of yellow stuff was under neuronal control, but the nerve innervating the hypobranchial
gland was not identified.
Since many secretory systems involve Ca“, these stimulation experiments were
also conducted in calcium-free ASW. Not only did secretion not occur, but the snail
virtually stopped moving during the experiments. When this snail was placed back in the
same solution containing 1OmM CaCl,, the snail quickly began to move again and
stimulation led to the secretion of YS. It was therefore concluded that calcium was required
in some fashion for the exudation of yellow stuff, although the sites of Ca* action were
unable to be determined.
A comparative electrical study was conducted using C. ligatum, a species that does
not display defensive secretions of any kind. Shocks were delivered to the same positions
described above but no secretion was seen.
DISCUSSION
Electrical Stimulation
Since the hypobranchial gland is located in the mantle cavity, physical contact of the
predator with the gland is not a method by which secretion of YS would be stimulated.
This implicates the presence of a neuronal connection between the gland and another body
region that is able to contact the predator. In order to test if such a connection exists,
electrical stimulation of the snail without its shell was employed. Shocks to the epipodial
tentacle region caused secretion where as shocks to the cephalic tentacles do not. This
suggests that not only is the secretion of YS under neuronal control, but the epipodial
tentacle region is involved.
Yellow cells and secretion
Yellow cells were found throughout the hypobranchial gland in the tissue
sections. However, there were certain areas that seemed to have a greater density of cells
than others, specifically within the folds of the gland (Fig. 2).
One of goals of this study is to discern a model for how these yellow cells secrete
their YS. Taking the electrical stimulation of the YS into account as well as the histological
analysis, it is possible to hypothesize as to the type of secretion that is occurring. In
visualizing YS, countless intact vesicles are seen. Conventional, or merocrine, secretion.
where a cell’s secretory vesicles fuse with the plasma membrane, is not very likely since
the contents of the vesicles, and not the vesicles themselves, are released (Alberts et al.,
1994; Maximow, 1930). Therefore, this type of secretion can be fairly decisively negated.
Another type of secretion, apocrine secretion, seems somewhat more plausible
(Maximow, 1930; Andrew, 1959). In this system, the top of the cell is weakened by
proteases from within the cell and eventually ruptures, allowing some of the cellular
contents to be released. Äfter release, such cells reseal and remain alive. In order for this
system to work, the release of proteases must be tightly controlled so as to keep the cell
viable. A normal characteristic of this type of secretion is a long delay between a stimulus
and the actual secretory event due to the tight control required. The first YS was seen being
secreted after 0.86 seconds (Figs. 5.5 & 5.6). Although, surely the system operates faster
than this since this time was calculated from the first moment that YS was seen moving
over the rectum. The bright color of the gland and the exudate did not allow for
differentiation prior to the time the YS had been secreted and transported away from the
hypobranchial gland. The time course necessary for apocrine secretion would seem to be
too long for such a rapid response.
There remains only one other general type of secretion that the snail could be using,
holocrine secretion (Andrew, 1959). In this type of process, cells are lysed and their entire
contents released. Since the entire cell is secreted, control over the amount of protease
action is not as tight as in apocrine secretion. Only the secretory cells with their apical
membranes bulging beyond the edge of the hypobranchial gland would secrete so that
when the cell lyses, the cellular contents empty into the mantle cavity (Fig. 3.1). Figure
3.2 shows a yellow cell with no apical membrane that seems like it is actively secreting at
the time of fixation.
Ciliated cells
Once the YS is secreted, it must move to the aperture of the shell to exert its effect
on the attacker. Cilia are used to accomplish this purpose. They seem to cover most of the
gland, including openings to minuscule crevices and seem be laid out in such a way as to
create channels of flow for the secreted YS over the hypobranchial gland (Fig. 4).
CONCLUSION
This study has been able to discern much about the secretion of YS from the
hypobranchial gland. There remain a few questions to be answered and experiments to be
run, however. Perhaps most importantly, a nuclear dye needs to be used to determine
whether there are nuclei in YS or not. If nuclei are found in significant numbers, this
would provide further evidence supporting the hypothesis that holocrine secretion is
occurring in the release of yellow stuff. SEM and TEM studies would be beneficial, as
well. Finally, since it has been determined that the secretion of YS is under neuronal
control, further histological and electrical stimulation studies should be conducted to
determine if there is a hypobranchial nerve and if so what neurotransmitter/hormone serves
as the trigger for this system.
ACKNOWLEDGMENTS
I would like to thank the entire Gilly lab and especially Professor W.F. Gilly, Dr.
Jonathan Sweedler, Dr. Thomas Preuss, Mat Brock, and Taylor Liu for their tremendous
efforts and time expenditure on my behalf. I would also like to thank Natalie Lu and
Professor Jim Watanabe for their help and support. This project was conducted for
Biology 175H at Hopkins Marine Station (Pacific Grove, CA). Permission is granted to
Stanford University to use the citation and abstract of this paper.
Literature Cited
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D. 1994. pp.626-
630 in Molecular Biology of the Cell. Garland Publishing, New York.
Andrew, Warren. 1959. p. 32 inTextbook of Comparative Histology. Oxford University
Press, New York.
Bryan, P.J., McClintock, J.B., and Hamann, M. 1997. Behavioral and Chemical
Defenses of Marine Prosobranch Gastropod Calliostoma canaliculatum in Response to
Sympatric Seastars. J. Chem. Ecol. 23:645-658.
Denny, M.W. 1989. pp. 337-366 in E. Chantler and N.A. Ratcliffe, eds. Mucus and
Related Topics, The Company of Biologists Limited, Cambridge, England.
Fretter, V. and Graham, A. 1962 p.122 in British Prosobranch Molluscs: Their Functional
Anatomy and Ecology, Adlard & Son Ltd., Dorking.
Hyman, Libbie H. 1967. p.195 in The Invertebrates: Mollusca I. McGraw-Hill, Inc. New
York.
Maximow, Alexander. 1930. ed. W. Bloom. p. 14 in A Textbook of Histology. W.B.
Saunders Co., Philadelphia.
Roller, R.A., Rickett, J.D., and Stickle, W.B. 1995. The hypobranchial gland of the
estuarine snail Stramonita haemastoma canaliculata (Gray) (Prosobranchia: Muricidae): a
light and electron microscopical study. American Malacological Bulletin 11(2):177-190.
Fig. 1
Fig. 2
Fig. 3
Fig. 3.1
Fig. 3.2
Fig. 4
Fig. 5
Fig. 5.1
Fig. 5.2
Fig. 5.3
Fig. 5.4
Fig. 5.5
Fig. 5.6
FIGURE LEGENDS
A photograph taken by fluorescence microscopy of YS. The intact vesicles
are notably fluorescent and visible.
Section of hypobranchial gland stained with multiple stain. The arrow
points to a cluster of yellow cells. This section was Zum thick.
3um sections stained with either toluidine blue or multiple stain viewed
under oil immersion.
A typical cross section of the hypobranchial gland stained with toluidine
blue. The smaller arrow points to a white vesicle-filled cell. The larger
arrow points to a typical yellow cell. The nucleus of this cell can be clearly
seen in the basal end and the apical end can be seen protruding into the
mantle cavity.
A cross section of the hypobranchial gland stained with multiple stain. The
arrow points to a cell with no apical membrane and what looks like YS
being secreted from it. It is hypothesized that this cell was secreting at the
time of fixation.
A section stained with multiple stain showing a ciliated cell (arrow) at the
opening of a deep cleft in the tissue. There is another ciliary cell located
directly above this one. This location is typical.
Frames from video recorded electrical stimulation experiments. All pictures
were obtained through video analysis software.
Frame showing hypobranchial gland immediately prior to direct electrical
stimulation.
Frame showing the hypobranchial gland 3.0 seconds after direct
stimulation. YS can be seen flowing from the gland (arrows).
Frame showing hypobranchial gland immeadiately prior to stimulation of
right cephalic tentacle. The rectum can be seen in the forefront.
Frame showing the hypobranchial gland after stimulation of the right
cephalic tentacle. No secretion can be detected.
Frame showing hypobranchial gland immediately prior to electrical
stimulation of epipodial tentacle region. The rectum is in the forefront of the
picture.
Frame showing the hypobranchial gland 3.0 seconds after stimulation of
epipodial tentacle region. YS can be seen flowing from the gland and over
the rectum in definite ciliary pathways.
Figure
Bar = 50um
Bar = 100um
Figure 2
Figure 3
Figure 3.1
Figure 3.2

All bars = 40um
Figure 4
Bar = 50 um
Figure 5.1
Figure 5.2
Figure 5
Figure 5.3
Figure 5.4
Figure 5.5
Figure 5.6