Josh Rapport: Mantle flap function in Olivella biplicata
Abstract: The neogastropod mollusk Olivella biplicata possesses an extension of the mantle
fold referred to as the mantle flap. Its function is undetermined. The structure extends from
mantle tissue dorsal to the siphon and lays flat against the exposed top surface of the animal's
shell, rarely covering more than approximately 20% of the shell surface. It may extend as far
back as the apical whorl of the shell and can be fully retracted. Excised flaps begin
regeneration within one week. The structure plays no part in the light sensing ability of the
nocturnal snail, but may affect its ability to sense water-borne chemicals. Olivella with their
mantle flaps artificially removed locate crushed Mytillus roughly one hour slower than
unmanipulated counterparts (P£0.01). Scanning Electron Microscopy (SEM) and light
microscopy reveal dense, motile cilia lining the lateral edges of the flap. SEM of the surface of
the structure in direct contact with shell makes visible possible sensory cilia. No evidence was
found for the potential role of the flap in shell cleaning. A program for future study directed at
elucidating the function of the mantle flap is suggested.
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Josh Rapport: Mantle flap function in Olivella biplicata
Introduction
The neogastropod snail Olivella biplicata lives on sandy substrate from the low intertidal
zone to as deep as 50m. It ranges from Vancouver Island, British Colombia to Bahia Magdalena,
Baja California, and can reach an abundance of up to 650 individuals per m’ (Abbott and
Hadderlie, 1980). The animal is nocturnal; it buries itself a few millimeters under the sand during
the day and comes up at night to scavenge for drift kelp and a variety of live and dead animals.
Previously reported behaviors for Olivella include: intertidal zonation (Edwards, D. C., 1969a),
reproduction and mate sensing (Edwards, D. C., 1968), a predator escape response (Phillips, D.
W., 1977a; Edwards, D. C., 1969b), and a light/dark response (Phillips, D. W., 1977b), and a
feeding response briefly mentioned by Phillips (1977b).
The snail possesses a large foot for crawling and burying in the sand and a siphon for
pulling water across a single gill and osphradium. The osphradium lies adjacent to the gill in the
mantle cavity and has been implicated in olfaction for other prosobranch species (Burke, W. R.
1964; Copeland, M., 1918). A flap-like extension of mantle tissue lays flat on top of the shell,
exposed unless disturbed, in which case it retracts. I have seen it extended in various orientations,
but how the flap moves into different orientations is not clear.
This study combined an ultrastructural analysis with a behavioral study to investigate the
role of the mantle flap. Reported here are possible roles the flap plays in light detection,
chemoreception, and shell cleaning.
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Josh Rapport: Mantle flap function in Olivella biplicata
Materials and Methods
Scanning Electron Microscopy (SEM and Light Microscopy (LM)
Mantle flaps were visualized using a variety of techniques, and then analyzed for
functional structures.
1 made squash preps of freshly excised mantle flaps for compound light microscopy.
Water-immersion lens allowed visualization of the tissue, without the negative effects of the
squash prep.
Mantle flaps were fixed for scanning electron microscopy by two methods employing
different fixatives. The best results were obtained with tissue fixed by the procedure given for
fixation of external tissues by Dobbs (1974-1978), which consisted of treatment with
gluteraldehyde without post-treatment in osmium tetroxide. Fixation in Parducz’ fixative as
described by Phillips (1976) involved treatment with mercuric chloride and osmium tetroxide.
The procedure lysed the tissue surface, so these preps were discarded for this study.
Behavioral Experiments
Olivella were collected subtidally from sandy substrate in the Hopkins Marine Life
Refuge, Pacific Grove, California. Specimens remained for a maximum of two weeks in a
49x49x32cm aquarium supplied with a constant flow of unfiltered sea water. Throughout the
study period, aquaria water temperatures paralleled environmental temperatures, and snails were
not fed.
For experiments involving snails without flaps, the structure was severed as close to the
shell aperture as possible. Snails were anesthetized in isotonic MgCl,. Survival rate for the
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Josh Rapport: Mantle flap function in Olivella biplicata
procedure after one month was 100%. In all experiments control snails with flaps were of
comparable size to those without flaps.
Light Response
To test the role of the flap in photo-detection, 10 Olivella were placed in each of 6
32x17x8cm plastic tubs, filled with 2cm of sand and supplied with a constant flow of sea water.
Half of the tubs contained snails without flaps. After snails had fully buried (1 hr.), tubs were
wrapped with foil, and snails were left in darkness for ½, 1, or 2 hours. I then removed the foil
and immediately tallied the number of snails visible. Additional counts were made at 2, 3 and 10
minutes after exposure. The experiment was repeated 3 times for both treatment groups - flap
and no flap - and treatment groups were systematically rotated between tubs to prevent
confounding effects of variation between different tubs. To account for varying activity levels
due to different ambient light intensities and time of day, I performed experiments under a range
of initial conditions, such as cloudy, sunny, morning, noon, and afternoon.
Feeding Response
Snails with and without flaps were placed in separate 34x30x13cm tubs, 41 individuals in
each. Several small, whole mussels were crushed and randomly dropped, shell and all, into each
tub. Empty mussel shells were placed in a third tub to control for disturbance. Snails were
monitored for activity over a period of 2 hours. Initially I counted the number of snails
performing various behaviors at given time intervals, however, for data analysis, I converted
these numbers to average times per snail to locate mussel meat.
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Josh Rapport: Mantle flap function in Olivella biplicata
Shell Cleaning
As a test for the role of the flap in shell cleaning, I left snails with and without flaps in
unfiltered sea water for approximately one week, and then checked for fouling by algae or
diatom growth. I also took 12 hours of time lapse video from one snail to see how it moved its
flap across its shell. The snail was filmed indoors in a small dish. I also checked flap
regeneration for snails operated on 1 and 3 weeks earlier.
Results
Scanning Electron Microscopy and Light Microscopy
An ambiguous network - either extracellular matrix or artifact - was strewn across both
the top and bottom surfaces of the flap. SEM of the bottom surface of the flap (Fig. 3a) revealed
a dense packing of relatively long (approx. 4um) cilia lining the lateral edges of the flap (Fig. 3b
and 3c). At high magnification, raised spots of concentrated, short (approx. 1.5um), possibly
sensory cilia or microvilli peppered the otherwise smooth bottom surface of the flap (Fig. 3d).
The cilia were seen under compound light microscope as well. Cilia visible at 100x beat
in uncoordinated waves. Small organic particles that contacted the cilia spun in place in squash
preps as well as water-immersion preps, caught by the small water currents created by the cilia.
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Josh Rapport: Mantle flap function in Olivella biplicata
Light/Dark Response
The number of snails visible at the surface after ½, 1, and 2 hours is plotted in Fig. 1. The
emergence rate of snails with flaps does not differ significantly from those without flaps
(ANOVA; p = 0.17). The subsequent reburial response (Fig. 2) again shows no significant
difference between snails with and without their flaps (ANOVA; p = 0.59)
Feeding Response
Snails did not respond consistently to food stimulants, but for one successful experiment
the number of snails feeding at given time intervals was compared between snails with flaps and
snails without flaps. Snails with their flaps located the food, on average, about one hour faster
than snails without flaps (T-test; p « 0.001). Empty-mussel-shell control snails were inactive
throughout the experiment.
Shell Cleaning And Flap Regeneration
No algae or diatom colonies developed on any snails’ shells in one week, and the snail in
the 12 hr. time-lapse recording did not move its flap appreciably. Regeneration had begun after
only 1 week (Fig. 4a). Äfter three weeks, flaps had about half regenerated (Fig. 4b).
Discussion
Excising the mantle flap does not affect Olivella's light sensing ability. This result
indicates that the flap is not light sensitive, and the light sensory ability resides elsewhere. A
comparison between this experiment and the light/dark response as reported first by Phillips
(1977b) implies that population density may affect individual snail behavior. Snail density in
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Josh Rapport: Mantle flap function in Olivella biplicata
Phillips’ experiment was 645 individuals per m’, which is at the upper limit of naturally
occurring densities (Abbott and Hadderlie, 1980). After 2 hours of artificially imposed darkness,
only 53% of Phillips’ snails had emerged. Snails in my experiment were packed about 1/3 as
densely, and 95% had emerged after only 1 hour. The effects of between-snail interactions may
have considerable consequences for behavioral studies, ecology, and other processes governed by
snail behavior.
The role of the flap in shell cleaning remains ambiguous. The negative results from the
fouling experiment and time lapse video does not exclude the possibility that over a longer
period, fouling and flap movement will occur. Nor have I tested whether the onset of fouling
stimulates the flap to clean the shell. Conversely, the nature of the snail's lifestyle may prevent
fouling. The sand could scrape the shell clean, or even polish it, or the snails' nocturnal pattern
could prevent algae from growing, since photosynthesis cannot occur at night. Further study is
needed.
In the feeding experiments, control snails located chemical stimulant sources faster than
snails without flaps, indicating that the flap has chemosensory capabilities. Although I was
unable to run controls for the effects of injury or treatment with MgCl,, snails manipulated for
the light/dark experiments showed no aversion to injury or MgCl,. In preliminary experiments,
after removing the flap, manipulated snails displayed normal burial and activity patterns. These
observations suggest that loss of flap functionality reduces the snails' sensitivity to chemical
stimulants.
The flap’s ultrastructure also suggests chemosensory function. The ciliated band around
the flap resembles the cilia found in the squid olfactory organ (Gilly and Lucero, 1991), in which
beating cilia hold chemical stimulants close to a central chemoreceptor. The flap has no obvious
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Josh Rapport: Mantle flap function in Olivella biplicata
central chemoreceptor, but sensory cells may reside underneath the motile cilia. The next step is
to take cross sections of the flap or remove the motile cilia with protease to analyze what kinds of
cells exist concealed by the ciliary mat.
Since the osphradium is most likely a chemosensory organ (Copeland, 1918; Burke,
1964), the flap probably enhances olfaction or provides a backup chemosensor. Olivella often
pull their siphons below the surface of the sand (personal observation), which may make it
difficult to draw water across the osphradium. Having a second chemosensor exposed directly to
the surrounding medium could optimize food sensing. This may be vital over the sparsely
inhabited sandy substrate where food is scarce (Ricketts et al., 1985).
The short regeneration time and low fatality for removal of the flap are consistent with
this model of the flap’s use. As exposed soft tissue, the flap is vulnerable to injury. If the animal
has evolved a need to risk the structure, we should anticipate that its loss would not jeopardize
the animal’s health. This is true as evidenced by zero fatality for flap removal. At the same time,
if the structure increases the animal’s chances of survival, we should, and do, see fast
regeneration.
Electrophysiological techniques may provide the best opportunity to establish the
chemosensory capabilities of the flap. Large, easily accessible nerves extend from the cerebral
ganglia (personal observation), providing the opportunity for nerve staining and visualizing
innervation patterns. Potentially, one could record activity from the nerves and determine what
stimulates action potentials, although whole cell recordings of flap neurons may be more feasible
(Gilly, W. F., personal communication).
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Josh Rapport: Mantle flap function in Olivella biplicata
Conclusion
The mantle flap of Olivella plays no role in sensing light, but probably does play a role in
food detection and olfaction in general. The latter is questionable, but makes sense given the
ecology of the open ocean sandy beach. To resolve whether the flap is olfactory, I recommend
fürther visualization of the ciliary band around the flap, and the nerves innervating it. In addition.
electrophysiological techniques could contribute valuable insights. If the flap were found to be
chemosensory, the next step would be to determine which stimulants cause the strongest
responses. The structure may have multiple functions, and though the flap does not appear to
clean the shell, I suggest further exploration of this option as well.
Acknowledgments
I would like to thank Dr. Stewart Thompson, Dr. William Gilly, and Dr. David Epel for their
help, insights, and consultations. I would especially like to thank Chris Patton for his expertise
with scanning electron microscopy and his patience. I am most grateful to Dr. Jim Watanabe for
his invaluable guidance and the many hours he devoted to this project.
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Josh Rapport: Mantle flap function in Olivella biplicata
Literature Cited
Abbott, D. P., and Hadderlie, E. C. (1980) Prosobranchia: marine snails. In Intertidal
Invertebrates Of California (ed. R. H. Morris, D. P. Abbott, and E. C. Hadderlie). pp. 290-92.
Stanford University Press. Stanford, California.
Burke, W. R. (1964). Chemoreception by Tegula funebralis. Veliger. 6 (Supp.). 17-20.
Copeland, M. (1918). The olfactory reactions and organs of the marine snails Alectrion obsoleta
(Say) and Busycon canaliculatum (Linn.). J. of Exp. Zool. 25(1). 177-227.
Dobbs, G. H., (1974-1978). Soft tissue of marine teleosts. In Principles And Techniques Of
Scanning Electron Microscopy (ed. M. A. Hayat). pp. 73-75. New York. Van Nostrand Reinhold
Co.
Edwards, D. C. (1968). Reproduction in Olivella biplicata. Veliger. 10. 297-304.)
Edwards, D. C. (1969a). Zonation by size as an adaptation for intertidal life in Olivella biplicata.
Amer. Zool. 9. 399-417.)
Edwards, D. C. (1969b). Predators on Olivella biplicata, including a species-specific predator¬
avoidance response. Veliger. 11. 326-33.)
Gilly, W. F. and Lucero, M. T. (1991). Behavioral responses to chemical stimulation of the
olfactory organ in the squid Loligo opalescens. J. Exp. Biol. 162. 209-229.
Phillips, D. W. (1976). A scanning electron microscope study of sensory tentacles on the mantle
margin of the gastropod Acmaea (Notoacmea) scutum. Veliger. 19. 266-71.
Phillips, D. W. (1977a). Avoidance and escape response of the gastropod mollusk Olivella
biplicata (Sowerby) to predatory asteroids. J. Exper. Mar. Biol. Ecol. 28. 77-86.
Phillips, D. W. (1977b). Activity of the gastropod mollusk Olivella biplicata in response to a
natural light/dark cycle. Veliger. 20. 137-43.)
Ricketts, E. F., Calvin, J., Hodgepeth, J. W. (1985). Open-coast sandy beaches. In Between
Pacific Tides; fifth edition (ed. Phillips, D. W.). pp. 249-265. Stanford University Press.
Stanford, California.
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Josh Rapport: Mantle flap function in Olivella biplicata
Figure Legends
Figure 1. Number of Olivella at the surface after being treated with a half hour, an hour, and two
hours of darkness. Snails with flaps were compared to snails without flaps, and no significant
difference was found (ANOVA; p = 0.17). A snail was counted as exposed if any portion of its
shell was visible for the numbers in this graph. Snails were also counted when half or more and
100% of their shell was exposed, and no significant difference was found.
Figure 2. Number of Olivella buried as a percentage of the number visible immediately after
exposing the snails to daylight. Counts were taken 2, 5, and 10 minutes after exposure to
daylight, and snails with flaps were compared to snails without flaps. No significant difference
was found (ANOVA; p = 0.59).
Figure 3. Length of time it took to find the mussel, for each snail that did find it, was converted
from the number of snails feeding at each time period. Note that snails were not timed
individually, but counted at set intervals. Thus the averages are most likely overestimated.
However, given the large significance (T-test; p « 0.001), the trend should hold for the actual
averages as well.
Figure 4. The ventral surface of the mantle flap of Olivella biplicata. This surface is in direct
contact with the shell in vivo. (A) The flap extends from the top left, where the structure is
attached to the mantle in the live animal (some severed mantle tissue is labeled M), down right to
the tip of the flap (T). (B) An enlargement of the boxed area in A reveals a band of cilia along the
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Josh Rapport: Mantle flap function in Olivella biplicata
edge of the mantle flap. (C) An enlargement of the boxed area in B shows a dense mat of cilia.
(D) A small area inward to the ciliary band, marked by the small circle in B, reveals a
concentration of microvilli, function unknown.
Figure 5. Photographs of regeneration for mantle flaps of three snails. (A) Regeneration 1 week
after excising the flap. (B) Regeneration 3 weeks after excising the flap. (C) Normal flap, for
comparison.
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Josh Rapport: Mantle flap function in Olivella Biplicata
Figure 5
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Josh Rapport: Mantle flap function in Olivella biplicata