OLFACTORY ULTRASTRUCTURE COMPARISON OF HERBIVOROUS AND
CARNIVOROUS TERRESTRIAL GASTROPODS

By

Amy Jo Albin

A Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

Master of Science

Zoology

2011

ABSTRACT

OLFACTORY ULTRASTRUCTURE COMPARISON OF HERBIVOROUS AND
CARNIVOROUS TERRESTRIAL GASTROPODS

By
Amy Jo Albin
The study of gastropod neuroanatomy and behavior has contributed
greatly to the understanding of various subjects across a wide range of phyla and
disciplines. Unfortunately, a vast number of these studies focus on only a few
model organisms, which may not be representative of gastropods as a whole.
One deficiency of the current research is the focus on herbivorous species while
all but ignoring what data carnivorous species could yield. This study aimed to
examine the ultrastructure of the three main chemosensory areas - the posterior
and anterior tentacle tips along with the labial palps - in two herbivorous and two
carnivorous species to see if the differences in structures could account for the
differences in foraging behavior. Though several previously undescribed
structures were noted and described - including a matrix layer, circular
membrane structures and ciliated tufts - these structures did not appear to
correlate with foraging behavior. Additional study is needed to discern the nature
of these structures and to further describe the possible differences between
herbivorous and carnivorous land snails.

Copyright Notice

Copyright by
Amy Jo Albin
2011

Dedication

I would like to dedicate this publication to my wonderful husband and my loving
parents who have supported me throughout this whole experience.

iv

Acknowledgements

I would like to thank all of the staff at the Center for Advanced Microscopy,
including Dr. Stanley Flegler, Carol Flegler, Alicia Pastor, Melinda Frame and
Ewa Danielewicz, for all of their help and guidance throughout my research.
Without your assistance I would not have made it half so far in this journey for my
degree. I would also like to thank my committee members, Heather Eisthen,
Weiming Li, and Alicia Pastor for advice and support. Lastly, I would like to thank
Dr. James Atkinson for allowing me this chance to pursue this degree and to gain
so many useful skills. Also, I would like to thank Dr. Ron Caldwell for collecting
and sending me the Haplotrema concavum specimens and for Dr. Eugene Keferl
for collecting and sending me the Euglandina rosea specimens.

v

Table of Contents

Chapter 1: Introduction…………………………………………………..……………..1
Chapter 2: Materials and Methods………………………………….…………………6
Chapter 3: Results…………………………………………………………….……….11
Chapter 4: Discussion………………………………………………..……………….36
Appendix ………………………………………………………………..……...………46
References …………………………………………………………………………….51

vi

List of Tables

Table 1: Table of Ultrastructural Elements by Species…………..………………..34

vii

List of Figures
Figure 1: General Anatomy of a Snail Head……………………………..……..……5
Figure 2: General Epithelial Cell Morphology………………………………..……..12
Figure 3: Detail of Matrix of Euglandina rosea……………………………………..15
Figure 4: Anguispira alternata SEM Images………………………...……….……..16
Figure 5: Anguispira alternata SEM Images of Ciliated Structures…………..…..17
Figure 6: Anguispira alternata TEM Images………………………………………..18
Figure 7: Anguispira alternata TEM Images of Ciliated Structures of the Labial
Palps……………………………………………………………………..……..19
Figure 8: Arion subfuscus SEM Images……………………………...…….……….21
Figure 9: Arion subfuscus TEM Images of the Posterior Tentacles……………...22
Figure 10: Haplotrema concavum SEM Image……………………………..………24
Figure 11: Haplotrema concavum TEM Images…………………………..………..25
Figure 12: Euglandina rosea SEM Images……………………….…………………27
Figure 13: Euglandina rosea SEM Images of Ciliated Structures………………..28
Figure 14: Euglandina rosea TEM Images of the Posterior Tentacles………..…31
Figure 15: Euglandina rosea TEM Images of Ciliated Structures of the Labial
Palps………………………………………………………….…………………32
Figure 16: Euglandina rosea TEM Images of Cellular Spaces Containing Matrix
Material in the Posterior Tentacles………………………….……………….33
Figure 17: Diagram of Ultrastructural Elements Examined Across Species….…35

viii

Chapter 1: Introduction
Escherichia coli, Chlamydomonas reinhardtii, Caenorhabditis elegans,
Drosophila melanogaster and Mus musculus is a short list of organisms familiar
to most scientifically minded persons. These few species belong to a small
group of organisms that are instrumental to numerous fields of research and are
close to many researchers’ hearts. They are model organisms; they are easily
cultured in labs, typically of small size, and have a short life cycle. These traits
lend themselves to the laboratory and experimental design; therefore these key
species are used by scientists worldwide in every imaginable aspect of research
including genetic analysis, behavioral studies and medical research. Large
amounts of data have been compiled on each of these species and great
advances in knowledge can be attributed to the use of model organisms.
Sometimes the use of the organism itself has spurred the growth of new fields.
However, there is a danger of having so much understanding of only a few, or in
some cases a single species. The danger of dependence on a few model
organisms rests on over generalization and overlooking biological phenomena
that could provide significant insights. What holds true for one species, may not
hold true in even closely related species, let alone apply to distantly related taxa.
Without proper investigation, false assumptions can stifle future scientific study.
A common example is a positive clinical test of a drug in mice may not yield the
same results with other rodents, primates or human subjects. The dangers of
biased models was recently discussed in Science (Wald and Wu, 2010).
Primarily male rodents were used for a wide range of studies because of their

1

ease of use and the increase in cost and time associated with including females
in the study design. They note one high profile problem that was created by the
bias: several drugs tested only on male rodents have been pulled from the
market by the FDA because of their adverse reaction in women.
Though less alarming to the general public, the field of gastropod research
is no exception when it comes to similar biases. Species like Aplysia californica,
Helix pomatia, Limax maximus, Cornu aspersus and Achatina fulica have been
used for decades for behavioral and neural research providing important
information. For example, the study of Aplysia’s giant neurons has led to a better
understanding of basic neuronal function in both invertebrates and vertebrates
(Chase, 2002). Another major component of gastropod research has been in the
field of learning and feeding behavior (Croll and Chase, 1977). The focus on A.
fulica in such research is driven by the fact that A. fulica is an agricultural pest
and invasive species (Civeyrel and Simberloff, 1996).
Gastropods are particularly well-suited for the study of chemoreception as
their feeding behavior relies heavily upon it. Gastropods sense their world
primarily through gustatory and olfactory means. Their eyes, if they have them at
all, range from patches of light sensitive simple optic cups to eyes containing
lenses. In most cases, light sensing areas seem to be mostly related to circadian
rhythms (Chase, 2002) and light/dark recognition (Andrew and Savage, 2000).
The field of gastropod vision has some of the same model organism issues as
gastropod chemoreception in that research underrepresents pulmonate species,
and those terrestrial gastropods that have been researched are all from a single

2

genus, Cornu (formerly in the genus Helix) (Zieger and Meyer-Rochow, 2008).
While chemosensory studies have been concerned with more than a single
genus, the number of species investigated has remained too low (Achatina,
Limax, Cornu). The chemosensory capabilities of snails has been studied
through various means, including lesions studies, studies that isolate and directly
stimulate the sensory areas, electrophysiological and behavioral studies (Chase
1986; Chase and Croll,1981; Ermentrout et al., 2004; Friedrich and Teyke, 1998).
These conclusions about behavior and sensory capabilities that are based on a
few herbivorous species have been generally assumed to fit other species.
However, those species where diet requires other methods for gathering food
may experience a different sensory world.
A hint that there may be some behavioral and physiological differences
between animals with carnivorous and herbivorous strategies can be found in the
work of Shearer and Atkinson (2001). They found that the herbivore Anguispira
alternata (Say) could navigate around scent-permeable barriers to find a food
source, while the carnivore Haplotrema concavum (Say) could not. Additionally,
the carnivore had a tendency to follow prey’s slime trails to a food source, while
the herbivore rarely followed conspecifics’ slime trails to a food source. Even a
marine carnivorous species, Navanax inermis, was found to track prey via slime
trails and not distance chemoreception (Murray, 1971). This suggests that there
may be some structural differences in sensory structures located in the three
main chemosensory organs in herbivorous and carnivorous land snails. In these
organisms the principal chemosensory structures are the tips of the large optic or

3

posterior tentacles, the tips of the short anterior tentacles and thickened areas
associated with the mouth, the labial palps (Chase, 2002). Therefore these are
logical places to perform a comparative study seeking structural components of
the differences in behavior between herbivorous and carnivorous gastropods
(Figure 1).
The aim of the research reported here was to examine the surface
epithelium of three key areas involved in snails’ chemoreception: the surface of
the posterior tentacle tips, anterior tentacle tips and labial palps of four terrestrial
snail species. Two herbivorous species, Anguispira alternata (Say) and Arion
subfuscus (Draparnaud), and two carnivorous species, Haplotrema concavum
(Say) and Euglandina rosea (Ferussac), were examined by means of
transmission electron microscopy (TEM), scanning electron microscopy (SEM)
and confocal laser scanning microscopy (CLSM) to better understand if snails
with different foraging behaviors present morphological differences in their
chemosensory structures.
TEM and SEM were used to study the structure of the endothelial surface
(Kunz and Haszprunar, 2001) of the cephalic tentacles in other gastropod
species.

4

e

e

pt

pt

m

at

at
lp

lp

Figure 1 General Anatomy of a Snail Head: This diagrammatical representation
of a snail’s head as seen from the front shows the posterior tentacles (pt),
anterior tentacles (at), labial palps (lp), mouth (m) and eyes (e).

5

Chapter 2: Materials and Methods
Specimen Collection
The herbivorous species, A. subfuscus and A. alternata, were collected in
local wood lots in Ingham County, Michigan, and used to create a lab colony.
The carnivorous species, H. concavum and E. rosea, were generously collected
by Dr. Ron Caldwell in Arkansas and Dr. Eugene Keferl in Georgia.

Cultures
Both herbivorous species were cultured in either clear plastic containers
(9.4cm x 15.0cm x 30.0 cm) or small glass aquaria (7.5cm x 16.0cm x 24.5cm).
Approximately ½ inch of potting soil was used as a substrate, while a piece of
broken flower pot was placed in the enclosure for cover. A small piece of wood or
dry leaf litter was also included to help maintain proper moisture levels and to
create a more natural environment. The carnivorous species were kept in similar
cultures, but with one individual per culture to eliminate the chance of
cannibalism.
Herbivorous species were fed sliced carrot and the soil moistened once a
week. Any uneaten carrot was removed at the end of the week before new food
was added. A. alternata was separated into three cultures: adult, juvenile and
young. When a juvenile grew large enough, it was moved into the adult culture.
Juveniles were also used to feed the carnivores (see below). The A. alternata
adult culture also produced fertile eggs several times during this period. Any live
young snails were removed from the adult culture and placed in a separate

6

young culture. When they grew to juvenile size, they were placed in the
appropriate culture. About a dozen specimens were used for this study. A.
subfuscus did not reproduce in the standard culture and was, therefore, used for
analysis within a month or two of collection. A. subfuscus was very abundant.
These were used to develop and standardize techniques for processing all
specimens.
The carnivores were fed either a single A. subfuscus or A. alternata every
2-3 weeks but still watered weekly. Occasionally other locally available
gastropods such as the garden slug Deroceras reticulatum were used to
supplement their diet. Due to limited numbers, two specimens of H. concavum
and three specimens of E. rosea were examined.

Anesthesia
Proper extension of the areas of interest (posterior and anterior tentacles
and labials palps) is of particular importance in the scanning electron microscope
(SEM) investigation of the morphology of the olfactory epithelium, and to a lesser
extent transmission electron microscopy (TEM) and confocal laser scanning
microscopy (CLSM). Several techniques were attempted before satisfactory
procedures were reached (several of these are described in the appendix).
A combination of the submersion in chilled water and 5% succinylcholine
chloride was devised for proper extension and anesthesia. Approximately 5
drops of the 0.5% succinylcholine chloride in a small finger bowl filled with chilled
water had a fairly good result, with some extension of the tentacles; the animal’s

7

reaction time was usually decreased sufficiently enough for careful dissection.
Interestingly, neither cold water nor 5% succinylcholine chloride seemed to have
any effect on E. rosea, which contracted in their shells and would not emerge
while immersed. Once removed, however, the E. rosea actively extended their
tentacles and started to explore the surrounding area. This allowed rapid
removal of the head with a sharp razor blade. Once the head was removed, if the
tentacles were partially retracted, it was possible to force extension with careful
use of a pair of fine forceps by slowly and gently moving from the base toward
the tip of the tentacle so as to not damage the tissue. It was essential not to use
the tips of the forceps, as they tended to crush or tear the tissue. The fixative
acrolein (Jongeblored et al., 1999) facilitated tentacle extension when used after
the removal of the head and before removal of mucus.

Scanning Electron Microscopy (SEM)
One challenge to producing clear imaging of gastropods is the mucus coat
that can block the electron beam from the surface of the organism. This was
particularly a problem with Arion subfuscus, which secretes copious amounts of
thick mucus when agitated. A method using 16% glycerol (Zaitseva and
Bocharova, 1981) to remove the mucus coat produced satisfactory results.
Removal of the mucus was improved by sonicating the samples for 5 minutes.
Because of the large size of the samples studied, three different fixation
techniques were used. Acrolein (Jongeblored et al., 1999) was used to initially
fix the cells and facilitate tentacle extension followed by 2.5% paraformaldehyde

8

and 2.5% glutaraldehyde in 0.1M sodium cocadylate buffer (pH = 7.4) to fix
proteins. After fixation, the samples were washed in 0.1M sodium cacodylate
buffer (pH = 7.4) and postfixed with 1% osmium tetroxide in 0.1M sodium
cacodylate buffer (pH = 7.4) to fix lipids. Once all fixation was completed, the
samples were dehydrated in an ethanol series and critical point dried. The
dehydration steps had to be increased because of the large size of the samples
to at least 6 hours. To increase resolution and minimize charging, the samples
were osmium coated using a NEOC-AT Osmium Coater (Meiwafosis Co., Ltd.,
Osaka, Japan). Samples were observed using JEOL 6300f and JEOL 6400V
Scanning Electron microscopes (JEOL, Japan).

Transmission Electron Microscopy (TEM)
Although not necessary for TEM, full extension of the tentacles made
interpretation easier. The main areas of interest (posterior tentacles, anterior
tentacles, and labial palps) were dissected and fixed with 2.5%
paraformaldehyde and 2.5% glutaraldehyde in 0.1M sodium cocadylate buffer
(pH = 7.4) and postfixed with 1% osmium tetroxide in 0.1M sodium cocadylate
buffer (pH = 7.4). After postfixation the samples were washed with sodium
cocadylate buffer to remove any excess osmium tetroxide. The samples were
then dehydrated using an acetone series and infiltrated and embedded in
Poly/Bed 812 epoxy resin (Polysciences, Warrington, PA). Standard resin
infiltration times were increased to 5-6 hours to insure complete infiltration. The
blocks containing the samples were polymerized at 60º C for 24 hours. Thick

9

and thin sections were obtained with a MTX Ultramicrotome (RMC, Boeckeler
Instruments, Tuscon, AZ) using a diamond knife.
The thick sections (1000 nm) were placed on a glass slide and stained
with Ready-to-Use Epoxy Tissue Stain® (Electron Microscopy Sciences, Hatfield,
PA) and were observed with a compound light microscope to select areas of
interest that would be observed with the TEM. Thin sections (~90 nm) were
collected on 200 mesh copper grids. These grids were then stained using 2%
uranyl acetate in 50% ethanol and lead citrate (Reynold’s formulation). Stained
grids were viewed on a JEOL100 CXII (JEOL, Japan) Transmission Electron
microscope at an accelerating voltage of 80 kV.

10

Chapter 3: Results
General Epithelial Cell Morphology – All Species
SEM imaging did not reveal any general cell morphology, even with
satisfactory mucus removal. Areas covered with microvilli appeared as a solid
mass, except where damaged areas allowed for a deeper view. In general, the
tips of the tentacles at low power appeared smoother than the surrounding tissue
(See Figures 4B, 8A, 10 and 12). There were only a few cases where general
morphological differences could be seen. Those cases include lone tufts of
microvilli or specialized ciliary structures that extended past the microvillar mass
(see Figures 5, 12C and 13).
TEM revealed more detailed characteristics. In general, at low
magnification, all of the tissue examined seemed to share the same basic
structures. The epithelial layer is a single layer of simple columnar cells (Figure
2). The distal edge of the cells contained a continuous brush border of microvilli.
Large nuclei, taking up roughly 1/2 to 1/3 of the cell, are most often located near
the basal edge of the cells. This single layer rested upon a basement membrane.
Neighboring epithelial cell membranes were tightly interdigitated. The thickness
of the epithelial layer varied and appeared to be the thickest at the distal tip of the
tentacle, while becoming thinner proximally (Figure 17). Epithelium containing a
microvillar brush border was found only at the distal end of the tentacles and
labial palps, and was not found on adjacent areas of the snail body. At the tip,
the surface bearing microvilli appeared to be smooth, but proximally

11

l
n

h
mt

m

Figure 2 General Epithelial Cell Morphology: TEM micrograph of the epithelial
cells showed cellular structures including the nucleus (n), heterochromatin (h),
microvilli (m), lysosomes (l), mitochondria (mt) from an E. rosea. The center cell
is uncharacteristically lightly stained but is shown because the lighter stain
makes the cellular structures easier to see. More typical staining can be seen on
its neighbors. Scale bar = 5µm.

12

invaginations were found, which coincide with the appearance of these structures
seen with the SEM.
The epithelial cells containing microvilli stained darkly in the TEM
preparation (uranyl acetate/lead citrate stain). Neighboring cells and tissues did
not appear to stain overly dark, so it is unlikely that the sections were stained
improperly. Although dark appearance occasionally made it difficult to make out
distinct cellular structures, heterochromatin was seen within the nucleus, and
cytoplasmic structures including smooth and rough endoplasmic reticulum,
lysosomes, Golgi apparatus and mitochondria were seen (Figure 2).

Anguispira alternata
SEM analysis of the surface of the tentacles showed that the tips
appear smoother than the surrounding tissue on both anterior and posterior pairs
(Figure 4). Areas where microvilli were separated showed tightly packed
microvilli, but they do not appear to be very long. Distinct structures were visible
on either side of the mouth on the labial palps. These structures were first clearly
visible at 300x magnification as lighter specks. At higher magnification, the
structures appeared to be cilia emerging from the microvilli border and were
grouped together in small clumps in a concentric circular pattern (Figure 5).
These groups of cilia did not have any overall pattern of appearance but were
rather irregularly scattered across the area.
The TEM revealed that the tip of the posterior tentacle contained microvilli
that extended from the cell surface at the epithelial cell edge through a

13

convoluted and tightly packed matrix-like layer. The matrix layer had a well
defined sharp edge, easily distinguished between the edge of the epithelial cells
and the brush border. The matrix material stained lighter than the microvilli and
internally had a grainy texture when compared with the dark, evenly stained
microvilli. A membrane can be seen around the individual strands of matrix
material and each microvillus (see Figure 2, Note: E. rosea is the example of
matrix material because of the clarity of the images). Where the microvilli
extended out of the matrix layer, they were discretely organized parallel to one
another at the outer surface of the tentacle tip. This matrix layer contained
circular structures consisting of concentric layers of cell membrane. The circular
structures were completely covered by the matrix layer and do not appear in any
other layer. Proximally along the tentacle, the matrix disappeared and regular
microvillar bush border was found to dominate the outer edge of the epithelial
cells. No circular structures were found along the tentacle shaft. Moving more
proximally, the brush border disappeared completely, leaving a smooth
membrane surface.
The above described matrix material was not found on the anterior
tentacle tips. Rather the microvillar brush border of the tip was somewhat longer
than that found on the epithelium of the tentacle shaft. Otherwise the anterior
tentacles were similar to the posterior tentacles.
The labial palps also contained a microvillar brush border. TEM images
revealed that the ciliary projections seen in the SEM micrographs contained the
typical 9+2 microtubular doublet pattern in cross section (Figure 5). The number

14

A

m
tm
b

B

Figure 3 Detail of Matrix of Euglandina rosea: TEM of the tight matrix. Image A
shows the concise edge of the tight matrix (tm) and the brush border (b). A single
microvillus can be seen emerging from the matrix and branching into the border
(m). In B the matrix material stains lighter and has a grainy texture (black arrow)
when compared with the microvilli (white arrow). The arrows also show the
membrane that binds the structures. A scale bar =500nm, B scale bar = 200nm.

15

A

B

C

Figure 4 Anguispira alternata SEM Images: Image A shows a whole mounted A.
alternata head. Note the smooth appearance of the partial right posterior (left
posterior tentacle is contracted) and both anterior tentacles. B is a left anterior
tentacle at higher magnification, while C shows the high magnification of another
A. alternata specimen of the smooth surface that has been fractured during
processing, revealing the underlying microvilli. A scale bar = 1mm; B scale bar =
100µm; C scale bar = 5 µm.

16

A

B

C

Figure 5 Anguispira alternata SEM Images of Ciliated Structures: A shows the
labial palp area at low magnification (box). B shows box A at higher
magnification, the cilia tufts look like light specks. C shows individual tufts
(arrows). A scale bar = 1mm; B scale bar = 100µm; C scale bar = 5 µm.

17

A

c
b
tm

B

b

Figure 6 Anguispira alternata TEM Images: Image A shows the tight matrix (tm)
of the posterior tentacles tip, complete with circular structure (c) just beneath the
brush border (b). Image B shows the general microvilli of brush border (b) which
is located proximal to the distal tip. A and B scale bar = 2µm.

18

lm

A

B
Figure 7 Anguispira alternata TEM Images of Ciliated Structures of the Labial
Palps: Image A shows the loose matrix (lm) with embedded ciliated tufts (arrow).
Notice the ring of microvilli around the tuft in the upper left corner. Image B is the
cross-section of a tuft, showing the internal structure of the 9+2 microtubule
doublet. A scale bar = 2µm; B scale bar = 500nm.

19

of cilia contained in each tuft and in each ring was not constant and no
numerical pattern could be easily deciphered. The total number of cilia in each
tuft varied from 5 to 15. The tufts also appeared to have a ring of microvilli
surrounding them. These microvilli were of slightly smaller diameter than those in
the nearby brush border but not associated with ciliary tufts. These ciliated tufts
and the microvilli extended to the surface through a matrix layer, similar to that
described above for the posterior tentacle. This matrix layer is much less
organized and not as densely packed as that described in the posterior tentacle.
The edge of this loose but uniform matrix layer was less sharply defined than that
of the posterior tentacle, unlike the closely packed nature of the tight matrix
which had a discrete outer layer of tightly packed parallel microvilli. The loose
layer also contained a few small circular structures consisting of concentric layers
of membrane; however, these were less numerous and smaller than those found
in the posterior tentacles. As described in the posterior tentacles, this loose
matrix layer disappeared proximally from the area with the ciliary tufts, leaving
only a microvillar brush border, which also shortened and disappeared
proximally.

Arion subfuscus
SEM analysis of A. subfuscus revealed what appeared to be a covering of
small microvilli on the surfaces of interest, similar to that seen in A. alternata
(Figure 6). No ciliary structures were seen in the areas examined, including the
labial palps. TEM revealed a substructure similar to A. alternata, with the

20

A

B

Figure 8 Arion subfuscus SEM Images: Image A shows a whole A. subfuscus
head with the tentacles in various states of extension. Image B is the typical
sensory surface, showing the tips of the microvillar brush border found at the tips
of all four tentacles and labial palps. A scale bar = 500 µm; B scale bar = 1 µm.

21

A

tm
b
c

B

C

Figure 9 Arion subfuscus TEM Images of the Posterior Tentacles: Image A
shows the tight matrix (tm), brush border (b) and circular structures (c) of the
posterior tentacle tip of A. subfuscus. Image B shows the typical non-matrix
containing microvilli proximal to A. C shows a cluster of microvilli. A and B scale
bar = 2 µm; C scale bar = 1 µm.

22

posterior tentacle containing a tight matrix layer with embedded large circular
membrane structures (Figure 9). Microvilli extended through the matrix at the tip,
emerging in parallel rows. Characteristically, the matrix thinned and disappeared
proximally; the microvilli reduced in size, and eventually they too disappeared.
The anterior tentacle contained no matrix or circular membrane structures,
only a microvillar border. Unlike in other species, microvilli occasionally seemed
to be grouped together, but this difference in orientation was not seen in SEM
and the clustered microvilli did not appear to be different from other microvilli in
the same area.
Unlike in A. alternata, the labial palps in A. subfuscus did not have any
matrix or ciliary structures. The labial palps appeared remarkably like the anterior
tentacles. The only structures seen extending from the cell surface were
microvilli.

Haplotrema concavum
Unfortunately, the only H. concavum specimen prepared for the SEM was
destroyed through a laboratory accident before much of any analysis could be
made. The only image collected using the SEM was that of the whole head. It
can be seen in Figure 10. The tips of the tentacles (but not the labial palps)
appeared smoother than the surface of the surrounding tissue. No other
differentiation in surface morphology could be seen at low magnification;
therefore, all additional analysis for H. concavum was done using only the TEM
data.

23

r

Figure 10 Haplotrema concavum SEM Image: SEM image of H. concavum
showing the smooth appearance of the tentacle tips. Mucous strands (arrows)
can easily be seen on some of the surface areas along with the open mouth
containing the radula (r) with long slicing teeth characteristic of carnivorous
species. Scale bar = 1mm.

24

A

c

b
tm

B

Figure 11 Haplotrema concavum TEM Images: Image A shows the matrix layer
found on the posterior tentacle tip. Note the highly organized top brush border (b)
over the convoluted tight matrix layer (tm) with circular structures (c). Image B
shows the normal brush border of the labial palp, anterior tentacle and posterior
tentacle proximal to the tip. A and B scale bar = 2 µm.

25

Cross sectional views of the tip of the posterior tentacle in H. concavum
revealed microvilli that extended from the surface of the epithelial cell and
extended through a dense convoluted middle tight matrix layer to emerge at a
highly organized top layer. In this tight matrix layer, circular structures with what
appeared to be many layers of concentric circular cell membranes are seen
(Figure 11), similar to those seen in the other species. Some microvilli are
branched near the middle-top layer border. As one examined medially towards
the body, the matrix layer diminished and normal non-matrix containing microvilli
appeared. Proximally, the microvilli shortened and eventually disappeared.
The anterior tentacle contained no matrix layer, but was covered in normal
microvilli border. No other structures are seen on the cell surface.
The labial palps of H. concavum also appeared to be without a matrix
layer. The microvilli are very similar in appearance to the anterior tentacle
surface. No additional cell surface structures were found.

Euglandina rosea
Overall, the SEM analysis revealed that the tips of the posterior and
anterior tentacle of E. rosea had a smooth texture similar to the other species
examined. At higher magnifications, similar microvillar brush surfaces were seen
(Figure 12). Tufts were occasionally seen on both the posterior and anterior
tentacles (Figure 12C). Unfortunately these areas were not seen in TEM samples
and therefore the internal structure could not be examined; thus it is unknown if

26

A

B

C

Figure 12 Euglandina rosea SEM Images: Image A shows a whole E. rosea head
with its tentacles at near full extension, the labial palps lay curled along the side
of the face with the pleats along the ventral surface (arrow). Image B shows the
microvilli at a disruption of the brush border. Image C shows a tuft from the
tentacle shaft, which was only seen in SEM. A scale bar = 2mm; B and C scale
bar =2 µm.

27

A

C

B
Figure 13 Euglandina rosea SEM Images of Ciliated Structures: Image A shows
a single labial palp. The highlighted area is where the ciliated tufts were located.
Note the difference in surface texture between the highlighted area (described as
pleated) and surrounding pebble-like surface. Image B shows the density of the
ciliated tufts. C shows the roughly circular grouping pattern of the cilia (arrows)
found in box B. A scale bar = 1000 µm; B scale bar = 10 µm; C scale bar = 2 µm.

28

they are longer microvilli or cilia. SEM images of the elongated labial palps
showed tufts of cilia arranged in concentric circular patterns like that of A.
alternata. The tufts appear in a concentrated strip along the entire leading
anterior-ventral edge of the labial palps (Figure 13). This edge, in the relaxed
state viewed during SEM analysis, exhibited a distinct texture when compared
with the surrounding skin. This area appears to have a vertical rectangular pleat
pattern, while the surrounding areas were irregular and pebble-like (Figure 13A).
Whether or not this particular epithelial folding pattern is maintained when the
labial palps are extended is unknown.
Using the TEM, the tip of the posterior tentacle is shown to have a thick,
tight and dense matrix at the tip through which long microvilli extend. The matrix
did not appear to contain any circular membrane structures as described in the
other species. Areas containing dense concentrations of short microvilli
completely covered by matrix were seen in the distal tip (Figure 14). Matrix
material also was seen within intercellular pockets contained in the cytoplasm of
the epithelial cells, some of which opened to the extracellular space (Figure 16).
These areas were only found on the posterior tentacles of E. rosea.
The anterior tentacle, unlike in the other species, was found to have a tight
matrix layer similar in density and structure to the posterior tentacle. More
proximal to the body, the matrix eventually disappeared and the microvilli
appeared shorter until completely absent.
The TEM revealed images of the labial palp structures that resembled
those found in A. alternata. The loose matrix of the labial palps appeared less

29

tightly organized and packed than those found in the tentacles. There appeared
to be no circular membrane structures in either the matrix or non-matrix
containing areas. Cross sections of the tufts show characteristic cilia morphology.
The ciliary roots extend into the cell, but the depth to which they penetrate could
not be determined (Figure 15). These tufts also appear to be surrounded by a
ring of thinner microvilli, similar to those seen in A. alternata. The ciliated tufts on
E. rosea do not appear to have a specific number of cilia, as those seen ranged
from 6 to 17. Distal to the surface of the matrix the microvilli are highly organized
and parallel to each other forming a brush border. The matrix is thinner close to
the base of the labial palp where disappears, leaving the brush border which also
becomes thinner and disappears proximally.
A comparison of the structures and areas found across the species
examined is summarized in Table 1.

30

A

b

tm

B

Figure 14 Euglandina rosea TEM Images of the Posterior Tentacles: Image A
shows the tight brush border (b) and matrix layer (tm) of the posterior tentacle tip,
but without circular structures. Note the short microvilli covered by matrix (arrow).
B shows the brush border without any matrix proximal to A. A and B scale bar = 2
µm.

31

A

lm

B

C

Figure 15 Euglandina rosea TEM images of Ciliated Structures of the Labial
Palps: A shows a cross sectional view of the ciliated tufts (arrows) above the
loose matrix (lm) of the labial palp. B shows the ciliated tufts cut longitudinally
(arrows). C shows a high magnification image of the cross section of the ciliated
tuft with stereotypical 9+2 doublet internal structures. A and B scale bar = 2 µm;
C scale bar = 200 nm.

32

A

B

Figure 16 Euglandina rosea TEM images of Cellular Spaces Containing Matrix
Material in the Posterior Tentacles: A and B: TEM images of E. rosea, showing
the possible cellular origin of the extracellular matrix found on the posterior
tentacles. These kinds of structures (arrows) with matrix looking material were
not found in any of the other species examined. A and B scale bar = 2µm.

33

Posterior Tentacle
Matrix

Circular
Structures

Microvillar
Border

Anterior Tentacle
Microvillar
Border

Matrix

Aa

Labial Palp
Matrix

Ciliated
Tufts

Microvillar
Border

Yes
Loose
Yes
Yes
Yes
No
Yes
Yes
tip
tip
tip
As
Yes
Yes
Yes
No
Yes
No
No
Yes
tip
Hc
Yes
Yes
Yes
No
Yes
No
No
Yes
tip
Er
Yes
Yes
Loose
Yes
No
Yes
Yes
Yes
tip
tip
tip
tip
Table 1 Table of Ultrastructural Elements by Species: This table shows which ultrastructural elements were found by
species and area of interest. Aa = Anguispira alternata, As = Arion subfuscus, Hc = Haplotrema concavum, Er =
Euglandina
rosea

34

Figure 17: Diagram of Ultrastructural Elements Examined Across Species
A. alternata

A. subfuscus

H. concavum

E. rosea

Posterior
Tentacle
Tip

Tentacle
Shaft

Proximal
to Body

Anterior
Tentacle
Tip

Labial
Palp

Figure 17: Key : Epithelial Cells

Microvilli Tight Matrix

Loose Matrix
35

Circular Structures

Ciliated Tuft

Exterior

Chapter 4: Discussion
The hypothesis that snails with similar feeding strategies would have
similar epithelial morphology was not supported in the species examined in this
study. Every sensory area examined had similar microvilli. Other structures not
previously described in land snails were found including a matrix layer with two
different densities, the circular membranous structures and tufts of cilia. These
three structures were found in different areas in different species (Table 1), but
they did not correlate with feeding behavior. Even though the original hypothesis
was not supported by the evidence found, many new and interesting findings
suggest further study.

Microvilli of the Tentacles and Labial Palps
In all of the species used, a general pattern was seen in the tentacles and
labial palps. The distal tip of the tentacles’ epithelial cells stain darkly and the tip
surface is smooth and includes a matrix layer in all of the posterior tentacles
examined. E. rosea also exhibits a matrix layer in the anterior tentacle. The
labial palps showed a similar staining pattern at the leading ventral edge. The
labial palps of A. alternata and E. rosea contain a loose matrix. The microvilli and
cilia (when present) seem to pass through the matrix layer. Once free of the
matrix, the microvilli align and form a tight brush border. The microvilli could
occasionally be traced from the edge of the cell through the matrix, sometimes
branching where they extend through the matrix (Figures 6A, 11A and 14A).
Proximal to the tip of the tentacle tip (and proximal to the leading edge of the

36

labial palps in species that have matrix) the matrix is reduced and replaced by a
brush border of microvilli. The microvilli also reduce in length proximally until only
a smooth microvilli-free epithelium remains.
Several functions for microvilli of sensory structures have been suggested.
They may serve as chemo- or mechanoreceptors, protection for receptors
deeper in the tissue or they could merely increase cell surface area (Chase,
2002). This range of the possible functions can suggest different interpretations
of the results. Carnivores appear to follow slime trails to find a food source and
do not follow scent cues carried through the air when hunting prey (Shearer and
Atkinson, 2001). If it is assumed microvilli are involved with chemoreception, then
it would be expected to find microvilli prominently on the labial palps and, to a
lesser extent on the anterior tentacle tips as these structures brush along the
ground during trail following. Conversely, the microvilli on the posterior tentacle
tips would be reduced or nonexistent as they almost never come in contact with
the ground when trail following. The presence and morphology of microvilli did
not correlate with the preconceived assumptions about the chemosensory areas.
This suggests the function of the microvilli may not be chemosensory but may be
mechanosensory or serve as a protective covering for underlying receptors. Until
more research is done and the exact function microvilli perform on the tips of
gastropod sensory structures is determined, these questions cannot be
satisfactorily answered nor can interpreting their presence or absence in a
behavioral context be assessed. This could be accomplished by tracing the
sensory neurons from patches of microvilli to see if they are processed in known

37

regions of the brain that pertain to olfaction like the procerebrum, though exactly
what olfactory information the procerebrum processes is debated (Chase, 2002).
Emin Oztas’ (2003) paper Neuronal Tracing compiles several tracing techniques
that could accomplish this task. A more plausible method to find if
chemoreception is occurring in the microvilli is to search for some of the proteins
needed for chemical signal transduction (Hofer et al, 1999). These proteins
could be tagged using immunogold labeling techniques and then viewed using
TEM. Physiological data then could be correlated with behavioral studies in
more herbivorous and carnivorous species to see if these possible
chemoreceptors are used for just locating food, locating potential mates/chemical
signals or a combination of both. Determining what kind of chemicals these
receptors specifically detect, while outside the scope of the above described
techniques, could also be another avenue of research to better understand the
sensory world of these organisms.

Matrix
A matrix layer at the distal tip of the posterior tentacles is present in all
species. E. rosea also had such a matrix on the tip of the anterior tentacles. A.
alternata and E. rosea were the only two to have matrix on their labial palp
regions, though this matrix appeared to be a less dense than that found on the
posterior tentacle (Table 1). When present, the matrix was only found on the
distal tip of the tentacles or leading edge of the labial palps, not along the shaft of
the tentacles or in the surrounding tissue of the labial palps. The matrix forms a

38

layer between the edge of the epithelial cells and the brush border of microvilli.
The matrix can be very tightly and densely packed material with discrete edges
or loosely organized in a semi-indistinct layer. The matrix layer may originate
from within the epithelium (Figure 16). However, evidence for this was found only
in E. rosea. Thus further investigation is needed to see if the origin is intracellular
and if this holds true for other species and other matrix containing areas.
Within the matrix, membranous circular or ciliary structures can be found.
In all but two instances (E. rosea posterior and anterior tentacle tips), the
presence of the matrix coincided with the presence of either circular or ciliary
structure. The matrix may serve as a cushion or support system for long
microvilli, perhaps also protecting the circular and ciliary structures from damage.
If the matrix adds a cushion between these structures and the environment, a
likely area to find the matrix would be the posterior tentacles’ tips, as they are
more likely to brush against objects first in the environment, given their length
and location. During hunting/trail following, the handlebar-shaped labial palps of
E. rosea are actively being touched to the ground and other species trail follow to
find mates etc; but the matrix was only found on the labial palps in half the
species examined. The anterior tentacles sometimes come in contact with the
ground during foraging by all of the species examined, but only E. rosea had
matrix in this area. An extracellular matrix containing tethers increased
sensitivity to sheering forces for mechanoreception in invertebrates such as
nematodes (Fritzsch et al, 2007). This may explain the presence of the matrix in
areas important to mechanoreception, but none have yet been described in this

39

capacity for gastropods. The matrix may also play a role in scent capture similar
to that of a coating of mucus. If the presence of a matrix layer could be correlated
with confirmed olfactory structures, this would support such a hypothesis.

Ciliated Tufts and Circular Structures
In addition to the previously undocumented matrix, the ciliated tuft
structures of the labial palps of A. alternata and E. rosea had not previously been
reported in either species. Both species had groups of cilia arranged roughly in
two rings (the unit here being called a ‘tuft’). The tuft was surrounded by another
ring of microvilli specifically associated with the tuft. The cilia extend through the
matrix layer and past the brush border. How deep the cilia and ciliary root extend
into the tissue is not clear. If subsequent research finds that the root extends
beyond the epithelial layer into neural tissue, this may strengthen the case for a
sensory role for these cells. These structures are found only on the labial palps
of A. alternata and E. rosea, and not in the tissue surrounding the oral cavity
itself. In E. rosea they are specifically located on the area of the labial palps that
comes in contact with the ground most often when the animal is actively trail
following. The function has yet to be determined, but examination of behavior
between species with these tufts and those without may help clarify their role.
Perhaps they are specifically used for circulating mucus for tasting as the animal
is trail following in general, either for hunting, following others to stationary food
sources or for finding receptive mates. It should be noted that only in E. rosea the
areas on the labial palps that contained tufts took on a pleated appearance, but

40

this was not the case for A. alternata. Whether these specialized cilia function in
a chemo- or mechanoreceptor role is unknown. Ciliary tufts were described by
Kunz and Haszprunar (2001) in the gastropod Patella caerulea; however, their
role was not fully investigated but was postulated to be used to circulate water.
Tracing these ciliary structures to neurons excited by odor cues would strengthen
the hypothesis that they are used in olfaction and not for generating water
currents. To generate currents, the cilia would have to be motile. Motile cilia
contain dynein arms which link the microtubule doublets and allow for movement.
(Cross and Mercer, 1993). Dynein arms can be imaged with TEM, but the clarity
of the images captured in this study were not sufficient to determine their
presence in these cilia.
Circular structures were also found in the matrix layer. They appear to be
layers of concentric membrane superficially similar to the layers of an onion.
These structures were found in the posterior tentacles of three out of the four
species examined (A. alternata, A. subfuscus and H. concavum). These
structures do not appear to be associated with cilia or the surrounding microvilli.
Superficially they resemble mechanoreceptors found in mammals or insects,
specifically Meissner’s or Pacinian corpuscles (Freeman and Bracegirdle, 1976),
although these structures lie deeper in the tissue and have a connection to the
nervous system. It is not believed that they are cellular debris or dead cells, given
their very specific location (only tentacle tips and outside of the cells) and their
highly structured internal layering. Behaviorally, the tentacles can be retracted
extremely quickly in response to even very light touch stimuli. Thus, it is

41

reasonable that these structures serve some kind of mechanoreceptor, but
further study is needed to fully characterize their function. Why these structures
are not seen in E. rosea remains a puzzle. Given the density and ease of
identification of these structures in the other species examined, it is unlikely that
they were missed via human error, though the low number of E. rosea samples
examined may have led to their being missed through random chance. Barring
that, E. rosea could have lost its circular structures through the course of its
evolution, thus specializing its behavior towards hunting. The eyes of E. rosea
are also located at the tentacle tip. The eyes of gastropods are not thought to be
image forming and are not associated with foraging or hunting behavior but with
phototaxis, circadian locomotion and detection of movement (Chase, 2002).
Protection of the eyes via mechanoreceptors may not have been as
evolutionarily important as developing specialized olfactory receptors and/or
structures that greatly improve food gathering abilities for some species. This
needs to be researched further by examining other known carnivorous species
such as those in the families Rhytidae (syn., Paraphantidae) and Streptaxidae.

Conclusion
The focus of research on model organisms has left gaps in the
understanding of gastropod chemosensory capabilities. It is no longer enough to
use a handful of species to generalize across a multitude of clades. This tactic
overlooks specialized structures, leaving the larger picture incomplete without
their possible behavioral and neuroanatomical significance. In depth study of the

42

ultrastructure of epithelial tissue seems to be overlooked in favor of gross
neuroanatomical data like that of Achantina fulica (Chase, 1986). Research
correlating ultrastructure or neuroanatomical findings to directly observable
behavior is rare. There are a few instances where specialized sensory structures
were directly linked to behavior. Murray (1971) found that direct chemical
stimulation of the Navanax’s anterior lateral folds evoked turning behaviors. Upon
further investigation, specialized cilia-containing structures called the phalliform
organ were discovered only in this area. Matera and Davis (1982) described
dense fields of cilia in behaviorally significant areas ending with a paddle-shaped
bulb on the rhinophores and tentacles of the marine gastropod Pleurobranchaea
californica. These were theorized to be chemoreceptors. With more unique
structures being discovered, particularly those containing cilia like the ciliated
tufts of E. rosea and A. alternata, the possible link to behavior needs to be more
concretely established. Conversely, these structures may relate more to mate
finding and recognition than to foraging for some species; thus a reexamination
of a wider range of behaviors and the presence of specialized structures is
warranted.
This connection between behavior and structure can be difficult to confirm
because of the nature of the delicate and time-consuming research techniques
needed to examine sensory areas. A multifaceted approach involving descriptive
morphology, behavior, neuroanatomical mapping and various molecular
biological surveys, including immuno-gold labeling for proteins needed for
chemical signal transduction, should be employed to create a truly

43

comprehensive understanding of the relationship among microvilli, specialized
ciliated structures and matrix. Also, discovering the origin of the matrix, either
extra- or intra-cellular, and its makeup may help explain its function and
relationship to sensory structures.
One explanation for the pattern of specialized structures seen in the
species examined may be the result of the close phylogenetic relationship
between A. alternata (Enodontidae) and E. rosea (Oleacinidae), while A.
subfuscus (Ariondae) and H. concavum (Haplotrematidae) are further apart
(Barker, 2001). The presence of loose matrix and ciliated tufts may have arisen
from sharing a common ancestor and not because of any behavioral differences.
The classification scheme used by Barker (2001), while including a wide range of
57 genetic and morphological characteristics including alimentary, renal,
neurological, muscular, genital, developmental, reproductive and cytological
characteristics, the scheme does not include any consideration of microvilli or
sensory areas other than eye position. This is probably because there are not
enough data available in enough different species to include it in the evaluation.
A second phylogenetic tree appears in Barker (2001), which uses the same
characteristics but also constrains the data to conform to the 28S rDNA
sequence phylogeny. In this version, the two closest related groups are those
that include E. rosea and A. subfuscus, not A. alternata. To clarify, an
investigation of other species, both closely and distantly related to E. rosea, A.
subfuscus and A. alternata is needed to see if they share similar structures
because of a shared common ancestor. Kunz and Haszprunar (2001) previously

44

suggested that a more thorough examination of ciliary tuft and sensory structure
morphology of the epidermis may help with phylogenetic reconstructions.
Expanding the focus from a few key species to a broader scope encompassing
many clades will create a clearer picture of the relationships and traits shared
among groups of organisms. Although the original hypothesis that differences in
sensory structures are correlated with differences in foraging behavior was not
borne out, analysis of a variety of different gastropod species from a wide range
of different clades is necessary to determine if such differences correlate with
phylogenetic position.

45

APPENDIX

46

Appendix
Unsatisfactory Methods
Anesthesia
The use of diluted chloretone in water (Lee, 1950) appeared to result in
satisfactory extension of the head and foot. However, when touched, the snail
immediately retracted into the shell. Chilling the solution to 10° Celsius helped to
slow the animal’s reaction times. Nevertheless, tentacle extension was only
moderately successful. Dissolving a few chloral hydrate crystals into water
containing the specimen (Lee, 1950) met with little success. A 5% solution of
ethanol in water (Flores, Salas and Saavedra, 1983) was used. Although there
was some relaxation with some tentacle extension while in the solution, removal
of the animal from the water to begin dissection resulted in contraction. Although
on some occasions, waiting for the animal to re-extend its tentacles was
possible, this usually required several hours, making it impractical. Methanol
was tried (Lee, 1950) with unsatisfactory extension. Mechanical restraint was
also considered, but abandoned. A commercially available topical anesthesia
(benzocaine) was tried, but was not successful. Use of liquid nitrogen did not
successfully preserve extension and caused major tissue damage visible with the
naked eye. Injection with Ringer’s solution plus 0.5% succinylcholine chloride
was used on several animals (Chase and Croll, 1981). This method worked well
for animals without shells, as the combination of anesthesia and increased
internal pressure from added fluids expanded the tentacles nicely. Animals with
shells were more difficult; if they retracted into the shell, any further benefit from

47

anesthesia was rendered useless. This method also seemed to have a high
mortality rate for those that did not extend well.

Mucus Removal
To remove the mucus coating, several methods were considered including
using glycosidase (Monteiro-Riviere and Jiang, 1986), glucosidase (Nilsson,
Hellstrom and Hedlund, 1992), and pronase (Saito et al., 2002). These methods
proved to be too cost prohibitive to pursue and may have the unwanted side
effect of damaging the microstructure of the underlying cells.

Confocal Laser Scanning Microscopy (CLSM)
1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI)
manufactured by Invitrogen (Carlsbad, California) was determined to be the best
neural tracing dye to study the path the olfactory signal may take into the brain.
Using an investigation of DiI penetration in cortical slices of rats (Kim et al., 2007)
as a basis, a study of what temperature and amount of fixation was best for
crystal dye penetration in the gastropod brain was performed.
An entire brain was dissected from an A. alternata. The cerebrum and
visceral loop were removed and bisected to test the following parameters on
identical tissue. The cerebral ganglia were placed in buffered Ringer’s solution,
with one half kept at room temperature (21°C) and the other cooled to 10°
Celsius. The visceral loop was used to test 1.5% paraformaldehyde solution in
0.1 M phosphate buffer with one half kept at room temperature and the other at

48

10º Celsius. Each quarter of the brain was placed in its fixative solution for one
hour. After this incubation, each section was quickly dried by carefully whisking
away the liquid with an absorbent Kimwipe, but not touching the tissue. The
tissue was then placed in a depression slide and a crystal of DiI was positioned
next to the cut end of the connective of interest with a small amount of petroleum
jelly: the optic connective for the cerebrum halves and the parietal nerves on the
visceral loop (depending on which one was longer and easier to manipulate). The
depression was then filled with treatment solution. A ring of petroleum jelly
secured the cover slip. This step proved to be very important not only for the use
with the inverted confocal microscope but also to prevent evaporation of the
liquid. The samples were examined after 12 hours and again after nine days.
Although the Ringer solution worked better than the paraformaldehyde, the
dyeing was inconsistent. Olympus FluoView FV1000 Laser Scanning Confocal
Microscope (Center Valley, PA) and Zeiss LSM 5 Pascal Laser Scanning
Confocal Microscope (Thornwood, NY) were used to image the results.
To increase consistency, a gel version of DiI was tested along with a one
hour fixation in 1.5% paraformaldehyde solution followed by mounting in buffered
Ringer’s on another cerebral ganglia preparation. This method yielded exciting
results, with dye seeming to be drawn into the brain rather consistently along the
connective, with definition of structures increasing up to two weeks post
incubation, with no bacterial growth. There did appear to be some lysis of cells at
this point, but it did not appear to affect the dye’s progress. Unfortunately, due to
the thickness of the brain, the fluorescence could not to be resolved once the

49

connective entered the cerebrum. A solution for this issue would be to fix, dye
and then embed the dyed brain; then, three-dimensionally scan several thickly
sliced brain sections and reconstruct the path of the dyed nerves through
stacking and reconstructing of the full brain. Such an analysis was not
undertaken in this study.

50

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