Download structure of the excretory system of hawaiian

J. Moll. Stud. (1999), 65, 61–72
© The Malacological Society of London 1999
S TRU CTU RE OF THE E XC RE TO RY S YST EM OF HAWAIIAN
NERITES (GAS TRO PODA: N ER ITOIDE A)
W .A. ESTA BRO OKS 1 *, E.A . K AY 1 a nd S .A. McC ARTH Y 2
1
University of Hawaii, Department of Zoology, Edmondson Hall-2540 the Mall, Honolulu, HI 96822, USA;
2
University of Hawaii, SOEST, Department of Oceanography, 1000 Pope Road Honolulu, HI 96822, USA
(Received 26 November 1997; accepted 15 May 1998)
ABSTRACT
Neritoidean gastropods are present in freshwater,
brackish and marine environments, which vary in
salinity and exposure to dehydration. In this study we
examine the structure of the excretory system of
Hawaiian nerites, which indicates possible processes
that enable these gastropods to survive in a wide
range of environments in the Hawaiian Islands.
As is true of other nerites studied to date, the main
excretory mechanism of Hawaiian nerites is through
filtration of the blood between podocytes in the auricle epicardium, resulting in production of an ultrafiltrate, which collects in the pericardial cavity. No
podocytes are present on the surface of the ventricle
of Hawaiian nerites. The reno-pericardial canal conveys the urine to the kidney, the epithelium of which
is composed mainly of acidophilic cells in marine
nerites. In brackish and fresh water species, basophilic cells are present in addition to the acidophilic
cells present in marine nerites. It is proposed that the
basophilic kidney cells allows non-marine nerites to
osmoregulate and produce a hyperosomotic urine at
low salinities. A bladder is present, and empties into
the mantle cavity near the gill by way of a ureter.
INTRODUCTION
In prosobranch gastropods, ion balance and
osmoregulation usually occur both across the
integument and by the action of specific tissues
and organs (Andrews, 1985). Elimination of
nitrogenous wastes by prosobranchs is typically
accomplished by the renal system, though it
may take place across the general body surface
in some species. In aquatic species, ammonia
diffuses across permeable surfaces, such as the
gills. The nephridia (kidneys) are paired in
‘primitive’ gastropods, bivalves and cephalopods (Martin & Harrison, 1966). Unlike the
kidneys of cephalopods and bivalves, the
paired kidneys of ‘primitive’ gastropods differ
*author to whom correspondence should be addressed. Present
address: 8944 Miller Lane, Vienna, VA 22182, USA
from one another in both structure and function. The right kidney is involved in elimination
of nitrogenous wastes as purines and the left
with resorption of organic solutes from urine
(Andrews, 1985). In two groups of prosobranchs, the nerites and caenogastropods, the
right kidney is absent and all renal functions
(including elimination of nitrogenous wastes)
are performed by the left kidney (Delhaye,
1974, 1976).
In most primitive gastropods, primary urine
is formed in the pericardial cavity by filtration
of the blood through the atrial wall (Andrews,
1976, 1981). Since Andrews & Little (1972) first
described podocytes in the cyclophorid Poteria,
they have been identified in many gastropod
species. From the pericardial cavity the urine
passes through the renopericardial canal to the
kidney, from which it is eliminated through the
ureter. The kidneys of prosobranchs vary
widely in structure and function.
Nerites are unique among the more ‘primitive’ gastropods in that these gastropods lack a
right kidney. Most excretory functions (undertaken by separate kidneys in the archaeogastropods) therefore probably take place in the
single remaining kidney, as in the caenogastropods. Perrier (1889) first described the structure of the nerite kidney. Lenssen (1902) and
Bourne (1908) corrected errors in Perrier’s initial descriptions and contributed additional
detail. Little (1972 and 1985) described the
function of the kidneys of marine, freshwater,
and terrestrial nerites and proposed evolutionary relationships between nerites, based on
their excretory physiology. Delhaye (1974)
published a comparative study of the excretory
and circulatory system of several neritoid
species, including the terrestrial helicinids. No
comparison of the excretory system of nerites
from the same area have been published.
Hawaiian nerites present an opportunity to
study renal adaptations in a group of closely-
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W.A. ESTABROOKS, E.A. KAY & S.A. McCARTHY
related marine, brackish and freshwater prosobranchs from the same geographical area.
MATERIAL AND METHODS
Five Hawaiian nerite species were studied. Their
habitat preferences are in Table 1. Specimens of
Nerita picea (Recluz, 1841) were collected from
supratidal basalt formations along the Waianae
coast, and from the southern coast of O’ahu. Nerita
polita Linnaeus, 1758 were obtained from intertidal
rocks near Kona, Hawai’i. The endemic Hawaiian
nerites Theodoxus cariosus (Wood, 1828) and Ner itina vespertina (Sowerby, 1849) were collected from
brackish pools near Honokohau Bay and Hookena,
Hawai’i. Specimens of the diadromous nerite Ner itina granosa (Sowerby, 1825) was collected from
shallow streams near Keanae, Mau’i.
All specimens were transported live to the laboratory and maintained in aquaria at 22–24°C at
ambient salinities. Specimens for routine histology
were removed from the shell, and the operculum
detached. Small specimens were fixed entire. For
large specimens (mainly specimens of Neritina
granosa), organs of interest (heart and viscera) were
removed and fixed separately. The radula was not
removed because it is located adjacent to the kidney
and heart, both of which are easily torn. Animals
were not decalcified prior to embedding. Specimens
were fixed in Bouin’s solution for 24–48 hours, dehydrated in a graded ethanol series, cleared in xylene,
and embedded in Paraplast. Serial sections were
stained with Delafield hematoxylin and eosin, Mallory’s connective tissue stain, Alcian blue (pH 1.0
and 2.8), or Periodic Acid-Schiff.
For transmission electron microscopy, animals
were maintained in aquaria for 5–7 days to purge the
gut. Gastropods were removed entire from shells,
and the organs and tissues dissected out at ambient
salinity. For Nerita picea and N. polita, 0.5 mm diameter pieces of organs/tissues were fixed for 1.5 h in
2.5% glutaraldehyde buffered in 0.1M Sorensen
phosphate buffer with 14% sucrose. Tissues of the
Figure 1. Diagram of the excretory system of Hawaiian nerites. Not drawn to scale. Abbreviations: au, auricle;
bl, bladder; ct, ctenidium; gk, glandular region of the kidney; pd, pericardial cavity; rc, reno-pericardial canal;
ut, ureter; vn, ventricle.
Table 1. Habitats of Hawaiian nerites.
Species
Geographical
distribution
Habitat
Exposure to
desiccation
Exposure to
fresh water
Nerita picea
common
Hawaii—endemic
Hawaii—endemic
Hawaii—endemic
rare (emerges
only at night)
never
never
never
rare
Theodoxus cariosus
Neritina vespertina
Neritina granosa
marine—high
intertidal
marine—low
intertidal
brackish polls
brackish pools
freshwater streams
common
Nerita polita
Hawaiian Islands
and Indonesia
Indo-West Pacific
common
common
immersed
EXCRETORY SYSTEM OF HAWAIIAN NERITES
63
Figure 2. A. Scanning electron micrograph of the heart (Nerita picea). The intestine passes through the lumen
of the ventricle. Scale bar 5 250 mm. B. The nerite heart (Nerita picea). There is a single ventricle and auricle.
Hematoxylin and eosin. Scale bar 5 100 mm. Abbreviations: au, auricle; ig, integument; in, intestine; pd, pericardial cavity; vn, ventricle.
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W.A. ESTABROOKS, E.A. KAY & S.A. McCARTHY
Figure 3. A. The wall of the ventricle (Nerita polita). Muscle fibres are arranged in a spiral manner. The
epicardium (arrow) incompletely covers the ventricle. Hematoxylin & eosin. Scale bar 5 30 mM. b. Scanning
electron micrograph of the surface of the ventricle (Theodoxus cariosus). The surface of the ventricle is relatively smooth, with nuclei of the squamous epicardium visible. Scale bar 5 5 mm. Abbreviations: cl, cardiac
lumen; my, myocardium; pd, pericardial cavity.
EXCRETORY SYSTEM OF HAWAIIAN NERITES
65
Figure 4. The wall of the auricle (Neritina granosa). The auricle surface is highly irregular, with globose cells
protruding into the pericardial cavity (arrows). Hematoxylin & eosin. Scale bar 5 30 mm. B. Scanning electron
micrograph of the auricle surface (Nerita picea). Scale bar 5 10 mm. C. Transmission electron micrograph of
pedicels of the podocytes of the auricle surface (Neritina granosa). Arrows indicate gaps between pedicels.
Scale bar 5 3 mm. Abbreviation: cl, cardiac lumen; pd, pericardial cavity.
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W.A. ESTABROOKS, E.A. KAY & S.A. McCARTHY
brackish-water species Theodoxus cariosus were
fixed in 2.5% glutaraldehyde buffered in 0.1M
Sorensen phosphate buffer (with 7% sucrose) for 1.5
h. Specimens of Neritina granosa were fixed in 2.5%
glutaraldehyde buffered in 0.02M Cacodylate buffer,
for 2 h. Sorenson’s phosphate buffer gave poor
results with specimens of this species.
Organ fragments of all species were post-fixed in
buffered 1% osmium tetroxide, and dehydrated in
a graded ethanol and acetone series. Specimens
were embedded overnight in low viscosity Spurr’s
Medium. Thin sections were made using a diamond
knife and stained with saturated uranyl acetate in
50% ethanol, and lead citrate, and examined on a
Zeiss EM-10 transmission electron microscope.
For scanning electron microscopy, specimens were
fixed in buffered 2.5% glutaraldehyde, post-fixed in
buffered 1% osmium tetroxide, and dehydrated in a
graded ethanol series. Specimens were critical point
dried, coated with gold-palladium and examined on a
scanning electron microscope.
RESULTS
There are few species-unique differences in the
morphology and structure of the excretory
system of the five species examined. For this
reason the general plan of the excretory system
of Hawaiian nerites is described, with unique
characteristics of species included where
appropriate. Figure 1 is a schematic diagram of
the excretory system of a ‘typical’ Hawaiian
nerite.
Heart
The heart lies in the crescent-shaped pericardial cavity on the post-torsional left side of the
visceral mass, and has a single, thin-walled
auricle and thick-walled ventricle (Figs 2A and
2B). A simple squamous epithelium incompletely lines the pericardial cavity, including
the heart and rectum (which passes through the
ventricle) (Figs 2B, 3A and 3B). The external
surface of the auricle has numerous invaginations, which are covered by interdigitating
podocytes (Fig 4A–C). Podocytes are absent
from the wall of the ventricle. The myocardium
of the auricle is thinner than that of the ventricle, though both have irregularly-oriented
bundles of muscle fibres (Figs 3A and 4A).
A single, large reno-pericardial canal (Figs
5A and 5B) extends from a horn of the
crescent-shaped pericardial cavity to the kidney and opens into the glandular region of the
kidney adjacent to the ureter. The wall of the
reno-pericardial canal adjacent to the pericardial cavity and kidney is thin; the opposite
wall (adjacent to the ureter) is much thicker.
Large (20–30 mm diameter) cells line the renopericardial canal for most of its length, but
are less abundant where it empties into the
kidney. Numerous long (60–100 mm) cilia extend from these large cells, into the canal
lumen. Sparse connective tissue is present
beneath the reno-pericardial canal epithelium.
Mucoid (‘goblet’) cells are present in the wall
of the reno-pericardial canal, and in the kidney
epithelium adjacent to the canal (Fig. 5B).
Glandular Region of the Kidney
The single kidney is located posteriorly and
dorso-laterally in the visceral mass, just
beneath the integument, adjacent to the heart.
The kidney, a roughly u-shaped, tubular organ,
has a glandular (proximal) region and a distal
non-glandular region (bladder). The renopericardial canal opens into the glandular
region (Figs 5A and 5B). Folds in the interior
wall of this region of the kidney occupy most of
the lumen of the organ. Urine passing through
the kidney does not pass through a distinct
tubule, as in vertebrates, but rather through a
series of interconnected passageways and
sinuses, eventually reaching the bladder. Few
blind sacs are present in the glandular region of
the kidney.
In the marine Hawaiian nerites (Nerita picea
and Nerita polita), the renal epithelial cells are
large (15–20 mm high), with a centrally to
basally located nucleus. These cells form a
continuous simple epithelium. Vacuoles and
vesicles occupy the apical and central parts of
the cell (Fig. 5C); mitochondria are present in
the central and basal parts of the cells. Invaginations are present on the basal membrane
surface. Densely-packed microvilli are present
on the apical (luminal) surface and form a
continuous, simple epithelium. The cytoplasm
does not stain darkly with routine light
microscopy staining procedures. Most cellular
inclusions lack carbohydrates, though some
vesicles are slightly PAS-positive, possibly
indicating the presence of small amounts of
carbohydrate. Blood vessels are present in the
sparse connective tissue in the anterior of the
glandular folds.
In the brackish-water Hawaiian nerites
(Theodoxus cariosus and Neritina vespertina)
and in the freshwater nerite Neritina granosa,
areas of basophilic renal cells are present in the
simple kidney epithelium (Fig. 6A). These cells
have centrally-located vacuoles and vesicles,
densely-packed infoldings of the cell mem-
EXCRETORY SYSTEM OF HAWAIIAN NERITES
67
Figure 5. A. The renopericardial canal in longitudinal section (Neritina granosa). Hematoxylin & eosin. Scale
bar 5 100 mm. B. Opening of the reno-pericardial canal into the glandular region of the kidney (Nerita polita).
Numerous mucus-containing cells (arrows) open into the kidney lumen and terminus of the reno-pericardial
canal. Hematoxylin & eosin. Scale bar 5 50 mm. C. Transmission electron micrograph of acidophilic kidney
cells (Nerita picea). Densely arranged microvilli (arrow) are present on the apical surface. Vesicles and vaculoes occupy most of the cytoplasm. The basal region of the cell has infoldings of the cell membrane, endoplasmic reticulum, and mitochondria. Scale bar 5 5 mm. Abbreviations: bl, bladder; gk, glandular region of the
kidney; pd, pericardial cavity; rc, reno-pericardial canal.
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W.A. ESTABROOKS, E.A. KAY & S.A. McCARTHY
Figure 6. Basophilic renal cells. A. Basophilic cells form a simple epithelium in the glandular kidney of brackish and freshwater species (Theodoxus cariosus). Hematoxylin & eosin. Scale bar 5 30 mm. B. Transmission
electron micrograph of the basal region of basophilic renal cells. Membrane infoldings and endoplasmic reticulum are densely-packed in this part of the cell (Neritina granosa). Scale bar 5 1 mm. C. Transmission electron
micrograph of the apical region of basophilic renal cells (Neritina granosa). The apical region of the basophilic
renal cell has microvilli, vesicles, and vacuoles. Scale bar 5 2 mm. Abbreviation: bl, bladder.
EXCRETORY SYSTEM OF HAWAIIAN NERITES
brane, and mitochondria and endoplasmic
reticulum in the basal region (Fig. 6B) and
microvilli on the apical surface (Fig. 6C). The
vesicles within the basophilic cells do not contain carbohydrates or muco-substances. The
basophilic cells are mainly present in the proximal part of the glandular region of the kidney,
with the acidophilic cells generally restricted to
the central and distal areas of the kidneys of
brackish and freshwater species. In the freshwater species N. granosa, basophilic renal cells
dominate the renal epithelium (Fig. 6A).
In all species, mucoid (‘goblet’) cells are
interspersed with the epithelial cells throughout the kidney, but are most abundant in the
vicinity of the terminus of the reno-pericardial
canal and opening of the ureter. These mucuscontaining cells are packed with spherical vesicles.
Bladder
The lumen of the glandular portion of the
kidney is continuous with the lumen of the
non-glandular (bladder) region. The bladder
curves around the periphery of the kidney
adjacent to the mantle cavity, pericardial cavity
and integument adjacent to the heart and is
lined by a simple cuboidal to columnar epithelium (Fig. 7A). The lumen of the bladder is
occasionally filled with single cells or sheets of
kidney cells (usually highly vacuolated) and
other cellular debris. Similar debris is present
in the glandular region, but not to the extent
observed in the bladder. The epithelial cells of
the bladder have sparse flagella on the apical
surface, and centrally and apically-located
cytoplasmic vesicles and vacuoles.
The bladder empties into the short ureter,
which is located at the distal end of the bladder.
The opening of the bladder to the ureter is
oriented perpendicular to the uropore, which
empties into the mantle cavity adjacent to the
single ctenidium. Clusters of mucus-containing
cells similar to the mucoid cells present in the
epithelium of the kidney folds are scattered
throughout the wall of the ureter, and open
into the ureter lumen (Fig. 7B). The ureter wall
is lined by a ciliated simple cuboidal to columnar epithelium (Fig. 7C). A thin layer of connective tissue is present at the base of the
lumen epithelium. Muscle fibres are present in
the wall of the ureter in the vicinity of the
uropore, and thin strands of connective tissue
scattered throughout the abluminal layer of the
ureter, adjacent to the basement membrane of
the ureter epithelium.
69
DISCUSSION
The auricle of the heart has been identified as
the site of ultrafiltration and production of a
primary filtrate in many gastropods. Due to the
presence of podocytes in the auricle of the
Hawaiian nerites examined, and absence from
the ventricle, it is likely that the auricle is the
sole site of blood ultrafiltration in these species.
Andrews and Little (1972) postulated that the
interdigitating processes of podocytes serve to
filter large molecules and circulating cells from
the blood serum filtered between the cells of
the epicardium into the cavity surrounding the
heart, the pericardial cavity. Auricular filtration is common among more primitive gastropods, and probably reflects the ancestral prosobranch condition (Andrews, 1988; Andrews &
Jennings, 1993). Podocytes have also been
identified in the ventricle of several species of
molluscs. The limpet Patella vulgata has podocytes on the surface of both the auricle and
ventricle (Okland, 1982). In cyclophorids, the
ventricle is also probably the main site of ultrafiltration, with sparsely distributed auricular
podocytes a minor contributor to production of
a blood filtrate (Andrews & Little, 1972).
Nerites, though primitive in many ways, have
only one kidney. Most primitive gastropods
have a right and left kidney, which are involved
in excretion and transport of substances, respectively. The epithelium of the right and left
kidneys differs, reflecting the different functions of the two organs. Caenogastropods have
a single bilobed kidney, which is probably
involved in secretion and absorption of substances, as well as elimination of nitrogenous
wastes (Martin, 1983). The purine excretion
function, performed by the right kidney in
diotocardians, occurs in the single kidney of
caenogastropods (Andrews, 1985). In contrast
to the single type of cells present in the diotocardian left kidney, the simple columnar epithelium lining the kidney of caenogastropods
(Andrews, 1981) is composed of interspersed
ciliated cells with numerous microvilli, and
vacuolated excretory cells. The former are
involved in bidirectional transepithelial movement of compounds and facilitation of circulation of the urine in the kidney lumen, while
the latter cells are involved in excretion. In
some marine diotocardians, excretory cells
have basal membrane projections that extend
into the underlying tissue.
The structure of the kidney of Hawaiian nerites is similar to that of marine and freshwater
nerites from other areas that were previously
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W.A. ESTABROOKS, E.A. KAY & S.A. McCARTHY
Figure 7. A. Simple cuboidal epithelial cells of the bladder (Neritina granosa). Bladder cells (arrows) are
cuboidal to columnar. Hematoxylin & eosin. Scale bar 5 30 mm. B. Goblet cells in the vicinity of the bladder
and ureter (Nerita polita). Mucoid cells (arrows) are densely arranged in the vicinity of the ureter and bladder.
Hematoxylin & eosin. Scale bar 5 30 mm. C. The ureter receives urine from the bladder and conveys it to the
mantle cavity (Theodoxus vespertinus). Ciliated simple columnar epithelium (arrow) lines the lumen of the
ureter. Hematoxylin & eosin. Scale bar 5 50 mm. Abbreviations: bl, bladder; ig, integument; mc, mantle cavity;
rc, reno-pericardial canal.
EXCRETORY SYSTEM OF HAWAIIAN NERITES
described by Delhaye (1974). The glandular
region of the nerite kidney has numerous
infoldings, which greatly increase its interior
surface area. In the marine nerites Nerita picea
and Nerita polita, the epithelium of the glandular region of the kidney is a simple epithelium,
composed mainly of tall, acidophilic, vacuolated cells with densely-packed microvilli, a
finely granular cytoplasm, and relatively few
basal infoldings. The ultrastructure of these
cells resembles that of the cells of the right
kidney of diotocardians, which are believed to
have an osmoregulatory function. It is likely
that these cells serve a similar function in
marine nerites.
Acidophilic cells similar to those present in
marine nerites are present in the brackish
nerites Theodoxus cariosus and Neritina ves pertina. In these two species, small numbers of
strongly basophilic epithelial cells are also present. These cells have deep basal infoldings,
which is are commonly observed in cells
involved in transport of substances. In the kidney of the freshwater species Neritina granosa,
basophilic epithelial cells dominate the glandular region and acidophilic cells limited to small
patches primarily adjacent to the reno-pericardial canal. Because there is a correlation
between ambient salinity and cell types present
in Hawaiian nerites, the basophilic renal cells
may represent an adaptation to low salinity
environments, perhaps functioning in osmoregulation and ion transport.
Mucoid cells are present in the renal epithelium of all of the Hawaiian nerite species examined, but are most abundant adjacent to the
reno-pericardial canal and ureter regions. They
are in greatest abundance in the marine nerite
Nerita picea. Mucoid cells were reported in all
species of nerites examined by Delhaye (1974).
In the terrestrial helinicids, the distal region of
the renal epithelium, which is smooth and lacks
excretory cells, probably serves as a bladder in
which urine is retained (Andrews, 1981; Delhaye, 1974). Mucoid secretions may function to
decrease the surface tension between the walls
of the kidney partitions, and the walls of the
reno-pericardial canal and the ureter. Semiterrestrial nerites such as N. picea are often
exposed to prolonged desiccation, so drying of
the mantle cavity (and therefore the uropore)
might occur. Mucopolysaccharides in the uropore may decrease the rate of desiccation. A
behavioral mechanism of N. picea may also
assist in minimizing the drying of mantle cavity
tissues, for when attached to the substrate,
these nerites hold a droplet of water in their
71
mantle cavity. This retained water probably
allows continued respiration via the single gill
in the mantle cavity and minimizes desiccation
of mantle cavity structures during prolonged
removal from seawater.
General conclusions
1. The gross morphology of the excretory system of Hawaiian nerites is similar to the
excretory system of other nerites, the heart
with a single ventricle and auricle, and spacious pericardial cavity, single kidney and
large ureter.
2. Podocytes, which produce an ultrafiltrate
(primary urine) by filtering blood between
their pedicels, are present on most of the
surface of the auricle, but are absent from
the thick-walled ventricle.
3. The nerite kidney has several main cell
types: large, acidophilic and basophilic secretory cells, mucoid cells (mainly near the
reno-pericardial canal) and flattened epithelial cells. The lumenal epithelium of the
kidney of the fresh-water nerite Neritina
granosa is dominated by basophilic cells
similar in many respects to the acidophilic
cells of the marine species. Brackish Hawaiian nerites have both acidophilic and basophilic renal cells. Basophilic cells probably
function in osmoregulation, acidophilic cells
in excretion.
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