AMER. ZOOL., 27:737-746 (1987)
Comparative Functional Morphology of the
Bivalve Excretory System1
M. PATRICIA MORSE
Biology Department and Marine Science Center,
Northeastern University, Nahant, Massachusetts 01908
SYNOPSIS. Combining injection techniques with ultrastructural observations, and relating
these findings to the more traditional physiological and morphological studies have shed
new light on the excretory mechanisms underlying the processes of ultrafiltration, secretion and reabsorption in some bivalve molluscs. These basic processes are further elucidated by comparing normal excretory tissues with those in bivalves that have been subjected to stress by pollutants in either the natural environment or under laboratory
experimentation. The process of ultrafiltration is size and charge dependent and occurs
at the filtration barrier at the base of the podocytes in the pericardial gland. Primary
urine may be modified by secretion (primarily from the kidney cells but also from the
podocytes), reabsorbtion in the kidney, and by the addition of hemocytes passing from
blood spaces through the epithelium into the lumen of the kidney. Numerous concrements
(granules, concretions and membranes) that result from lysosomal activities in the podocytes, kidney cells and hemocytes along with the fluid are excreted into the mantle cavity.
INTRODUCTION
In bivalve molluscs the major excretory
organs are the pericardial glands, associated with the pericardial cavity, and the
paired kidneys. This complex is coelomic
in origin and functions to rid the bivalves
of molecules and ions that are nonessential
to their metabolism and unavailable for
deriving energy. Specialized cells within this
system function to filter, transport, accumulate and excrete these products into the
mantle cavity. Peculiar to bivalves is the
accumulation of numerous granular and
crystalline deposits or concrements (Florey,
1966), primarily by cellular lysosomal systems in kidney and pericardial gland cells.
While these concrements are secreted into
the primary urine and eventually excreted
from the bivalves, utilizable substances are
reabsorbed and either stored in cells or
recycled by the circulatory system. The circulatory system plays an important role in
transport of wastes to the initial site of urine
formation, the pericardial glands, and to
the sinuses of the kidneys. Hemocytes
phagocytize foreign matter and, particularly in animals from pollutant-stressed
environments, often pass through epithe-
1
Past-Presidential Address presented at the Annual
Meeting of the American Society of Zoologists, 2730 December 1986, at Nashville, Tennessee.
Hal cell layers by diapedesis, thereby being
immediately lost from the bivalves.
A general pattern of excretory flow starts
with delivery of metabolic by-products by
the blood to the pericardial gland (Fig. 1).
Here, the pericardial gland cells (podocytes), with their protoplasmic extensions,
the pedicels, and the underlying basal lamina form an ultrafiltration barrier. The
urine, including concrements from podocytes, flows through the renopericardial
ducts into the kidney lumina. There, reabsorption and secretion modify the urine
before it exits through the kidney openings
into the mantle cavity.
The bivalve excretory system was among
those excretory systems well-described in
the late 19th and early 20th century. Studies by Grobben (1888, 1890) and White
(1942) on the pericardial glands and
Odhner (1912) on the kidneys provided a
firm basis for subsequent advances in our
understanding of function. Utilizing dissections and light microscopy they were
able to accurately suggest sites of excretion
that are only now being fully defined.
About the same time, physiologists, for
example Picken (1937) and Potts (1954),
began to measure filtration and secretion
rates in bivalves. Of particular importance
in understanding filtration was the use of
freely filterable, uncharged, inulin to study
ultrafiltration (see review by Martin, 1983).
737
738
M. PATRICIA MORSE
1
PERICARDIAL GLAND
PODOCYTE
PERICARDIAL
CAVITY
blood
sinus
Pedicels
Basal lamina
4
PERICARDIAL
FLUID
via
auricular
circulation
4
BLOOD
KIDNEY
RENOPERICARDIAL
DUCT
via
renal ^
circulatiorr
unne
blood
sinus
Lumen
concrements
blood'cell
-\
KIDNEY
OPENING
FIG. 1. Generalized pathway of excretory products from the blood through the bivalve excretory system
into the mantle cavity.
PAST-PRESIDENTIAL ADDRESS
Physiologists also turned their attention to
direct measurement of the hemodynamic
forces necessary for bivalve ultrafiltration
(Florey and Cahill, 1977; Hevert, 1984).
Much of the methodology leading to our
understanding of primary urine formation
in molluscs can be traced to the development of electron microscopy and to techniques utilized by Farquhar and her colleagues (see review by Farquhar, 1982) in
early investigations of the vertebrate glomerulus. Sites for ultrafiltration have been
described in major groups of molluscs (gastropods—Andrews and Little, 1971;
Andrews, 1985; chitons—0kland, 1980,
1981; cephalopods—Schipp and Hevert,
1981; Schipp et al, 1985; bivalves—Pirie
and George, 1979; Meyhofer et al., 1985).
Less is known about the ultrastructure or
physiology associated with reabsorption
and secretion, the secondary processes that
modify the primary urine (see review by
Martin, 1983). Most of our knowledge
about excretion in bivalves comes from
research groups that addressed the mechanisms involved when bivalves are exposed
to pollutants, especially metals (see reviews
by George, 1980, 1982; George and
Viarengo, 1985, with emphasis on the mussel, Mytilus edulis; and Robinson et al., 1985,
with emphasis on the quahog, Mercenaria
mercenaria).
It is on these foundations that studies on
bivalve excretory organs in our laboratory
are based. Our approach is comparative;
we utilize experimental tracer techniques
and cytochemical methods with electron
microscopy, stress bivalves with various
pollutants and measure changes, and look
at a variety of species to compare cells and
tissues in both normal and stressed animals. I wish to share our evidence as to the
processes used by bivalves to transport,
accumulate and excrete non-metabolizable
excretory products.
There is general agreement that the protobranchs are the primitive group of
bivalves. Recently, Allen (1985) combined
all other bivalves into a second subclass,
Lamellibranchia. However, for our discussion, the informal terms, protobranchs,
pteriomorphs (epifaunal mussels, scallops)
739
and heterodonts (infaunal siphonate clams,
quahogs) will be used.
ULTRAFILTRATION
In ultrastructural studies, we have found
pericardial glands in two basic positions in
the pericardial cavity, either associated with
the surface of the auricle or as evaginations
of the dorsal wall lying in a blood sinus
between the outer mantle epithelium and
inner pericardial cavity lining. In the first
type (characteristic of protobranchs and
pteriomorphs), the filtrate passes directly
from the blood of the auricle through the
ultrafiltration barrier at the base of the
podocytes into the pericardial cavity. In
the latter type (characteristic of heterodonts), the filtrate passes from the blood
sinus through the ultrafiltration barrier
into the pericardial gland lumen, through
ducts into the pericardial cavity. Another
difference noted in all specimens examined
is that the podocytes of protobranchs and
pteriomorphs have relatively smooth cell
surfaces whereas those of heterodonts have
numerous microvilli on their luminal surfaces (Meyhofer et al., 1985) (Fig. 2).
In all three bivalve groups, the ultrafiltration barrier has a similar configuration.
At the basal area of the podocyte, cytoplasmic extensions branch into minute
pedicels, and the pedicels interdigitate to
form a basal network. The blood sinus is
separated from this network by a single
layered, somewhat diffuse basal lamina.
The pedicel network and the basal lamina
together represent the ultrafiltration barrier (Meyhofer et al., 1985) (Fig. 3). Substructural elements between the oval to
round pedicels leave pores and slits in the
filter. The width of the ultrafiltration slit
is approximately 20 nm. In addition, extracellular collagen-like filaments often extend
toward the filter on the blood side of the
basal lamina.
As described for the glomerulus filter in
vertebrates (Farquhar, 1982), the bivalve
ultrafiltration barrier has two potential
mechanisms, size and charge, for admission or restriction of particles (proteins,
etc.) from the blood and urinary spaces.
Physiological studies by Deyrup-Olsen and
740
M. PATRICIA MORSE
FIG. 2. Podocytes from the pericardial gland of Mya arenaria. M, microvilli; US, urinary spaces; BS, blood
spaces.
Martin (1982) on land slugs and Hevert
(1984) on bivalves indicate that the process
of ultrafiltration is size-dependent. Caulfield and Farquhar (1976) have demonstrated anionic binding sites on the
basement membrane of the vertebrate
ultrafilter that, by nature of the negative
charge, may affect particle passage. In
bivalves it is not known whether the slits
between pedicels as described in gastropods by Boer and Sminia (1976) or the basal
lamina as reported in gastropods by
Andrews (1979) is the principle filter of the
system. To address these questions and further characterize the bivalve ultrafiltration
system, a series of opaque tracers and cy tochemical dyes were injected into the blood
of several bivalve species and the tissues
examined by electron microscopy. Our
results to date indicate that particles of a
mol wt of 40,000 daltons (horseradish peroxidase) are filtered through the barrier
PAST-PRESIDENTIAL ADDRESS
741
FIG. 3. Pedicels (P) and underlying basal lamina (BL) in the pericardial gland of Chlamys hastata.
and that large particles (ferritin, 400,000
mol wt) are retained at the basal lamina
(Meyhofer et al., 1985). In the pericardial
gland of the protobranch, Acila castrensis,
the blood pigment, hemocyanin that has a
mol wt of 250,000 daltons (Morse et al.,
1986) is also held back at the basal lamina.
It is our opinion that the principal filter is
the basal lamina (Meyhofer and Morse,
1986) and this conclusion agrees with
Andrew's (1979) results for gastropods.
The cationic dye, ruthenium red, stains
anionic binding sites on the bivalve basal
lamina and is indicative of negative charges
associated with the bivalve filter (Meyhofer
etal, 1985).
In the urinary spaces, the primary urine
undergoes modifications by reabsorption
and secretion. Although physiological evidence is lacking, the presence of microvilli
on the surface of the podocytes is suggestive of a site of absorption. That podocytes
are sites of cellular secretion is evidenced
by the presence of lysosomal organelles and
observations of podocyte vacuolar contents in the pericardial cavity, as we have
seen in Chlamys hastata and Mercenaria mercenaria. These ultrastructural characteristics in other bivalves (Meyhofer et al., 1985)
confirm the earlier obervation of Pirie and
George (1979) that podocytes are active in
excretory processes.
The modified urine is swept from the
pericardial lumen into the kidney by welldeveloped cilia of the renopericardial ducts.
Here, secondary processes, reabsorption of
filterable materials and secretion from the
kidney cells, form the final excretory products.
RESORPTION AND SECRETION
The bivalve kidneys, in their simplest
form, consist of a pair of tube-like organs
that connect the pericaridial cavity with
the mantle cavity. Often called the "organ
of Bojanus," the kidneys are really glan-
742
M. PATRICIA MORSE
. \
FIG. 4. Proximal epithelium of the kidney of Mercenaria mercenaria. Note the numerous basal infoldings (BI)
and the microvilli (M) and mitochondria (MI) at the apicies of the cells.
dular coelomoducts that are differentiated
into proximal arms that lead from the pericardial cavity and distal arms that lead to
the paired kidney openings near the bases
of the gills. Variations on this theme
include, compact vs. diffuse character of
the kidney, degrees of folding of the body
wall, differentiation of the proximal and
distal epithelium, degree of fusion of the
paired kidneys and modifications into a
bladder-like condition found in freshwater
bivalves (see Odhner, 1912). In all cases,
the kidney epithelium has an extensive
blood supply, and numerous hemocytes are
visible beneath the epithelium in the blood
spaces.
Our studies indicate that the protobranch kidneys (Yoldia limatula and Acila
castrensii) are tubular and diffuse and have
little epithelial differentiation (Morse and
Meyhofer, unpublished observations).
Among pteriomorphs (Chlamys hastata and
Placopecten magellanicus) the kidney pair is
completely separated and the tissue is compact, with numerous folds of the epithelium that are differentiated into proximal
and distal regions. In the heterodont, Mercenaria mercenaria, the kidneys are globular
in shape, with fusion of the paired lumina,
numerous body folds and proximal and distal regions of the epithelium differentiated.
The proximal epithelium is characterized by columnar cells with regular microvilli, a few cilia, deep basal infoldings, few
vacuoles and basal nuclei (Fig. 4). In Mercenaria mercenaria, a large amount of glycogen was found in these cells although its
origin is not clear. However, due to the
ultrastructural characteristics (microvilli,
PAST-PRESIDENTIAL ADDRESS
743
BS
FIG. 5. Distal epithelium of the kidney of Mercenaria mercenaria. A large extracellular concrement (EC) is
seen in the kidney lumen. V, vacuole; H, hemocyte; BS, blood sinus.
deep basal infoldings) in common with
other resorptive epithelial cells in other
organisms (Berridge and Oschmann, 1972),
the proximal region is considered the major
site of reabsorption in the kidney. This
is in agreement with Tiffany's (1974) description of similar cells in another species
of quahog.
The distal epithelium is characterized by
irregular cells with expanded apices and
numerous vacuoles containing concrements of varying size, shape and number
(Fig. 5). The cells have scattered microvilli
on the surfaces and basal nuclei. The lyso-
somal-vacuolar system is the major degradative system within the kidney cells. It
is the source of the numerous concrements, including lipofuchsin granules,
membrane remnants, concretions and
other electron-dense materials. These are
secreted from the inflated apicies into the
kidney lumen. In bivalve species there are
differences in both the kinds of concrements formed and the amounts stored in
the tertiary lysosomes at any one time. The
process of secretion appears to be cyclic
with accumulation of wastes in the vacuoles
often reaching giant proportions before
744
M. PATRICIA MORSE
they are shed into the lumen. During dis- 1986). In cadmium exposure experiments
section, kidneys of 20 living scallops (Placo- on Mercenaria mercenaria, waste-laden hempecten magellanicus), were examined. They ocytes accumulate in large numbers in the
varied in coloration from opaque white blood spaces in the kidney tissue and evithrough varying shades of tan, to deep dence supports the view that these cells
brown depending on stages of elimination pass through the intracellular spaces into
of the distal epithelial concrements (Morse, the kidney lumen by diapedesis (Morse and
unpublished observations). This excretory Robinson, unpublished results). Furtherpathway, well-documented by George more, limits to tolerance of stress (seen in
(1982), is also characteristic of the podo- Mercenaria mercenaria after a 10 day expocytes and the hemocytes. It is undoubtedly sure to 50 ppm cadmium) were apparent
an important pathway for pollutants as well in the breakdown of the kidney epithelium
as normal excretory products, and yet we that parallels histological observations on
know very little about the natural cycles of bivalves from stressed environments
materials through the system or the effects reported by Yevich and Yevich (1985).
pollutants might have on component Thus important insights as to the normal
organelles.
functional morphology of the excretory
In the quahog, Mercenaria mercenaria, in system have been developed through these
common with a few other bivalves such as pollution studies.
Tridacna gigas, there are two types of conThere is always a danger to assume that
cremental accumulations in the kidney: the what is seen in one bivalve is common to
usual intracellular concrements and large all species; comparative studies are reextracellular granules that appear to be quired. We need more investigations of
formed by a conglomeration of intracel- cellular and biochemical processes as we
lular granules plus other materials (Fig. 5). continue to unravel the patterns of bivalve
In our dissections of large numbers of excretion.
quahogs, these extracellular granules are
ACKNOWLEDGMENTS
easily visible and impart a dark coloration
to the kidney depending on their concenColleagues and former students have
tration within the kidney lumen.
contributed to this compilation through
We investigated the effects of cadmium informal discussion, encouragement and
on the infaunal bivalve, Mercenaria merce- their own scientific contributions. In parnaria, and found, as in previous studies, ticular I wish to thank Dr. Vera Fretter,
that the kidneys are the major organs where Professor Alastair Graham and Dr. Elizapollutants accumulate. Details of the tim- beth Andrews for discussions of molluscan
ing of this process, the regulating forces excretory systems, and Professor Dr. Ernst
and limits of its function for ridding the Florey who imparted excitement and interanimal of varying pollutant burdens are yet est in my work and infused encourageto be determined. In addition, the role of ment. To several former students, Drs.
intracellular and extracellular metal-bind- William Robinson, Carol Moore and Lindy
Eyster, Edgar Meyhofer and Greg Seiler,
ing proteins needs to be addressed.
Transport of the waste products to sites whose work is often incorporated into this
of excretion involves the blood, i.e., the paper, I owe particular gratitude. I wish to
hemolymph and its contained hemocytes. acknowledge that some of the original
Long known to be important in the excre- work in this paper has been supported by
tory process, hemocytes play an active role the Physiological Ecology Division of
in scavenging waste materials and elimi- the Department of Energy (DE-AC02nating them from the bivalve body (see 77EV04580) and that some of the research
review by Cheng, 1981). Our studies indi- was done at the Friday Harbor Laboratocate that in clams (Mya arenaria) taken from ries of the University of Washington.
a known polluted site (New Bedford HarFinally, I wish to dedicate this paper to
bor), there is a significant increase in gran- Dr. Dora Henry (Research Professor, Uniulocytes in the blood (Seiler and Morse, versity of Washington), a "barnacle" spe-
PAST-PRESIDENTIAL ADDRESS
cialist, who has unselfishly devoted many
hours to her fellow scientists—from
graduate students to full professors—furthering their use of correct English language in scientific writing. This is contribution #157, Marine Science Center,
Northeastern University.
745
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Hevert, F. 1984. Urine formation in the lamellibranchs: Evidence for ultrafiltration and quantitative description. J. Exp. Biol. 111:1-12.
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