Proc. malac. Soc. Land. (1972) 40, 115
NIGEL F. MATHERS
Department of Zoology, Chelsea College of Science and Technology,
London, SAV.IO*
INTRODUCTION
The technique of feeding animals with normal food materials which have been
labelled with a radioactive isotope, and of tracing the path of the resultant breakdown
products through the digestive tract and body tissues by means of autoradiography,
has been used in a number of phyla. However, it has been little used with bivalve
molluscs.
Radioactive isotopes used in investigations on feeding and filtration of marine
bivalves have been employed by Chipman and Hopkins (1954) with Phosphorus32
concerned with the filtration of Pecten irradians; Allen (1962) with P 32 in connection
with the filtration and feeding of Venus mercenaria and Mya arenaria; and Goreau,
Goreau and Yonge (1966) tracing Carbon11 labelled zooxanthellae in Tridacna
elongata. Recently Goreau et al. (1970) traced the path of C " labelled zooxanthellae
through the digestive tract of Fungiacava eilatetisis. They reported that 24 hr after
labelling the zooxanthellae all organs and tissues of the bivalve were also labelled,
indicating metabolic incorporation of the products derived from the disassociation
of the zooxanthellae. However, neither of the processes of digestion nor the means
of transport of the resultant products were identified.
In the experiments described here autoradiographs of sectioned tissues of Ostrea
edulis were developed at regular intervals after feeding on activated algal cells, in
an attempt to assess the times involved, and to investigate the successive processes
of absorption, digestion and translocation of food material. Care was taken to ensure
that as far as possible only the algal cells were activated and that the medium in
which they were suspended was not contaminated with radioactive isotope in solution.
Recently it has been suggested that feeding in Dreissena polymorpha and Cardium
edule appears to be a discontinuous rather than a continuous process (Morton, 1969,
1970). To ensure that the oysters used in the experiments here reported were all in
*Present address: Department of Zoology, University College, Galway, Eire.
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THE TRACING OF A NATURAL ALGAL FOOD
LABELLED WITH A CARBON 14 ISOTOPE
THROUGH THE DIGESTIVE TRACT OF
OSTREA EDULIS L.
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PROCEEDINGS OF THE MALACOLOGICAL SOCIETY
a 'feeding' phase each was attached, via wax cotton, to a heart lever which recorded
all movements of the shell valves on revolving kymograph drums. Only at such times
as it was evident that the animals were actively adducting, and therefore presumably
filtering and feeding, were the experiments conducted.
Experiment 1
2 hr
4 hr
6 hr
Experiment 2
15 min
30 min
45 min
60 min
Experiment 3
10 min
20 min
40 min
60 min
90 min
120 min
To each algal culture was added 1 ml of an aqueous solution of sodium bicarbonate
labelled with C14 (supplied by The Radiochemical Centre, Amersham), with a radioactive concentration of 1 mGi/ml. The G14O2 produced was assimilated by the algal
cells over a period of time and samples were taken of the algal cultures, from which
the algal cells were separated by centrifugation. Both algal cells and aO'l ml sample
of the medium were extracted and the radioactive pulse counts/100 sec recorded on
a scintillation counter. This procedure was repeated until an optimum count was
recorded for the algal cells (see Fig. 1).
. l400rf
o I2OO:
o IOOO1
I
800
o
o
J 600
1 400
200
10
12 14 16
Time (hr)
18
I
I
I
20
22
24
26
FIG. 1. The uptake of C11 in a concentration of 1 mCi/ml of aqueous solution of Cu-labelled
sodium bicarbonate, by the algal cells in the culture of Dunaliella tertiolecta used in experiment 2.
4
(-) mother liquor; •
• , algae.
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METHODS
The algal cultures used in experiments 1 and 2 were of Dunaliella tertiolecta
(Ghlorophyceae) and in experiment 3 a culture of Isochrysis galbana (Ghrysophyceae)
was used. The feeding periods allowed for the oysters in the experiments were:
MATHERS: DIGESTION IN OSTREA
117
I D E N T I F I C A T I O N O F G" IN T H E D I G E S T I V E
DIVERTIGULA
Ten minutes after the commencement of feeding C14 was noted in the lumina of
the primary and secondary ducts with a brush-border epithelium. This epithelium
contained a number of vacuoles which appeared to contain a homogeneous scattering
of G14 activity. This was interpreted as being the products of extra-cellular digestion
in the lumen of the stomach. These vacuoles were larger in size and fewer in number
towards the base of the cells and suggested movement in this dirction. The lumina
of the ciliated gutters of the primary ducts contained no C14, and there were no
food vacuoles in the epithelium of this region.
At the 10-, 15- and 20-min stages (see Fig. 2a) only a small number of the tubules of
the diverticula showed C14 activity. Carbon14 labelled algal cells were phagocytosed
by the digestive cells of the tubules, but never by the crypt cells. The tubules themselves were all well formed with a tri- or quadri-partite lumen. Such algal cells as
were phagocytosed appeared in vacuoles in the cytoplasm of the digestive cells as
small clusters of G14 activity.
Very little C14 was noted in the connective tissue surrounding the stomach and
digestive diverticula. However, a few amoebocytes in the connective tissue, and the
majority of amoebocytes contained in the lumen of the blood vessels, were heavily
labelled with C14. The walls of these blood vessels showed only a low level of activity.
In the 30- and 40-min stages there was a general increase in activity in the ducts
and tubules of the diverticula. No whole algal cells were seen to be phagocytosed
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At such time the whole culture was centrifuged, washed twice with clean, filtered
seawater and on the third centrifugation the medium replaced by an equal volume
of clean sea water. Scintillation readings were taken again at this point and it was
found that the contamination of the water was less than 1%, the algal cells retaining
99% of the activity.
Before commencing the experiments the oysters, which had previously been
scrubbed and starved for 1 week, were each placed in a litre of clean sea water in a
large tank of water maintained at 18° G, and were left for 3 hr to await the establishment of phasic adduction indicated on the kymograph drum. After this settling
period a known volume of water was removed and replaced by an equal volume of
activated algal suspension. On completion of the predetermined feeding periods the
oysters were quickly removed and carefully dissected to avoid contamination; the
tissues were fixed in Duboscq's alcoholic Bouin, dehydrated and embedded in ester
wax.
Sections were cut at 5 /i.m and floated on water on slides previously cleaned in
chromic acid, washed and coated in a gelatin solution. Each slide was vertically
dipped into a liquid emulsion (Ilford K5 Nuclear Research Emulsion) and after a
2-hr drying period the slides were stored in light-proof boxes in a refrigerator at
4° G for 8 days. The emulsion was developed using Kodak D 19 B developer and
fixed in Johnson's Fix-Sol. The slides were lightly stained with haematoxylin-eosin.
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PROCEEDINGS OF THE MALACOLOGICAL SOCIETY
FIG. 2. (a) Autoradiograph of a transverse section through a primary duct of 0. edulis after being allowed to feed on algae labelled with a C14 isotope for 20 min. All stippling represents C u
activity; in this autoradiograph present in the non-ciliated lumen and epithelial vacuoles.
(b) Autoradiograph of a transverse section through a digestive tubule of 0. edulis after being
allowed to feed on algal cells labelled with a C14 isotope for 40 mins. All stippling represents C14
activity in the autoradiograph. Algal cells shown being phagocytosed and within epithelial
cells, (c) Autoradiograph of a transverse section through a digestive tubule of O. edulis after
being allowed to feed on algal cells labelled with a C14 isotope for 120 min. All stippling in
the diagram represents C14 activity. G14 present in vacuoles in the epithelium and in spherules
in the lumen of the tubule. A, Amoebocyte; AC, active algal cell; BB, brush-border epithelium ; BFV, basal food vacuole; BG, basal granules; C, cilia; CG, crypt cell; CG, ciliated gutter;
C14V, vacuole with C14 activity; DC, digestive cell; DFV, distal food vacuole; DV, distal
vacuole: L, lumen; MB, muscle band; N, nucleus of epithelial cell; PNB, peripheral nuclear
band; S, spherules; WM, waste matter.
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by the vacuoles in the brush-border epithelial cells of the ducts and it appeared
that the food material taken in here was either finely particulate or soluble, indicating
previous extra-cellular digestion.
There was an overall increase in the size of the lumina of the tubules compared
with earlier stages. The digestive cells were more vacuolated distally and the cytoplasm and nuclei of the cells concentrated in a basal position. An increase in the
number of G14 clusters in the distal regions of the digestive cells was obvious, and
also in the numbers of large vacuoles in the basal regions of these cells. The crypt
cells showed no G11 activity nor the presence of amoebocytes or vacuoles (see Fig. 2b).
There was also an increase in the amount of C 11 stored in the connective tissue.
Amoebocytes in the connective tissue and also in the lumina of the blood vessels
showed the same respective degrees of activity as before.
At the 60-min stage there was a further general increase in C 14 activity in the
epithelial cells and amoebocytes. In the brush-border epithelial cells of the ducts
small vacuoles were still present distally, and these appeared to move down the cells
to fuse and form a smaller number of large basal vacuoles. Although the lumen of
the ciliated gutter remained free of activity, a band of G14 was now present in the
extreme distal regions of these ciliated epithelial cells; presumably it represented a
supply of nutriment to an area of intense activity.
The digestive cells of the tubules were beginning to round off distally and these
regions were highly vacuolated and contained brown pigmented 'waste' material as
well as phagocytosed algal cells. The C14 activity in the connective tissue noted in
earlier stages continued to increase.
The 90-min stage appeared to be the most critical. The lumina of the ducts bordered
by a brush-border epithelium were now empty of G14 material, although a few
vacuoles in the cells were still active. The distal band of C14 activity was still evident
underlying the cilia of the 'gutter' cells, and for the first time active material (some
of it pigmented) was found in the lumen of the ciliated gutter. This was presumably
rejected material moving out from the tubules towards the lumen of the stomach.
The distal ends of the digestive cells of the tubules were almost completely devoid
of cytoplasm and all that remained was very vacuolated and contained brown pig-
119
MATHERS: DIGESTION IN OSTREA
(a)
BG
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120
P R O C E E D I N G S OF T H E MALACOLOGIGAL S O C I E T Y
DISCUSSION--The autoradiographs showed that algae were present' in the lumina of the ducts
(excluding the ciliated gutter) only 10 min after being introduced into the water.
The level of G14 activity in the lumina.of the ducts increased up to the 30-min stage,
remained constant until" after the 90-min stage and ..then started to decrease. After
10 min the lumen of the main ducts bounded by the brush-border epithelium was
very active, as were the vacuoles in the epithelium itself. The lumen and epithelium
of the ciliated gutter^ on the other hand, remained free of activity. This gives support
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mental material and some C14 activity. Digestive cells at this stage were ready to
fragment distally and to nip off spherules from their apices. These nucleate spherules
containing waste material were occasionally seen free in the lumina of the tubules
and in the ciliated gutters of the primary ducts. G" continued to accumulate in the
connective tissue and there was a great increase in the number of amoebocytes containing C11 in the haemocoele and in the connective tissue. All amoebocytes in the
blood vessels remained heavily labelled. A low level of activity was noted in the
blood and in the walls of the blood vessels.
The sequence of breakdown of the tubules continued into the 120-min stage (see
Fig. 2c). The nucleate excretory spherules were nipped off distally from the digestive
cells and were liberated into the lumina of the tubules, which were now very large.
These spherules usually contained amoebocytes, waste material and also exhibited
some G14 activity. Large vacuoles and algal cells were still observed in the basal and
distal regions respectively of the digestive cells. The crypt cells remained free of
vacuoles and amoebocytes, and the little C14 activity present was concentrated in a
narrow band underlying the basal granules of the cilia.
Although C14 was absent in the lumina of the ducts, the epithelial cells of the
primary (with the exception of the ciliated gutter cells) and secondary ducts showed
signs of having recently absorbed soluble food material—small vacuoles were still
evident distally and appeared to migrate basally and fuse to form larger vacuoles.
Amoebocytes were still found free in the cytoplasm and in the vacuoles of the
epithelial cells of the ducts.
Excretory spheres were visible in the lumen of the ciliated gutter of the primary
ducts and were judged to be moving towards the lumen of the stomach. The epithelial
cells of the gutter did not form food vacuoles and with the exception of the distal
band of C", associated with the bases of the cilia, the level of activity remained low.
The level of activity in the connective tissue was the same as at the 90-min stage, as
were the levels of activity in the amoebocytes, both in the blood vessels and in the
haemocoele.
The 4- and 6-hr stages were very similar to the 2-hr stage described above, the
main differences being a slightly higher level of C14 activity in the digestive cells
and connective tissues and also generally a greater degree of breakdown of the cells
themselves.
MATHERS: DIGESTION IN OSTREA
121
The epithelium of the ciliated gutter of the primary ducts invariably lacked the
food vacuoles and C " activity which were identified in the brush-border epithelium
of the same ducts.
After a feeding period of 10 min C14 activity was noted in a small number of food
vacuoles in the digestive cells of a few tubules. This supports Yonge's theory (1926)
of the phagocytosis of food material by the digestive cells and its subsequent intracellular digestion. It offers no support to the theory of Mansour (1946, 1949),
Mansour-Bek (1946) and Mansour and Zaki (1947), who worked on a number of
species of bivalves, that the larger vacuolated cells of the tubules are not digestive
but solely secretory.
On the completion of 60 min feeding, all the tubules showed C14 activity. The
observations on the tubules of O. edulis during feeding were very similar to those
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for the concept of a counter-flow movement of food particles from the stomach to
the digestive diverticula by way of the main ducts. Such a theory was suggested
by Owen (1955) who observed that the cilia of the gutter of the primary ducts beat
actively away from the digestive tubules and towards the stomach; he suggested
that this active exhalant current created an inhalant counter-current in the nonciliated region of the ducts.
Even at the 10-min stage many vacuoles, all containing G14 activity, were present
in the brush-border epithelium of the ducts. Numerous small vacuoles appeared
distally and then appeared to move basally through the cells where they eventually
fused with similar vacuoles to form a smaller number of large vacuoles. The contents
of all these vacuoles were interpreted as being of a soluble nature or of microscopic
granules—no algal clusters of C 14 activity were observed at any time. It would
therefore appear that within 10 min extra-cellular enzymes had acted on the algae
in the lumen of the stomach to produce a soluble food material or to liberate
membrane-bound constituents of the cytoplasm of algal cells. Although no direct
evidence was available it seems very likely that these large vacuoles liberate
their contents directly into the haemocoele. This supposition is supported by
the rapid appearance of G14 activity free in the haemocoele in the early
stages of feeding. Although amoebocytes were present in the vacuoles, and free in
the haemocoele, only a small percentage of them was labelled with C " . It is suggested
that once these 'partially digested' products have been released into the haemocoele
surrounding the digestive diverticula they are passed in the blood spaces to the
pericardial gland, heart and to the blood vessels, and in these regions they are phagocytosed by amoebocytes for subsequent distribution around the body. Support for
this theory is gained from the observation of blood vessels showing all the enclosed
amoebocytes with a high C14 activity, but only a small proportion of amoebocytes
outside the blood vessels, in the haemocoele and connective tissue, similarly labelled.
The walls of the blood vessels themselves were also labelled, but usually at only a low
level. Although this opposes Yonge's concept (1926) of wandering amoebocytes carrying food material through the wall of the gut and tubules into the blood vessels, it
still supports the idea that the amoebocytes are of primary importance in the translocation of food around the body.
122
PROCEEDINGS OF T H E MALAGOLOGICAL SOCIETY
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made on Lasaea rubra by Morton (1956). The digestive cells of the tubules phagocytosed whole algal cells distally and formed vacuoles which moved basally and
coalesced, the contents becoming diffuse. As was observed in the epithelial cells of
the ducts, the end products of digestion in the basal vacuoles appeared to be released
directly into the haemocoele. The distal regions of the digestive cells became more
vacuolated with time and accumulated pigmented waste material. The nipping off
and release of excretory spherules into the lumina of the tubules commenced after
60-90 min and was very evident after 2 hr. Associated with the breakdown of the
cells was the appearance of amoebocytes in the lumina of the tubules and the increasing abundance of waste material labelled with C14 in the lumina of the ciliated
gutters of primary ducts—indicating that this is indeed a rejectory tract as suggested
by Owen (1955). After 6 hr 75% of the tubules were breaking down—this measure
of coordination may have been attributable to the 6-day 'fast' prior to the experiments, and it may well be that in nature breakdown is a continuous, staggered process.
At no time did the crypt cells contain any food vacuoles or C14 activity, except for
a narrow band of G14 associated with the basal granules of the cilia. Accordingly
they are judged to play no part in phagocytosis and intracellular digestion.
The excretory spherules as well as containing waste material may also contain
enzymes which could be released into the stomach for extracellular digestion if the
spherules ruptured in the lumen of the stomach. Thus the early occurrence of soluble
food material available for pinocytosis into the vacuoles of the epithelium of the
ducts can be attributed to extracellular digestion in the lumen of the stomach partly
by enzymes previously released from the digestive diverticula and partly by enzymes
liberated by dissolution of the crystalline style.
After feeding for 2 hr the lumina of the ducts, with the exception of the ciliated
gutters of the primary ducts, were free from activity while the tubules still retained
a high level of C14. The absence of C14 in the ducts at this stage was probably the
result of the oysters having filtered all the labelled algae that were in suspension in
the sea water.
During the 6 hr covered by the experiments there was a continual increase in
activity in the connective tissue surrounding the digestive diverticula—indicating the
importance of this region as a food storage area. There was also an increase in the
number of amoebocytes exhibiting C14 activity which were free in the haemocoele
and connective tissue.
All experiments, including that lasting 6 hr, were conducted while the animals
were attached to revolving kymograph drums, and in all cases were seen to be
actively adducting. Since adduction of the valves is associated with nitration and
feeding, and since autoradiographs of the digestive diverticula throughout the experiments showed intracellular digestion, it must be concluded that in Ostrea edulis
feeding, digestion and the extrusion of waste spherules from the digestive tubules
are all carried out while the animal is adducting. It also appears that the oysters
can continue feeding at the same time that intra-cellular digestion is occurring in
the digestive diverticula.
It may well be that O. edulis is adapted to estuarine life by the ability to com-
M A T H E R S : D I G E S T I O N IN OSTREA
123
mence feeding at such times as the tidal stream supplies suitable nutritious material,
which might only be for a short time, and in this case discontinuous feeding, as
opposed to continuous feeding, could be an adaptation to an estuarine mode of life.
(1) Algal cells entered the primary and secondary ducts of the digestive diverticula
within 10 min from the commencement of feeding.
(2) Soluble, or finely particulate, products of extracellular digestion of the algae
were absorbed by the brush-border epithelial cells of the ducts into vacuoles which
passed down to the basement membrane of the cells and probably discharged their
contents directly into the haemocoele.
(3) Intact algal cells were phagocytosed by the digestive cells of the tubules within
10 min after the commencement of feeding. These accumulated in large vacuoles in
the basal regions of the digestive cells; the majority of these vacuoles contained at
least one amoebocyte.
(4) In the early stage of feeding the ciliated gutter of the primary ducts remained
free of G14 activity. However, after 90 min G14 and waste material were evident in
the gutter—giving direct support for a two-way movement of material in the primary
ducts of the digestive diverticula.
(5) After a short period of experimental feeding all amoebocytes enclosed within
the walls of blood vessels were heavily labelled with G14 as were a few outside in the
surrounding connective tissue. The function of the amoebocytes appears to be
primarily the translocation of food material.
(6) No support was gained for Mansour's theory (1946, 1949) in which the large
vacuolated cells of the tubules were attributed with a secretory, as opposed to a
digestive, role.
(7). The crypt cells of the tubules remained free of radioactivity and played no
role in intracellular digestion.
(8) A sequence of events was observed in the tubules which was similar to that
described by Morton (1956) in Lasaea rubra.
(9) The release of 'excretory' spherules was observed from the distal regions of
the digestive cells of the tubules. These pass into the stomach lumen where their
rupture may result in the release of digestive enzymes capable of effecting extracellular digestion.
(10) The connective tissue surrounding the digestive diverticula and stomach was
identified as an important food storage region.
(11) Both inter- and intracellular digestion of food material occurs while the
oysters are still actively adducting, and therefore while filtering and feeding.
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SUMMARY
The purpose of this series of experiments was to allow experimental animals, Ostrea
edulis, to feed upon a natural food, unicellular algae, which had been labelled with
Carbon14, and then to trace the course of the food at different time intervals through
the digestive diverticula. The following observations were made :
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PROCEEDINGS OF T H E MALACOLOGICAL
SOCIETY
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ACKNOWLEDGMENTS
During the course of the investigations the writer has been supported by a research
. studentship awarded by Chelsea College of Science and Technology.
I would like to express my gratitude to Professor R. D. Purchon, under whose
direction the investigation was carried out, for his encouragement and advice, and
for his assistance in the preparation of this manuscript. Thanks are also due to Dr
S. R. C. Hughes of the Department of Chemistry for his advice concerning radioactive
isotopes, to Mr J. A. Baylie and technical staff for their practical assistance, and
also to Professor G. Owen for critically reviewing this paper.